European Journal of Nuclear Medicine and Molecular Imaging

Tryptophan (Trp) is the precursor for serotonin synthesis and other biologically relevant metabolites. We evaluated the novel radiotracer 7-[18F]Fluorotryptophan (7-[18F]FTrp) to assess its biodistribution, dosimetry, and potential for imaging brain Trp metabolism in humans. Six healthy volunteers underwent whole-body PET/CT imaging over 5.5 hours following intravenous injection of 7-[18F]FTrp. An additional four subjects underwent dynamic brain PET imaging for 2 hours. Time-activity curves (TACs) were extracted for source organs using VOIs defined on co-registered CT and PET images, and dosimetry was calculated using OLINDA software. The radiotracer showed rapid uptake and distribution, with highest activity observed in the liver, pancreas, salivary glands, in combination with urinary excretion. Brain pharmacokinetic analyses with image-derived input function (IDIF) determined that Patlak analyses were the best fit for brain image analyses. Brain uptake was modest, with highest region-specific accumulation in the pineal gland, which is a known site for serotonin synthesis. The estimated effective dose was within the expected range for 18F-labeled compounds (14.1 {+/-} 0.2 Sv/MBq). Our findings indicate that 7-[18F]FTrp is safe for human use, demonstrates favorable kinetics for studying both brain and peripheral Trp metabolism, and warrants further exploration in patients with serotonin metabolism disorders.

BackgroundB7-H4 is a cell surface ligand overexpressed by tumors to inhibit T cell functions and evade the immune system. B7-H4 is minimally expressed in normal tissues but is highly expressed by various cancer cells and tumor-associated macrophages (TAM). Despite its importance as an immune checkpoint inhibitor, no imaging techniques specifically targeting B7-H4 have been established. To close this gap, we sought to assess the ability of a monoclonal antibody (mAb) based immunoPET radiotracer to visualize B7-H4 in human and murine prostate cancer models. MethodsAnti-B7-H4 mAb clone 2H9 was functionally characterized for binding to the human and mouse B7-H4 protein. The antibody was conjugated with chelator p-SCN-Bn-Deferoxamine (DFO) and labeled with radioisotope Zirconium-89 (89Zr) to obtain immunoPET tracer 89Zr-2H9-mAb. The biolayer interferometry method was used to test the binding kinetics of DFO-2H9-mAb compared to that of parental 2H9 mAb. A group of six athymic nude mice with human DU145 prostate tumor xenograft underwent MicroPET imaging after tail vein injection of [~]150{micro}Ci 89Zr-2H9-mAb or non-binding 89Zr-Isotype-mAb. Next, immunocompetent C57BL/6J mice with TRAMP-C2 tumors each were injected with either PBS (n=8), cold 2H9 mAb (10mg/kg) to block B7-H4 (n=6), or chlodronate liposome (15mg/kg) to cause total macrophage depletion (n=6), followed by 89Zr-2H9-mAb MicroPET imaging. An ex vivo biodistribution assay was performed after 144 hr post radiotracer injection. Tumor radiotracer binding, quantified as a percentage injected dose per gram (%ID/g), was compared between different experimental groups using two-way ANOVA with Bonferroni or Tukey corrections. ResultsImmunoconjugation yielded a 2.59 {+/-} 0.08 chelator-to-antibody ratio, and the binding of DFO conjugated 2H9-mAb was similar to that of parental 2H9 mAb, with unaffected affinity in targeting B7-H4 protein moiety. The radiochemical purity of 89Zr-2H9-mAb tracer was yielded >95% with an average specific activity of 5{micro}Ci/{micro}g antibody. DU145 tumor xenografts demonstrated significantly stronger radiotracer binding at 24, 48, 72, 96, and 120 hr than the non-binding isotype control group. In TRAMP-C2 tumor xenografts, the radiotracer binding in B7-H4 blocked tumors was significantly lower than in the non-blocked PBS-injected group. Macrophage depletion resulted in a significant decrease in tumor binding compared to the control group. 89Zr-2H9-mAb could efficiently distinguish tumors with high sensitivity, showing a high correlation between PET imaging and bio-distribution. More importantly, the immunohistochemistry of the harvested tumor revealed no significant difference between the three groups, as discernible through in vivo PET imaging. ConclusionThis study highlights the potential of B7-H4 immunoPET imaging for monitoring immunotherapy response. With the emerging potential of B7-H4 blocking as an immunotherapeutic, immunoPET imaging could be readily expanded to patient stratification and therapy monitoring. B7-H4 imaging could augment our understanding of B7-H4 dynamics in response to various therapeutic interventions in clinical trials. The new B7-H4 immunoPET probe is, in principle, clinically translatable.

