The cathepsin K (CatK) enzyme is abundantly expressed in osteoclasts, and CatK inhibitors have been developed for the treatment of osteoporosis. In our effort to support discovery and clinical evaluations of a CatK inhibitor, we sought to discover a radioligand to determine target engagement of the enzyme by therapeutic candidates using positron emission tomography (PET). L-235, a potent and selective CatK inhibitor, was labeled with carbon-11. PET imaging studies recording baseline distribution of [11 C]L-235, and chase and blocking studies using the selective CatK inhibitor MK-0674 were performed in juvenile and adult nonhuman primates (NHP) and ovariectomized rabbits. Retention of the PET tracer in regions expected to be osteoclast-rich compared with osteoclast-poor regions was examined. Increased retention of the radioligand was observed in osteoclast-rich regions of juvenile rabbits and NHP but not in the adult monkey or adult ovariectomized rabbit. Target engagement of CatK was observed in blocking studies with MK-0674, and the radioligand retention was shown to be sensitive to the level of MK-0674 exposure. [11 C]L-235 can assess target engagement of CatK in bone only in juvenile animals. [11 C]L-235 may be a useful tool for guiding the discovery of CatK inhibitors.

Purpose Programmed cell death-1 receptor (PD-1) and its ligand (PD-L1) are the targets for immunotherapy in many cancer types. Although PD-1 blockade has therapeutic effects, the efficacy differs between patients. Factors contributing to this variability are PD-L1 expression levels and immune cells present in tumors. However, it is not well understood how PD-1 expression in the tumor microenvironment impacts immunotherapy response. Thus, imaging of PD-1-expressing immune cells is of interest. This study aims to evaluate the biodistribution of Zirconium-89 ( 89 Zr)-labeled pembrolizumab, a humanized IgG4 kappa monoclonal antibody targeting PD-1, in healthy cynomolgus monkeys as a translational model of tracking PD-1-positive immune cells. Procedures Pembrolizumab was conjugated with the tetrafluorophenol-N-succinyl desferal-Fe(III) ester (TFP-N-sucDf) and subsequently radiolabeled with 89 Zr. Four cynomolgus monkeys with no previous exposure to humanized monoclonal antibodies received tracer only or tracer co-injected with pembrolizumab intravenously over 5 min. Thereafter, a static whole-body positron emission tomography (PET) scan was acquired with 10 min per bed position on days 0, 2, 5, and 7. Image-derived standardized uptake values (SUV mean ) were quantified by region of interest (ROI) analysis. Results 89 Zr-N-sucDf-pembrolizumab was synthesized with high radiochemical purity (> 99 %) and acceptable molar activity (> 7 MBq/nmol). In animals dosed with tracer only, 89 Zr-N-sucDf-pembrolizumab distribution in lymphoid tissues such as mesenteric lymph nodes, spleen, and tonsils increased over time. Except for the liver, low radiotracer distribution was observed in all non-lymphoid tissue including the lung, muscle, brain, heart, and kidney. When a large excess of pembrolizumab was co-administered with a radiotracer, accumulation in the lymph nodes, spleen, and tonsils was reduced, suggestive of target-mediated accumulation. Conclusions 89 Zr-N-sucDf-pembrolizumab shows preferential uptake in the lymphoid tissues including the lymph nodes, spleen, and tonsils. 89 Zr-N-sucDf-pembrolizumab may be useful in tracking the distribution of a subset of immune cells in non-human primates and humans. Trial Registration ClinicalTrials.gov Identifier: NCT02760225

PURPOSE:In vivo imaging of programmed death ligand 1 (PD-L1) during immunotherapy could potentially monitor changing PD-L1 expression and PD-L1 expression heterogeneity within and across tumors. Some protein constructs can be used for same-day positron emission tomography (PET) imaging. Previously, we evaluated the PD-L1-targeting Affibody molecule [18F]AlF-NOTA-ZPD-L1_1 as a PET tracer in a mouse tumor model of human PD-L1 expression. In this study, we evaluated the affinity-matured Affibody molecule ZPD-L1_4, to determine if improved affinity for PD-L1 resulted in increased in vivo targeting of PD-L1. PROCEDURES:ZPD-L1_4 was conjugated with NOTA and radiolabeled with either [18F]AlF or 68Ga. [18F]AlF-NOTA-ZPD-L1_4 and [68Ga]NOTA-ZPD-L1_4 were evaluated in immunocompromised mice with LOX (PD-L1+) and SUDHL6 (PD-L1-) tumors with PET and ex vivo biodistribution measurements. In addition, whole-body PET studies were performed in rhesus monkeys to predict human biodistribution in a model with tracer binding to endogenous PD-L1, and to calculate absorbed radiation doses. RESULTS:Ex vivo biodistribution measurements showed that both tracers had > 25 fold higher accumulation in LOX tumors than SUDHL6 ([18F]AlF-NOTA-ZPD-L1_4: LOX: 8.7 ± 0.7 %ID/g (N = 4) SUDHL6: 0.2 ± 0.01 %ID/g (N = 6), [68Ga]NOTA-ZPD-L1_4: LOX: 15.8 ± 1.0 %ID/g (N = 6) SUDHL6: 0.6 ± 0.1 %ID/g (N = 6)), considerably higher than ZPD-L1_1. In rhesus monkeys, both PET tracers showed fast clearance through kidneys and low background signal in the liver ([18F]AlF-NOTA-ZPD-L1_4: 1.26 ± 0.13 SUV, [68Ga]NOTA-ZPD-L1_4: 1.11 ± 0.06 SUV). PD-L1-expressing lymph nodes were visible in PET images, indicating in vivo PD-L1 targeting. Dosimetry estimates suggest that both PET tracers can be used for repeated clinical studies, although high kidney accumulation may limit allowable radioactive doses. CONCLUSIONS:[18F]AlF-NOTA-ZPD-L1_4 and [68Ga]NOTA-ZPD-L1_4 are promising candidates for same-day clinical PD-L1 PET imaging, warranting clinical evaluation. The ability to use either [18F] or [68Ga] may expand access to clinical sites.

Deposition of hyperphosphorylated and aggregated tau protein in the central nervous system is characteristic of Alzheimer disease and other tauopathies. Tau is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification, and O-GlcNAcylation of tau has been shown to influence tau phosphorylation and aggregation. Inhibition of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc moieties, is a novel strategy to attenuate the formation of pathologic tau. Here we described the in vitro and in vivo pharmacological properties of a novel and selective OGA inhibitor, MK-8719. In vitro, this compound is a potent inhibitor of the human OGA enzyme with comparable activity against the corresponding enzymes from mouse, rat, and dog. In vivo, oral administration of MK-8719 elevates brain and peripheral blood mononuclear cell O-GlcNAc levels in a dose-dependent manner. In addition, positron emission tomography imaging studies demonstrate robust target engagement of MK-8719 in the brains of rats and rTg4510 mice. In the rTg4510 mouse model of human tauopathy, MK-8719 significantly increases brain O-GlcNAc levels and reduces pathologic tau. The reduction in tau pathology in rTg4510 mice is accompanied by attenuation of brain atrophy, including reduction of forebrain volume loss as revealed by volumetric magnetic resonance imaging analysis. These findings suggest that OGA inhibition may reduce tau pathology in tauopathies. However, since hundreds of O-GlcNAcylated proteins may be influenced by OGA inhibition, it will be critical to understand the physiologic and toxicological consequences of chronic O-GlcNAc elevation in vivo. SIGNIFICANCE STATEMENT MK-8719 is a novel, selective, and potent O-linked N-acetylglucosamine (O-GlcNAc)-ase (OGA) inhibitor that inhibits OGA enzyme activity across multiple species with comparable in vitro potency. In vivo, MK-8719 elevates brain O-GlcNAc levels, reduces pathological tau, and ameliorates brain atrophy in the rTg4510 mouse model of tauopathy. These findings indicate that OGA inhibition may be a promising therapeutic strategy for the treatment of Alzheimer disease and other tauopathies.

Respiratory syncytial virus (RSV) is the most common viral cause of bronchiolitis and pneumonia in children twelve months of age or younger and a significant cause of lower respiratory disease in older adults. As various clinical and preclinical candidates advance, cotton rats (Sigmodon hispidus) and non-human primates (NHP) continue to play a valuable role in RSV vaccine development, since both animals are semi-permissive to human RSV (HRSV). However, appropriate utilization of the models is critical to avoid mis-interpretation of the preclinical findings. Using a multimodality imaging approach; a fluorescence based optical imaging technique for the cotton rat and a nuclear medicine based positron emission tomography (PET) imaging technique for monkeys, we demonstrate that many common practices for intranasal immunization in both species result in inoculum delivery to the lower respiratory tract, which can result in poor translation of outcomes from the preclinical to the clinical setting. Using these technologies we define a method to limit the distribution of intranasally administered vaccines solely to the upper airway of each species, which includes volume restrictions in combination with injectable anesthesia. We show using our newly defined methods for strict intranasal immunization that these methods impact the immune responses and efficacy observed when compared to vaccination methods resulting in distribution to both the upper and lower respiratory tracts. These data emphasize the importance of well-characterized immunization methods in the preclinical assessment of intranasally delivered vaccine candidates.