The synaptic vesicle protein SV2C, predominantly found in the basal ganglia, has been associated with Parkinsons disease through genetic studies. It plays a crucial role in regulating dopamine release and has been shown to be disrupted in PD animal models and brain tissues from PD patients. In the context of PD-related synaptopathy, SV2C may serve as a potential imaging target for monitoring disease progression and response to treatment. [18F]UCB-F is a radioligand binding to SV2C developed by UCB. Preliminary autoradiography and PET studies in rats showed that [18F]UCB-F displays a brain distribution consistent with the expression of SV2C in vitro but does not display any specific binding in vivo. This study was therefore designed to further investigate the affinity and selectivity of [18F]UCB-F for SV2C and to examine the in vitro and in vivo properties of the radioligand in non-human primates. In vitro binding studies were performed to measure the affinity of UCB-F to SV2A, SV2B, and SV2C. Insilico modeling was used to assess the binding mode and energy of UCB-F. Autoradiography studies on rat and non-human primate (NHP) brain tissues were performed to confirm that [18F]UCB-F showed similar distribution in rat and NHP tissue. Finally, PET studied in NHPs were performed to examine the in vivo pharmacokinetic properties of [18F]UCB-F. [18F]UCB-F was successfully synthesized from the corresponding precursor with high yield. Autoradiography on brain slices from rats and NHPs demonstrated specific binding of [18F]UCB-F in the pallidum, striatum, substantia nigra, and brainstem, consistent with the known brain expression of SV2C. In NHPs, [18F]UCB-F rapidly crossed the blood-brain barrier, reaching peak uptake values of 2.8 %ID in NHP1 and 2.1 %ID in NHP2 at 4 minutes post-injection. The tracer wasrapidly washed out from the brain, with no clear regional distribution. Radiometabolite analysis revealed the formation of only more polar radiometabolites, with approximately 15% of unchanged radioligand remaining in plasma at 15 minutes post-injection. In vitro and in-silico studies demonstrated that the affinity of [18F]UCB-F decreased by approximately one factor of magnitude with increase of temperature from 4{degrees} to 37{degrees} C. This temperature-related decrease of the affinity for SV2C together with rapid in vivo radiometabolism might explain the discrepancy between in vitro and in vivo performance of [18F]UCB-F. Overall, these findings suggest that [18F]UCB-F is not a suitable PET radioligand for imaging SV2C. Further research is needed to identify alternative candidates with improved in vivo stability and brain retention.

Gabapentin, a selective ligand for the 2{delta} subunit of voltage-dependent calcium channels, is an anticonvulsant medication used in the treatment of neuropathic pain, epilepsy and other neurological conditions. We recently described two radiofluorinated derivatives of gabapentin (trans-4-[18F]fluorogabapentin, [18F]tGBP4F, and cis-4-[18F]fluorogabapentin, [18F]cGBP4F) and showed that these compounds accumulate in the injured nerves in a rodent model of neuropathic pain. Given the use of gabapentin in brain diseases, here we investigate whether these radiofluorinated derivatives of gabapentin can be used for imaging 2{delta} receptors in the brain. Specifically, we developed automated radiosynthesis methods for [18F]tGBP4F and [18F]cGBP4F and conducted dynamic PET imaging in adult rhesus macaques with and without preadministration of pharmacological doses of gabapentin. Both radiotracers showed very high metabolic stability, negligible plasma protein binding and slow accumulation in the brain. [18F]tGBP4F, the isomer with higher binding affinity, showed low brain uptake and could not be displaced whereas [18F]cGBP4F showed moderate brain uptake and could be partially displaced. Kinetic modeling of brain regional time-activity curves using a metabolite-corrected arterial input function shows that a 1-tissue compartment model accurately fits the data. Graphical analysis using Logan or multilinear analysis 1 produced similar results as compartmental modeling indicating robust quantification. This study advances our understanding of how gabapentinoids work and provides an important advancement towards imaging 2{delta} receptors in the brain.

BackgroundBispecific antibodies exploiting receptor-mediated transcytosis offer a promising strategy to overcome limited blood-brain barrier permeability in Alzheimers disease (AD) therapy and imaging. Lecanemab-Fab8D3 (Lec-Fab8D3), a bispecific anti-amyloid beta (A{beta}) antibody engineered for enhanced brain delivery, holds potential as a companion immunoPET imaging diagnostic with the novel lecanemab immunotherapy. This study aimed to compare three radionuclides--zirconium-89 (89Zr), copper-64 (64Cu), and iodine-124 (124I)--for PET imaging with Lec-Fab8D3 to study its in vivo brain distribution and evaluate its potential as an AD companion diagnostic. MethodsLec-Fab8D3 was conjugated to DFO* or NODAGA for 89Zr and 64Cu radiolabeling, respectively, or directly radioiodinated with 124I. PET imaging was performed in the Tg-ArcSwe mouse model of A{beta} pathology and wild-type (WT) littermates at multiple time points post administration of the radiolabeled antibody, followed by ex vivo biodistribution, autoradiography, and A{beta} quantification to assess brain uptake, specificity, and distribution of the radiolabeled Lec-Fab8D3. ResultsRadiolabeled Lec-Fab8D3 variants showed retained binding properties with high radiochemical purity and yields. PET imaging demonstrated cortical brain uptake of all three tradiotracers in Tg-ArcSwe mice, with [89Zr]Zr-DFO*-Lec-Fab8D3 and [124I]I-Lec-Fab8D3 showing the best discrimination between Tg-ArcSwe and WT mice at 48-72 h post-injection. The highest absolute brain retention, combined with a lower brain-to-cerebellum ratio, was observed in both Tg-ArcSwe and WT mice that received the radiometal-labeled (89Zr and 64Cu) antibody, likely due to the residualizing nature of radiometals. Ex vivo analyses confirmed PET findings, and immunostaining demonstrated co-localization of Lec-Fab8D3 with A{beta} deposits. ConclusionsImmunoPET imaging with bispecific Lec-Fab8D3 enables specific detection of brain A{beta} pathology in an AD mouse model. 89Zr was superior to 64Cu due to a more compatible half-life, while 124I displayed higher regional contrast than both radiometals, despite lower overall brain signal. The combined findings from radiometal- and iodine-based immunoPET will enhance our understanding of intra-brain distribution of bispecific antibodies. Furthermore, this highlights the importance of the choice of radiolabeling strategy and how it will impact the outcome of immunoPET with bispecific A{beta} antibodies.