Programmed death ligand 1 (PD-L1) is an immune regulatory ligand that binds to the T-cell immune check point programmed death 1. Tumor expression of PD-L1 is correlated with immune suppression and poor prognosis. It is also correlated with therapeutic efficacy of programmed death 1 and PD-L1 inhibitors. In vivo imaging may enable real-time follow-up of changing PD-L1 expression and heterogeneity evaluation of PD-L1 expression across tumors in the same subject. We have radiolabeled the PD-L1–binding Affibody molecule NOTA-ZPD-L1_1 with 18F and evaluated its in vitro and in vivo binding affinity, targeting, and specificity. Methods: The affinity of the PD-L1–binding Affibody ligand ZPD-L1_1 was evaluated by surface plasmon resonance. Labeling was accomplished by maleimide coupling of NOTA to a unique cysteine residue and chelation of 18F-AlF. In vivo studies were performed in PD-L1–positive, PD-L1–negative, and mixed tumor-bearing severe combined immunodeficiency mice. Tracer was injected via the tail vein, and dynamic PET scans were acquired for 90 min, followed by γ-counting biodistribution. Immunohistochemical staining with an antibody specific for anti–PD-L1 (22C3) was used to evaluate the tumor distribution of PD-L1. Immunohistochemistry results were then compared with ex vivo autoradiographic images obtained from adjacent tissue sections. Results: NOTA-ZPD-L1_1 was labeled, with a radiochemical yield of 15.1% ± 5.6%, radiochemical purity of 96.7% ± 2.0%, and specific activity of 14.6 ± 6.5 GBq/μmol. Surface plasmon resonance showed a NOTA-conjugated ligand binding affinity of 1 nM. PET imaging demonstrated rapid uptake of tracer in the PD-L1–positive tumor, whereas the PD-L1–negative control tumor showed little tracer retention. Tracer clearance from most organs and blood was quick, with biodistribution showing prominent kidney retention, low liver uptake, and a significant difference between PD-L1–positive (percentage injected dose per gram [%ID/g] = 2.56 ± 0.33) and –negative (%ID/g = 0.32 ± 0.05) tumors (P = 0.0006). Ex vivo autoradiography showed excellent spatial correlation with immunohistochemistry in mixed tumors. Conclusion: Our results show that Affibody ligands can be effective at targeting tumor PD-L1 in vivo, with good specificity and rapid clearance. Future studies will explore methods to reduce kidney activity retention and further increase tumor uptake.

Positron emission tomography (PET) using radiolabeled biomolecules is a translational molecular imaging technology that is increasingly used in support of drug development. Current methods for radiolabeling biomolecules with fluorine-18 are laborious and require multistep procedures with moderate labeling yields. The Al18F-labeling strategy involves chelation in aqueous medium of aluminum mono[18F]fluoride ({Al18F}2+) by a suitable chelator conjugated to a biomolecule. However, the need for elevated temperatures (100-120 °C) required for the chelation reaction limits its widespread use. Therefore, we designed a new restrained complexing agent (RESCA) for application of the AlF strategy at room temperature. Methods. The new chelator RESCA was conjugated to three relevant biologicals and the constructs were labeled with {Al18F}2+ to evaluate the generic applicability of the one-step Al18F-RESCA-method. Results. We successfully labeled human serum albumin with excellent radiochemical yields in less than 30 minutes and confirmed in vivo stability of the Al18F-labeled protein in rats. In addition, we efficiently labeled nanobodies targeting the Kupffer cell marker CRIg, and performed µPET studies in healthy and CRIg deficient mice to demonstrate that the proposed radiolabeling method does not affect the functional integrity of the protein. Finally, an affibody targeting HER2 (PEP04314) was labeled site-specifically, and the distribution profile of (±)-[18F]AlF(RESCA)-PEP04314 in a rhesus monkey was compared with that of [18F]AlF(NOTA)-PEP04314 using whole-body PET/CT. Conclusion. This generic radiolabeling method has the potential to be a kit-based fluorine-18 labeling strategy, and could have a large impact on PET radiochemical space, potentially enabling the development of many new fluorine-18 labeled protein-based radiotracers.