Routine clinical use of absolute PET quantification techniques is limited by the need for serial arterial blood sampling for input function and more importantly by the lack of automated pharmacokinetic analysis tools that can be readily implemented in clinic with minimal effort. PET/MRI provides the ability for absolute quantification of PET probes without the need for serial arterial blood sampling using image-derived input functions (IDIFs). Here we introduce caliPER, a modular and scalable software for simplified pharmacokinetic modelling of PET probes with irreversible uptake or binding based on PET/MR IDIFs and Patlak Plot analysis. caliPER generates regional values or parametric maps of net influx rate (Ki) using reconstructed dynamic PET images and anatomical MRI aligned to PET for IDIF vessel delineation. We evaluated the performance of caliPER for blood-free region-based and pixel-wise Patlak analyses of [18F] FDG by comparing caliPER IDIF to serial arterial blood input functions and its application in imaging brain glucose hypometabolism in Frontotemporal dementia. IDIFs corrected for partial volume errors including spill-out and spill-in effects were similar to arterial blood input functions with a general bias of around 6-8%, even for arteries <5 mm. The Ki and cerebral metabolic rate of glucose estimated using caliPER IDIF were similar to estimates using arterial blood sampling (<2%) and within limits of whole brain values reported in literature. Overall, caliPER is a promising tool for irreversible PET tracer quantification and can simplify the ability to perform parametric analysis in clinical settings without the need for blood sampling. HighlightsO_LIcaliPER is an adaptable image processing software for extracting image-derived input functions and generating parametric maps of irreversible PET tracer uptake. C_LIO_LIAnatomical (T1-weighted/T2-weighted/time-of-flight angiography) MRI carefully aligned to PET provides a robust approach for delineation of vessels on PET, eliminating the need for serial blood sampling for input functions. C_LIO_LIApplication of caliPER in modelling glucose uptake in patients with Frontotemporal dementia, demonstrates the feasibility of absolute quantification of cerebral metabolic rate of glucose in clinical populations. C_LI

We utilized positron emission tomography (PET) imaging in vivo to map the spatiotemporal biodistribution/expression (protein density) of class-IIa histone deacetylases (class-IIa HDACs) in the brain. Herein, we report an improved radiosynthesis of [18F]-NT160 using 4-hydroxy-TEMPO which led to a significant improvement in radiochemical yield and molar activity. PET imaging with [18F]-NT160, a highly potent class-IIa HDAC inhibitor with sub-nM affinity for HDAC4 and 5 isoforms, led to high-quality and high-contrast images among various brain regions. [18F]-NT160 displayed excellent pharmacokinetic and imaging characteristics: brain uptake is high in gray matter regions, leading to high-quality PET images; tissue kinetics are appropriate for an 18F tracer and specific binding for class-IIa HDACs is demonstrated by self-blockade. Higher uptake with [18F]-NT160 was observed in the hippocampus, thalamus, and cortex while there was relatively lower uptake in the cerebellum and striatum. Overall, our current studies with [18F]-NT160 will likely facilitate the development and clinical translation of class-IIa HDACs of the next generation of PET tracers for imaging and targeted therapy of cancer and the diseases of the central nervous system (CNS).

PurposeThe radionuclide pair cerium-134/lanthanum-134 (134Ce/134La) was recently proposed as a suitable diagnostic counterpart for the therapeutic alpha-emitter actinium-225 (225Ac). The unique properties of 134Ce offer perspectives for developing innovative in vivo investigations not possible with 225Ac. In this work, 225Ac- and 134Ce-labeled tracers were directly compared using internalizing and slow-internalizing cancer models to evaluate their in vivo comparability, progeny meandering, and potential as a matched theranostic pair for clinical translation. Despite being an excellent chemical match, 134Ce/134La has limitations to the setting of quantitative positron emission tomography imaging. MethodsThe precursor PSMA-617 and a macropa-based tetrazine-conjugate (mcp-PEG8-Tz) were radiolabelled with 225Ac or 134Ce and compared in vitro and in vivo using standard (radio)chemical methods. Employing biodistribution studies and positron emission tomography (PET) imaging in athymic nude mice, the radiolabelled PSMA-617 tracers were evaluated in a PC3/PIP (PC3 engineered to express a high level of prostate-specific membrane antigen) prostate cancer mouse model. The 225Ac and 134Ce-labeled mcp-PEG8-Tz were investigated in a BxPC-3 pancreatic tumour model harnessing the pretargeting strategy based on a trans-cyclooctene-modified 5B1 monoclonal antibody. ResultsIn vitro and in vivo studies with both 225Ac and 134Ce-labelled tracers led to comparable results, confirming the matching pharmacokinetics of this theranostic pair. However, PET imaging of the 134Ce-labelled precursors indicated that quantification is highly dependent on tracer internalization due to the redistribution of 134Ces PET-compatible daughter 134La. Consequently, radiotracers based on internalizing vectors like PSMA-617 are suited for this theranostic pair, while slow-internalizing 225Ac-labelled tracers are not quantitatively represented by 134Ce PET imaging. ConclusionWhen employing slow-internalizing vectors, 134Ce might not be an ideal match for 225Ac due to the underestimation of tumour uptake caused by the in vivo redistribution of 134La. However, this same characteristic makes it possible to estimate the redistribution of 225Acs progeny noninvasively. In future studies, this unique PET in vivo generator will further be harnessed to study tracer internalization, trafficking of receptors, and the progression of the tumour microenvironment. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/591165v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@ca3cb6org.highwire.dtl.DTLVardef@157eac6org.highwire.dtl.DTLVardef@f6ac6dorg.highwire.dtl.DTLVardef@b24d87_HPS_FORMAT_FIGEXP M_FIG C_FIG Redistribution of progeny. Investigating the 225Ac and 134Ce decay chain. This figure was created with BioRender.

Demyelination, the loss of the insulating sheath of neurons, causes failed or slowed neuronal conduction and contributes to the neurological symptoms in multiple sclerosis, traumatic brain and spinal cord injuries, stroke, and dementia. In demyelinated neurons, the axonal potassium channels Kv1.1 and Kv1.2, generally under the myelin sheath, become exposed and upregulated. Therefore, imaging these channels using positron emission tomography can provide valuable information for disease diagnosis and monitoring. Here, we describe the novel tracer for Kv1 channels [11C]3-methyl-4-aminopyridine ([11C]3Me4AP). [11C]3Me4AP was efficiently synthesized via Pd(0)-Cu(I) co-mediated Stille cross-coupling of a stannyl precursor containing a free amino group. Evaluation of its imaging properties in rats and nonhuman primates showed that [11C]3Me4AP has a moderate brain permeability and slow kinetics. Additional evaluation in monkeys showed that the tracer is metabolically stable and that a 1-tissue compartment model can accurately model the regional brain time-activity curves. Compared to the related tracers [18F]3-fluoro-4-aminopyridine ([18F]3F4AP) and [11C]3-methoxy-4-aminopyridine ([11C]3MeO4AP), [11C]3Me4AP shows lower initial brain uptake, which indicates reduced permeability to the blood-brain-barrier and slower kinetics, suggesting higher binding affinity consistent with in vitro studies. While the slow kinetics and strong binding affinity resulted in a tracer with less favorable properties for imaging the brain than its predecessors, these properties may make 3Me4AP useful as a therapeutic.

Determination of a drugs biodistribution is critical to ensure it reaches the target tissue of interest. This is particularly challenging in the brain where invasive sampling methods may not be possible. Here, a pretargeted imaging methodology is disclosed that utilizes bioorthogonal click chemistry to determine the distribution of an antisense oligonucleotide in the living brain following intrathecal dosing. A novel PET tracer, [18F]BIO-687, bearing a click-reactive trans-cyclooctene (TCO) was discovered and tested in conjunction with a Malat1 antisense oligonucleotide (ASO) conjugated with a methyltetrazine (MeTz). PET imaging in rats demonstrated that the tracer possesses good kinetic properties for CNS imaging and can react to form a covalent linkage with high specificity to the MeTz-conjugated-ASO in vivo. Further, the amount of tracer reacted by cycloaddition with the Tz was determined to be dependent on the concentration of ASO-MeTz in tissue, as determined through comparison of the imaging signal with the LC-MS of the tissue homogenate. The system was evaluated in cynomolgus monkeys, with PET imaging showing favorable tracer kinetics and specific binding to the ASO in vivo. These results demonstrate that the tracer [18F]BIO-687 can image intrathecally-delivered ASO distribution in the brain, and future studies should leverage this technology to evaluate ASO distribution in human subjects to study distribution. One Sentence SummaryDistribution of an intrathecally administered antisense oligonucleotide can be imaged using a pretargeted approach in the living brains of non-human primates.