5'-Adenosine monophosphate-activated protein kinase (AMPK) is a master regulator of energy homeostasis in eukaryotes. Despite three decades of investigation, the biological roles of AMPK and its potential as a drug target remain incompletely understood, largely because of a lack of optimized pharmacological tools. We developed MK-8722, a potent, direct, allosteric activator of all 12 mammalian AMPK complexes. In rodents and rhesus monkeys, MK-8722-mediated AMPK activation in skeletal muscle induced robust, durable, insulin-independent glucose uptake and glycogen synthesis, with resultant improvements in glycemia and no evidence of hypoglycemia. These effects translated across species, including diabetic rhesus monkeys, but manifested with concomitant cardiac hypertrophy and increased cardiac glycogen without apparent functional sequelae.

The folate receptor (FR) has been established as a promising target for imaging and therapy of cancer (FR-α), inflammation, and autoimmune diseases (FR-β). Several folate based PET radiotracers have been reported in the literature, but an 18F-labeled folate-PET imaging agent with optimal properties for clinical translation is still lacking. In the present study, we report the design and preclinical evaluation of folate-PEG12-NOTA-Al18F (1), a new folate-PET agent with improved potential for clinical applications. Radiochemical synthesis of 1 was achieved via a one-pot labeling process by heating folate-PEG12-NOTA in the presence of in situ prepared Al18F for 15 min at 105 °C, followed by HPLC purification. Specific binding of 1 to FR was evaluated on homogenates of KB (FR-positive) and A549 (FR-deficient) tumor xenografts in the presence and absence of excess folate. In vivo tumor imaging with folate-PEG12-NOTA-Al18F was compared to imaging with 99mTc-EC20 using nu/nu mice bearing either KB or A549 tumor xenografts. Specific accumulation of 1 in tumor and other tissues was assessed by high-resolution micro-PET and ex vivo biodistribution in the presence and absence of excess folate. Radiosynthesis of 1 was accomplished within ∼35 min, affording pure radiotracer 1 in 8.4 ± 1.3% (decay corrected) radiochemical yield with ∼100% radiochemical purity after HPLC purification and a specific activity of 35.8 ± 15.3 GBq/mmol. Further in vitro and in vivo examination of 1 demonstrated highly specific FR-mediated uptake in FR+ tumor, with Kd of ∼0.4 nM (KB), and reduced accumulation in liver. Given its facile preparation and improved properties, the new radiotracer, folate-PEG12-NOTA-Al18F (1), constitutes a promising tool for identification and classification of patients with FR overexpressing cancers.

UNLABELLED:Folate-receptor-targeted PET radiotracers can potentially serve as versatile imaging agents for the diagnosis, staging, and prediction of response to therapy of patients with folate-receptor (FR)-expressing cancers. Because current FR-targeted PET reagents can be compromised by complex labeling procedures, low specific activities, poor radiochemical yields, or unwanted accumulation in FR negative tissues, we have undertaken to design an improved folate-PET agent that might be more amenable for clinical development. For this purpose, we have synthesized a folate-NOTA-Al(18)F radiotracer and examined its properties both in vitro and in vivo.METHODS:Radiochemical synthesis of folate-NOTA-Al(18)F was achieved by incubating (18)F(-) with AlCl3 for 2 min followed by heating in the presence of folate-NOTA for 15 min at 100 °C. Binding of folate-NOTA-Al(18)F to FR was quantitated in homogenates of KB and Cal51 tumor xenografts in the presence and absence of excess folic acid as a competitor. In vivo imaging was performed on nu/nu mice bearing either FR+ve (KB cell) or FR-ve (A549 cell) tumor xenografts, and specific accumulation of the radiotracer in tumor and other tissues was assessed by high-resolution micro-PET and ex vivo biodistribution in the presence and absence of excess folic acid. Image quality of folate-NOTA-Al(18)F was compared with that of (99m)Tc-EC20, a clinically established folate-targeted SPECT imaging agent.RESULTS:Total radiochemical synthesis and purification of folate-NOTA-Al(18)F was completed within 37 min, yielding a specific activity of 68.82 ± 18.5 GBq/μmol, radiochemical yield of 18.6 ± 4.5%, and radiochemical purity of 98.3 ± 2.9%. Analysis of FR binding revealed a Kd of ∼1.0 nM, and micro-PET imaging together with ex vivo biodistribution analyses demonstrated high FR-mediated uptake in an FR+ tumor and the kidneys.CONCLUSIONS:Folate-NOTA-Al(18)F constitutes an easily prepared FR-targeted PET imaging agent with improved radiopharmaceutical properties and high specificity for folate receptor expressing tumors. Given its improved properties over (99m)Tc-EC20 (i.e., higher resolution, shorter image acquisition time, etc.), we conclude that folate-NOTA-Al(18)F constitutes a viable alternative to (99m)Tc-EC20 for use in identification, diagnosis, and staging of patients with FR-expressing cancers.