Cerenkov (or Cherenkov) luminescence occurs when charged particles exceed the phase velocity of a given medium. Cerenkov has gained interest in preclinical space as well as in clinical trials for optical visualization of numerous radionuclides. However, Cerenkov intensity has to be inferred from alternative databases with energy emission spectra, or theoretical fluence estimates. Here we present the largest experimental dataset of Cerenkov emitting isotopes recorded using the IVIS optical imaging system. We report Cerenkov measurements spanning orders of magnitude normalized to the activity concentration for 21 Cerenkov emitting isotopes, covering electron, alpha, beta minus, and positron emissions. Isotopes measured include Carbon-11, Fluorine-18, Phosphorous-32, Scandium-47, Copper-64, Copper-67, Gallium-68, Arsenic-72, Bromine-76, Yttrium-86, Zirconium-89, Yttrium-90, Iodine-124, Iodine-131, Cerium-134, Lutetium-177, Lead-203, Lead-212, Radium-223, Actinium-225, and Thorium-227. We hope this updating resource will serve as a rank ordering for comparing isotopes for Cerenkov luminescence in the visible window and serve as a rule of thumb for comparing Cerenkov intensities in vitro and in vivo. MethodsAll Cerenkov emitting radionuclides were either produced at Memorial Sloan Kettering Cancer Center (Carbon-11, 11C; Fluorine-18, 18F; Iodine-124, 124I), from commercial sources such as Perkin Elmer (Phosphorous-32, 32P; Yttrium-90, 90Y), Bayer (Radium-223, 223Ra, Xofigo), 3D-Imaging (Zirconium-89, 89Zr), Nuclear Diagnostic Products (Iodine-131, 131I), or from academic collaborators at Washington University at St. Louis (Copper-64, 64Cu), University of Wisconsin (Bromine-76, 76Br), MD Anderson Cancer Center (Yttrium-86, 86Y), Brookhaven National Laboratory (Arsenic-72, 72As; Thorium-227, 227Th), or Oak Ridge National Laboratory (Cerium-134, 134Ce, Actinium-225, 225Ac), and Viewpoint Molecular Targeting (Lead-203, 203Pb; Lead 212, 212Pb). All isotopes were diluted in triplicate on a black bottomed corning 96 well plate to several activity concentrations ranging from 0.1-250 Ci in 100-200 L of Phosphate Buffered Saline. Cerenkov imaging was acquired on a single Perkin-Elmer Spectrum In-Vivo Imaging System (IVIS) at field of view c with exposures ranging up to 15 minutes or lower provided no part of the image intensity was saturated, or that the activity significantly changed during the exposure. Experimental radiances on the IVIS were calculated from regions of interest drown over each 96 well, and then normalized for the activity present in the well, and the volume the isotope was diluted into.

ObjectivesDisialoganglioside 2 (GD2), overexpressed by cancers such as melanoma and neuroblastoma, is a tumor antigen for targeted therapy. The delivery of conventional IgG antibody technologies targeting GD2 is limited clinically by its co-expression on nerves that contributes to toxicity presenting as severe neuropathic pain. To improve the tumor selectivity of current GD2-targeting approaches, a next-generation bispecific antibody targeting GD2 and B7-H3 (CD276) was generated. MethodsDifferential expression of human B7-H3 (hB7-H3) was transduced into GD2+ B78 murine melanoma cells and confirmed by flow cytometry. We assessed the avidity and selectivity of our GD2-B7-H3 targeting bispecific antibodies (INV34-6, INV33-2, and INV36-6) towards GD2+/hB7-H3- B78 cells relative to GD2+/hB7-H3+ B78 cells using flow cytometry and competition binding assays, comparing results an anti-GD2 antibody (dinutuximab, DINU). The bispecific antibodies, DINU, and a non-targeted bispecific control (bsAb CTRL) were conjugated with deferoxamine for radiolabeling with Zr-89 (t1/2 = 78.4 h). Using positron emission tomography (PET) studies, we evaluated the in vivo avidity and selectivity of the GD2-B7-H3 targeting bispecific compared to bsAb CTRL and DINU using GD2+/hB7-H3+ and GD2+/hB7-H3- B78 tumor models. ResultsFlow cytometry and competition binding assays showed that INV34-6 bound with high avidity to GD2+/hB7-H3+ B78 cells with high avidity but not GD2+/hB7-H3+ B78 cells. In comparison, no selectivity between cell types was observed for DINU. PET in mice bearing the GD2+/hB7-H3- and GD2+/hB7-H3+ B78 murine tumor showed similar biodistribution in normal tissues for [89Zr]Zr-Df-INV34-6, [89Zr]Zr-Df-bsAb CTRL, and [89Zr]Zr-Df-DINU. Importantly, [89Zr]Zr-Df-INV34-6 tumor uptake was selective to GD2+/hB7-H3+ B78 over GD2+/hB7-H3- B78 tumors, and substantially higher to GD2+/hB7-H3+ B78 than the non-targeted [89Zr]Zr-Df-bsAb CTRL control. [89Zr]Zr-Df-DINU displayed similar uptake in both GD2+ tumor models, with uptake comparable to [89Zr]Zr-Df-INV34-6 in the GD2+/hB7-H3+ B78 model. ConclusionThe GD2-B7-H3 targeting bispecific antibodies successfully improved selectivity to cells expressing both antigens. This approach should address the severe toxicities associated with GD2-targeting therapies by reducing off-tumor GD2 binding in nerves. Continued improvements in bispecific antibody technologies will continue to transform the therapeutic biologics landscape. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/595624v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@11afee9org.highwire.dtl.DTLVardef@1558607org.highwire.dtl.DTLVardef@1d23d6eorg.highwire.dtl.DTLVardef@1bf1d63_HPS_FORMAT_FIGEXP M_FIG C_FIG