1165 Objectives Folate receptor (FR) has been identified as an important target for cancer therapy. A successful clinical FR targeting PET agent would facilitate the identification of small FR positive lesions and reduce scan time, which are key advantages over [99mTc]-EC20 SPECT clinical imaging. In this study, we designed and evaluated a new folate-PET agent, folate-PEG12-NOTA-Al[18F], and compared it to another folate targeting PET tracer folate-NOTA-Al[18F]. Methods Folate-PEG12-NOTA-Al[18F] synthesis was accomplished via one-pot labeling procedure by heating folate-PEG12-NOTA with in situ prepared ([18F]AlF)2+ for 15 min at 105 °C, followed by HPLC purification. Female 6-8 week-old nu/nu mice were implanted with KB (high FR expressors), Cal51 and A549 (low FR expressors) cell xenografts. Specific binding of the radiotracer to the FR was evaluated in homogenates of KB and Cal51 tumor xenografts, with in vivo imaging performed in mice bearing either KB or A549 tumor xenografts. Specific uptake of the radiotracer in tumor and other tissues was evaluated by micro-PET imaging and ex vivo biodistribution in the presence and absence of excess folic acid (FA). Results Total radiochemical synthesis including radioactive HPLC purification of folate-PEG12-NOTA-Al[18F] was completed within 35 min, affording pure radiotracer in ~ 25-30% radiochemical yield with ~ 100% radiochemical purity and a specific activity of 1320 ± 460 Ci/mmol. Analysis of FR binding revealed Kd of 0.4 nM and 1.2 nM, and binding potential (Bmax / Kd) of 603 and 11 in KB and Cal51 xenografts, respectively. Uptake of folate-PEG12-NOTA-Al[18F] was higher in KB xenografts (2.33 ± 0.13 SUV) compared to A549 (0.53 ± 0.06 SUV), and the uptake could be blocked by FA. Folate-PEG12-NOTA-Al[18F] showed higher FR-mediated uptake in FR positive tumor and reduced accumulation in liver compared to folate-NOTA-Al[18F]. Conclusions Folate-PEG12-NOTA-Al[18F] synthesis was achieved through one-step radiolabeling strategy which is efficient and reproducible. The tracer demonstrated higher specific binding to the FR positive tumor and improved biodistribution compared to folate-NOTA-Al[18F]. There is potential for further development of folate-PEG12-NOTA-Al[18F] as a PET imaging agent and diagnostic tool targeting FR positive tumors for clinical study.

529 Objectives PD-L1 (Programmed Death Ligand 1) is a 40 kDa immune regulatory ligand that binds to PD-1 (Programmed Death 1), which functions as an immune checkpoint and is expressed on activated immune cell types including T cells, B cells, natural killer (NK) cells and many tumor-infiltrating lymphocytes (TILs).[1] PD-L1 binding to PD-1 deactivates these cytotoxic T cells, and as a consequence the expression of PD-L1 in tumors is correlated to immune suppression and poor prognosis. Therefore it may be feasible to use tumor expression of PD-L1 as a predictive marker for anti-PD-1 therapy. The purpose of this work was to evaluate the feasibility of using PET as and in vivo tool to image PD-L1. For this, the human PD-L1 specific mAb 22C3 was chosen as our proof of concept molecule. Methods The anti-human PD-L1 mAb 22C3 and isotype matched control mAb 27F11 were conjugated with DOTA and subsequently radiolabeled with [64Cu]. MicroPET imaging studies were carried out in SCID mice implanted with the LOX human malignant melanoma cell line, known to express PD-L1, 48 hr. after administering either [64Cu]DOTA-22C3 (n=4) or [64Cu]DOTA-27F11 (n=4). Blocking studies were also performed in which the tumor bearing mice received 200 µg of unlabeled 22C3 1 hr. prior to administration of [64Cu]DOTA-22C3 (n=4). In all cases following the 48 hr. imaging scan, the mice were euthanized and biodistribution studies performed. Along with imaging studies, an in vitro homogenate binding assay using LOX xenografts homogenates was performed to measure the binding potential for both [64Cu]DOTA-22C3 and [64Cu]DOTA-27F11. Finally, autoradiographic and PD-L1 IHC staining were performed on the LOX tumors to see if tracer uptake correlated to regions of high PD-L1 expression. Results Displaceable and saturable binding of [64Cu]DOTA-22C3 was observed from the in vitro homogenate binding assay, with [64Cu]DOTA-22C3 binding to a single site with high affinity (Kd = 0.4 nM). For [64Cu]DOTA-27F11 binding in tumor tissue was minimal and not saturable, indicating that there was no measureable specific binding. ComparisonComparison of autoradiography and HC staining of tumor slices confirmed that PD-L1 expression patterns matched [64Cu]DOTA-22C3 binding patterns. A similar correlation was not observed for [64Cu]DOTA-27F11. From the PET Images, tumor uptake in the mice that received [64Cu]DOTA-22C3 was clearly visualized, with uptake higher than [64Cu]DOTA-27F11. In addition, pretreatment with the unlabeled 22C3 reduced tumor uptake of [64Cu]DOTA-22C3 mAb to levels comparable to [64Cu]DOTA-27F11. Biodistribution data at 48 hr. confirmed the PET imaging analysis with tumor uptake of [64Cu]DOTA-22C3 being significantly higher than [64Cu]DOTA-27F11 (p Conclusions The results of this study demonstrate that a PET ligand can specifically target PD-L1-expressing tumors in a mouse model, and support the hypothesis that PD-L1 can be imaged in vivo in the clinic using PET.

1362 Objectives Imaging of the folate receptor (FR) is important for assessing treatment options of patients with FR-expressing cancers, and has been primarily accomplished with the FR-targeting SPECT tracer [Tc-99m]-EC20 (Fisher, et al., 2008). However, clinical PET provides superior spatial resolution, shorter scan times, and greater dynamic imaging capabilities than SPECT. The PET tracer Folate-NOTA-Al-[F-18] has been shown to successfully target FR in vivo (Meng, et al., 2016). In this study, the in vivo biodistribution of Folate-NOTA-Al-[F-18] was compared to [Tc-99m]-EC20, and radiation dosimetry estimates for humans were obtained. Methods Rhesus monkeys (N=3) were scanned with both radiotracers. Monkeys were fasted and maintained under anesthesia with propofol. Following a CT scan, PET data were acquired for 180 minutes following IV administration of Folate-NOTA-Al-[F18] (137-178 MBq, 1.9-2.5 μg), while SPECT data were acquired for 150 min, starting 60 min after administration of [Tc-99m]-EC20 (390-399 MBq, 39-48 μg). Venous blood samples were taken throughout the study and metabolite-corrected blood levels of radiotracer were calculated. Regions of interest (ROI) were drawn for liver, kidney cortex, lumbar spine, lung, small intestine, spleen, bladder, and heart muscle, and average radiotracer uptake from ~60-150 minutes for each ROI was calculated. Absorbed radiation doses were calculated using OLINDA/EXM v1 (Organ Level Internal Dose Assessment) with adult human model inputs. Results PET and SPECT images revealed similar biodistribution for the two radiotracers (Table 1). Based on a mixed-effect model analysis, significant differences in accumulation between radiotracers were detected for spleen, (p= 0.02), heart muscle (p = 0.03), and lumbar spine (p = 0.05). Dosimetry analysis of Folate-NOTA-Al-[F-18] found that the critical organ for males was the testes, while for females the urinary bladder wall was the critical organ. A conservative estimate of the recommended maximum allowable dose of folate-NOTA-Al-[F-18] for clinical research subjects (U.S) is 197 MBq per dose and 328 MBq per year for males, and 235 MBq per dose and 704 MBq per year for females. Conclusions Folate-NOTA-Al-[F-18] showed similar biodistribution to [Tc-99m]-EC20 in Rhesus monkey, and an analysis of radiation dosimetry indicated that human studies will be feasible. These results add support for the PET radiotracer Folate-NOTA-Al-[F-18] as a viable alternative [Tc-99m]-EC20 for FR imaging.