INTRODUCTIONDoxil/Caelyx is a PEGylated liposomal formulation of the chemotherapeutic doxorubicin used in the clinic for Kaposis sarcoma, advanced ovarian cancer, progressive multiple myeloma and metastatic breast cancer. Talidox(R), a smaller doxorubicin PEGylated liposome is undergoing clinical trials and has been proposed as an improvement on previous liposomal formulations for the treatment of advanced solid tumors. We aimed to validate an easily translatable radiolabeling method using zirconium-89 (89Zr) that enables quantitative whole-body PET imaging of these formulations to study their biodistribution and pharmacokinetics. METHODS[89Zr][Zr(oxinate)4] was produced using a kit-based approach followed by use as a direct radiolabeling agent of the liposomal formulations. DFT studies were performed to elucidate the mechanism behind the radiolabeling stability observed within the liposomes. Purified 89Zr-labelled Doxil/Talidox(R) liposomes (5 mg/kg doxorubicin dose) were administered in female BALB/c mice bearing 4T1 tumors. PET/CT imaging was acquired at 20 min, 24 h, 48 h, and 72 h, followed by post-mortem biodistribution at 72 h. RESULTS and DISCUSSIONBoth formulations were radiolabeled efficiently with high stability in serum in vitro for 72 h. In vivo, both formulations showed high tumor uptake at 72 h (18.5 {+/-} 2.4 % IA/g for Doxil and 20.2 {+/-} 2.3 % IA/g for Talidox). In general, ex vivo biodistribution showed similar uptake values for both formulations with high spleen/liver uptake and low bone uptake, confirming stability. Talidox(R) showed significantly lower spleen uptake and higher uptake in bone than Doxil. DFT studies confirmed that doxorubicin can form complexes with 89Zr that are more stable than [89Zr][Zr(oxinate)4], explaining the radiolabeling mechanism and stability results in vitro and in vivo. CONCLUSIONSClinically available PEGylated liposomes containing doxorubicin can be efficiently radiolabelled with 89Zr for PET imaging studies, using a clinically translatable radiolabelling method. HighlightsO_LIDoxorubicin-containing liposomes can be labeled with the positron-emitting radionuclide 89Zr with no impact on their original physicochemical properties. C_LIO_LIRadiolabeling is stable in vivo and enables imaging and biodistribution studies of the liposomes using positron emission tomography (PET). C_LIO_LIThe radiolabeling method is clinically translatable and would allow early assessment of existing and novel doxorubicin liposome biodistribution in humans or personalized medicine (nanotheranostic) approaches. C_LI

Immunotherapies, especially the checkpoint inhibitors such as anti-PD-1 antibodies, have transformed cancer treatment by enhancing immune systems capability to target and kill cancer cells. However, predicting immunotherapy response remains challenging. 18F-AraG is a molecular imaging tracer targeting activated T cells, which may facilitate therapy response assessment by non-invasive quantification of immune cell activity within tumor microenvironment and elsewhere in the body. The aim of this study was to obtain preliminary data on total-body pharmacokinetics of 18F-AraG, as a potential quantitative biomarker for immune response evaluation. MethodsThe study consisted of 90-min total-body dynamic scans of four healthy subjects and one non-small cell lung cancer (NSCLC) patient, scanned before and after anti-PD-1 immunotherapy. Compartmental modeling with Akaike information criterion model selection were employed to analyze tracer kinetics in various organs. Additionally, seven sub-regions of the primary lung tumor and four mediastinal lymph nodes were analyzed. Practical identifiability analysis was performed to assess reliability of kinetic parameter estimation. Correlations of SUVmean, SUVR (tissue-to-blood ratio), and Logan plot slope (KLogan) with total volume-of-distribution (VT) were calculated to identify potential surrogates for kinetic modeling. ResultsStrong correlations were observed between KLogan and SUVR values with VT, suggesting that they can be used as promising surrogates for VT, especially in organs with low blood-volume fraction. Moreover, the practical identifiability analysis suggests that the dynamic 18F-AraG PET scans could potentially be shortened to 60 minutes, while maintaining quantification accuracy for all organs-of-interest. The study suggests that although 18F-AraG SUV images can provide insights on immune cell distribution, kinetic modeling or graphical analysis methods may be required for accurate quantification of immune response post-therapy. While SUVmean showed variable changes in different sub-regions of the tumor post-therapy, the SUVR, KLogan, and VT showed consistent increasing trends in all analyzed sub-regions of the tumor with high practical identifiability. ConclusionOur findings highlight the promise of 18F-AraG dynamic imaging as a non-invasive biomarker for quantifying the immune response to immunotherapy in cancer patients. The promising total-body kinetic modeling results also suggest potentially wider applications of the tracer in investigating the role of T cells in the immunopathogenesis of diseases.

Aim/IntroductionDifferentiating malignant from inflammatory uptake on 18F-FDG PET/CT remains a major diagnostic challenge, as standardized uptake value (SUV) lacks specificity. Dynamic acquisitions with Patlak analysis can separate metabolized from unmetabolized tracer, potentially improving discrimination. We evaluated whether short-duration dynamic FDG PET/CT with Patlak parametric imaging provides complementary information beyond SUV for distinguishing malignancy from inflammation. Materials and MethodsTwenty-seven patients undergoing oncologic PET/CT (breast, lung, or gastrointestinal cancer) were included, yielding 96 lesions (69 malignant, 27 inflammatory). Short dynamic acquisitions (20 min) were motion-corrected and analysed to generate influx rate (Ki) and distribution volume (Vd) maps. Lesions were segmented on SUV images (40% SUVmax), and radiomic features were extracted from SUV, Ki, and Vd maps. Exploratory data analysis, linear modelling, and dimensionality reduction assessed separability. A Random Forest classifier was trained with crossvalidation, integrating Synthetic Minority Oversampling (SMOTE) to address class imbalance. An independent validation cohort of 15 lesions (13 inflammatory, 2 malignant) was tested. ResultsMalignant lesions showed higher SUVmean (5.8 vs. 2.8 g/ml) and Ki (1.95 vs. 0.75 ml/min/100ml), whereas inflammatory lesions demonstrated higher Vd (44.7 vs. 35.1%). No single feature provided reliable thresholds. Logistic regression achieved 89% accuracy but suffered from quasi-separation, confirming limited linear discriminability. Random Forest classification yielded robust performance (cross-validated AUC-ROC 0.876; AUC-PR 0.948). With G-mean thresholding, inflammation was detected with high recall (0.93) but recall for malignancy was lower (0.74). Feature importance highlighted SUV and Ki variance, as well as Ki/ Vd ratios, as strongest predictors. In the external validation set, accuracy reached 0.80, with inflammation reliably identified (precision 0.85, recall 0.85). ConclusionShort dynamic Patlak imaging combined with machine learning improves the characterization of malignant versus inflammatory uptake beyond SUV alone. By decomposing FDG up-take into metabolized (Ki) and unmetabolized (Vd) fractions, this approach provides physiologically meaningful separation of tracer behaviour. While sensitivity for malignancy requires further optimization, our findings establish a reproducible framework for future more extensive research on clinical interpretation of parametric imaging in oncologic PET.

Background: Prostate-specific membrane antigen (PSMA) PET/CT is central to prostate cancer staging and theranostic workflows. To our knowledge, no direct within-patient comparison of [18F]FC303 ([18F]Florastamin) and [68Ga]Ga-PSMA-11 has been reported. We performed a preliminary paired method-comparison study under non-harmonized acquisition protocols. Patients and Methods: Twenty patients with histologically confirmed prostate cancer underwent [68Ga]Ga-PSMA-11 PET/CT (185 +/- 37 MBq, 60 +/- 10 min) followed by [18F]FC303 PET/CT (370 +/- 37 MBq, 105 +/- 15 min) on the same PET/CT system within each patient (median interval, 29.5 days). Index targets were anatomically matched to the biopsied or surgically sampled lesion or target region. The primary malignant set included 18 histologically malignant targets; two histology-negative or indeterminate targets were included only in sensitivity analysis. Fixed [68Ga]Ga-PSMA-11-first scan order and the 45-min uptake-time difference were central interpretive constraints. Results: Across five predefined reference organs, [18F]FC303 showed lower SUVmean than [68Ga]Ga-PSMA-11 (all Benjamini-Hochberg-adjusted p < 0.001; [68Ga]/[18F]FC303 geometric mean ratio [GMR], 1.29-3.89). In the primary malignant set, [18F]FC303 lesion SUVmax was lower than [68Ga]Ga-PSMA-11 (median, 11.3 vs 18.1; paired median difference, -5.50; 95% CI, -6.85 to -2.90; Wilcoxon p = 8.4 x 10-4), with strong rank correlation (Spearman {rho} = 0.90). Passing-Bablok regression yielded {beta} = 1.13 (95% CI, 1.04-1.45), and log-Bland-Altman GMR (FC303/[68Ga]) was 0.75, consistent with proportional non-interchangeability. Tumor-to-liver and tumor-to-mediastinum ratios did not differ significantly (GMR, 1.17 [95% CI, 0.94-1.45] and 0.96 [0.80-1.15], respectively); the study was not powered for equivalence. The n = 20 sensitivity analysis showed consistent directionality. Conclusions: Under non-harmonized acquisition conditions, [18F]FC303 showed lower physiologic reference-organ SUVmean and malignant target-region SUVmax than [68Ga]Ga-PSMA-11, whereas tumor-to-liver and tumor-to-mediastinum ratios were not significantly different. Absolute SUVs were not interchangeable; [68Ga]Ga-PSMA-11-derived SUV thresholds should not be directly transferred to [18F]FC303 without tracer-specific calibration.