1198 Objectives Folate receptor (FR) has been identified as an important target for cancer therapy due to its limited distribution in normal tissues but overexpression on a variety of tumor types. The SPECT tracer [99mTc]-EC20 has been used as an imaging companion diagnostic in clinical trials, but a PET tracer that targets folate receptor would provide significant sensitivity, acquisition speed and image quality improvements over a SPECT tracer. For this purpose, we have synthesized a folate PET radiotracer and evaluated it in murine xenograft tumor models. Methods Folate-NOTA-Al[18F] was prepared by incubating folate-NOTA with in situ prepared ([18F]AlF)2+ for 15 min at 100 °C. KB (high FR expressors), Cal51 and A549 (low FR expressors) cells were implanted in 6-8 week-old immune compromised nude mice. Dynamic 90 min scan were acquired in micro-PET camera with 200-400 µCi tracer IV alone, or folic acid (FA, 50 or 100 µg) immediately before tracer in KB or A549 tumored mice (n=3-5 each group). Mice were euthanized immediately after the scan, and tissues were collected, weighed, and counted in gamma counter. Homogenate binding assay was performed to measure the binding potential of the tracer in KB and Cal51 xenografts. The results were compared with data from a [99mTc]-EC20 study. Results Total radiochemical synthesis and purification of folate-NOTA-Al[18F] was completed within 37 min, yielding a specific activity of 1860 ± 500 Ci/mmol, with a radiochemical yield of 40-50%, and radiochemical purity of ~ 98%. Uptake of folate-NOTA-Al[18F] was higher in KB xenografts (2.84 ± 0.76 SUV) as compared to A549 (0.64 ± 0.16 SUV), and the uptake could be blocked by FA. Folate-NOTA-Al[18F] showed a higher binding potential (Bmax / Kd) in KB (730) as compared to Cal51 (33) xenografts. The biodistribution of folate-NOTA-Al[18F] was comparable to that of [99mTc]-EC20. Conclusions Folate-NOTA-Al[18F] constitutes an easily prepared PET agent with high specificity for FR expressing tumors and a comparable biodistribution to [99mTc]-EC20. Folate-NOTA-Al[18F] therefore has the potential as a viable alternative to [99mTc]-EC20 for clinical studies in patients with FR-positive tumors.

1133 Objectives Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract disease in children and seniors worldwide. Despite the prevalence of the disease, there is currently no RSV vaccine, and therapeutic options are limited. Common practices for intranasal (IN) delivery in rodents provide a range of outcomes that could result in ambiguous interpretation. We have since refined methods to retain vaccine inoculum solely in the upper respiratory tract (URT) while providing sufficient immune responses to protect against viral challenge. Non-human primates are equally invaluable to assess novel RSV vaccines preclinically, with African green monkeys (C. aethiops) commonly used for evaluating efficacy and safety of RSV vaccines. We evaluated whether traditional intranasal immunization methodologies utilized in non-human primate studies result in vaccine deposition in both the upper and lower respiratory tract, providing a pathway to an immune response that would not be available in a clinical setting. Methods African Green monkey (N=6) were dosed intranasally with either 250 µL or 100 µL RSV vaccine radiolabeled with 64Cu. Monkeys were fasted and anesthetized with Ketamine/Xylazine followed by Propofol. A 30 minute PET scan of the URT was started followed immediately by IN delivery of vaccine. A 5 minute per bed whole body scan was acquired 1 hour post-dose to determine distribution to the lower respiratory tract. Blood samples were taken prior to scanning and 28 days post-dose to determine immunogenicity. PET images were analyzed to determine the percent of injected radiotracer in nasal cavity, pharynx/larynx, trachea, and lungs. Results PET analysis results are in Table 1. This analysis revealed substantial lung accumulation (up to 34%) in animals receiving the 250 µL dose, while animals receiving the 100 µL dose had very low lung accumulation (2% or less). Serum titers of the vaccine determined animals were positive at the highest dilution factor at 28 days post-dose for both 100 µL and 250 µL doses. Conclusions PET results demonstrated that nasal administration of 250 µL of 64Cu-RSV leads to variable, but substantial lung accumulation. Reduction of dose to 100 µL resulted in substantial reduction of pulmonary immunization accumulation with no loss of immunogenicity.