Accurate quantification of tau binding from 18F-PI-2620 PET requires kinetic modeling and an input function. Here, we implemented a non-invasive Image-derived input function (IDIF) derived using the state-of-the-art total-body uEXPLORER PET/CT scanner to quantify tau binding and tracer delivery rate from 18F-PI-2620 in the brain. Additionally, we explored the impact of scan duration on the quantification of kinetic parameters. Total-body PET dynamic data from 15 elderly participants were acquired. Time-activity curves from the grey matter regions of interest (ROIs) were fitted to the two-tissue compartmental model (2TCM) using a subject-specific IDIF derived from the descending aorta. ROI-specific kinetic parameters were estimated for different scan durations ranging from 10 to 90 minutes. Logan graphical analysis was also used to estimate the total distribution volume (VT). Differences in kinetic parameters were observed between ROIs, including significant reduction in tracer delivery rate (K1) in the medial temporal lobe. All kinetic parameters remained relatively stable after the 60-minute scan window across all ROIs, with K1 showing high stability after 30 minutes of scan duration. Excellent correlation was observed between VT estimated using 2TCM and Logan plot analysis. This study demonstrated the utility of IDIF with total-body PET in investigating 18F-PI-2620 kinetics in the brain.

The traditional design of PET target engagement studies is based on a baseline scan and one or more scans after drug administration. We here evaluate an alternative design in which the drug is administered during an on-going scan (i.e., a displacement study). This approach results both in lower radiation exposure and lower costs. Existing kinetic models assume steady state. This condition is not present during a drug displacement and consequently, our aim here was to develop kinetic models for analysing PET displacement data. We modified existing compartment models to accommodate a time-variant increase in occupancy following the pharmacological in-scan intervention. Since this implies the use of differential equations that cannot be solved analytically, we developed instead one approximate and one numerical solution. Through simulations, we show that if the occupancy is relatively high, it can be estimated without bias and with good accuracy. The models were applied to PET data from six pigs where [11C]UCB-J was displaced by intravenous brivaracetam. The dose-occupancy relationship estimated from these scans showed good agreement with occupancies calculated with Lassen plot applied to baseline-block scans of two pigs. In summary, the proposed models provide a framework to determine target occupancy from a single displacement scan.

Functional Positron Emission Tomography (fPET) provides a method to track molecular dynamics in the human brain. With a radioactively labelled glucose-analogue, [18F]-flurodeoxyglucose (FDG-fPET), it is now possible to index the dynamics of glucose metabolism with temporal resolutions approaching those of functional magnetic resonance imaging (fMRI). This direct measure of glucose uptake has enormous potential for understanding normal and abnormal brain function, and probing the effects of metabolic and neurodegenerative diseases. Further, new advances in hybrid MR-PET hardware makes it possible to capture fluctuations in glucose and blood oxygenation simultaneously using fMRI and FDG-fPET.\n\nThe temporal resolution and signal-to-noise of the FDG-fPET images is critically dependent upon the administration of the radioactive tracer. In this work we present two alternative continuous infusion protocols and compare them to a traditional bolus approach. We detail a method for acquiring blood samples, time-locking PET, MRI and experimental stimulus, and administrating the non-traditional tracer delivery. By applying a visual stimulus, we demonstrate cortical maps of the glucose-response to external stimuli on an individual level with a temporal resolution of 16-seconds.\n\nSummaryRadiotracer infusion protocols for positron emission tomography (PET) provide improved temporal resolution over bolus administration. Here, we describe radiotracer administration for two protocols, constant infusion and bolus plus infusion protocol. We compare this to the standard bolus administration protocol. Using [18-F] fluorodeoxyglucose PET (FDG-PET) as an example, we show that temporal resolutions of approximately 16sec are achievable using these protocols.

Positron emission tomography (PET) has become an essential clinical tool for diagnosing neurodegenerative diseases with abnormal accumulation of proteins like amyloid-{beta} or tau. Despite many attempts, it has not been possible to develop an appropriate radioligand for imaging aggregated -synuclein in the brain for diagnosing, e.g., Parkinsons Disease. Access to a large animal model with -synuclein pathology would critically enable a more translationally appropriate evaluation of novel radioligands. We here establish a pig model with cerebral injections of -synuclein preformed fibrils or brain homogenate from postmortem human brain tissue from individuals with Alzheimers disease (AD) or dementia with Lewy body (DLB) into the pigs brain, using minimally invasive surgery and validated against saline injections. In the absence of a suitable -synuclein radioligand, we validated the model with the unselective amyloid-{beta} tracer [11C]PIB, which has a high affinity for {beta}-sheet structures in aggregates. Gadolinium-enhanced MRI confirmed that the blood-brain barrier was intact. A few hours post-injection, pigs were PET scanned with [11C]PIB. Quantification was done with Logan invasive graphical analysis and simplified reference tissue model 2 using the occipital cortex as a reference region. After the scan, we retrieved the brains to confirm successful injection using autoradiography and immunohistochemistry. We found four times higher [11C]PIB uptake in AD-homogenate-injected regions and two times higher uptake in regions injected with -synuclein-preformed-fibrils compared to saline. The [11C]PIB uptake was the same in non-injected (occipital cortex, cerebellum) and injected (DLB-homogenate, saline) regions. With its large brains and ability to undergo repeated PET scans as well as neurosurgical procedures, the pig provides a robust, cost-effective, and good translational model for assessment of novel radioligands including, but not limited to, proteinopathies.