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Background: Osteosarcoma (OS) is an aggressive pediatric cancer with unmet therapeutic needs. Glutaminase 1 (GLS1) inhibition, alone and in combination with metformin, disrupts the bioenergetic demands of tumor progression and metastasis, showing promise for clinical translation. Materials and Methods: Three positron emission tomography (PET) clinical imaging agents, [ 18 F]fluoro-2-deoxy-2-D-glucose ([ 18 F]FDG), 3'-[ 18 F]fluoro-3'-deoxythymidine ([ 18 F]FLT), and (2S, 4R)-4-[ 18 F]fluoroglutamine ([ 18 F]GLN), were evaluated in the MG63.3 human OS xenograft mouse model, as companion imaging biomarkers after treatment for 7 d with a selective GLS1 inhibitor (CB-839, telaglenastat) and metformin, alone and in combination. Imaging and biodistribution data were collected from tumors and reference tissues before and after treatment. Results: Drug treatment altered tumor uptake of all three PET agents. Relative [ 18 F]FDG uptake decreased significantly after telaglenastat treatment, but not within control and metformin-only groups. [ 18 F]FLT tumor uptake appears to be negatively affected by tumor size. Evidence of a flare effect was seen with [ 18 F]FLT imaging after treatment. Telaglenastat had a broad influence on [ 18 F]GLN uptake in tumor and normal tissues. Conclusions: Image-based tumor volume quantification is recommended for this paratibial tumor model. The performance of [ 18 F]FLT and [ 18 F]GLN was affected by tumor size. [ 18 F]FDG may be useful in detecting telaglenastat's impact on glycolysis. Exploration of kinetic tracer uptake protocols is needed to define clinically relevant patterns of [ 18 F]GLN uptake in patients receiving telaglenastat.

Background: The activities of MYC, the androgen receptor, and its associated pioneer factors demonstrate substantial reprogramming between early and advanced prostate cancer. Although previous studies have shown a shift in cellular metabolic requirements associated with prostate cancer progression, the epigenetic regulation of these processes is incompletely described. Here, we have integrated chromatin immunoprecipitation sequencing (ChIP-seq) and whole-transcriptome sequencing to identify novel regulators of metabolism in advanced prostate tumors characterized by elevated MYC activity., Results: Using ChIP-seq against MYC, HOXB13, and AR in LNCaP cells, we observed redistribution of co-bound sites suggestive of differential KMT2A activity as a function of MYC expression. In a cohort of 177 laser-capture microdissected foci of prostate tumors, KMT2A expression was positively correlated with MYC activity, AR activity, and HOXB13 expression, but decreased with tumor grade severity. However, KMT2A expression was negatively correlated with these factors in 25 LuCaP patient-derived xenograft models of advanced prostate cancer and 99 laser-capture microdissected foci of metastatic castration-resistant prostate cancer. Stratified by KMT2A expression, ChIP-seq against AR and HOXB13 in 15 LuCaP patient-derived xenografts showed an inverse association with sites involving genes implicated in lipid metabolism, including the arachidonic acid metabolic enzyme PLA2G4F. LuCaP patient-derived xenograft models grown as organoids recapitulated the inverse association between KMT2A expression and fluorine-18 labeled arachidonic acid uptake in vitro., Conclusions: Our study demonstrates that the epigenetic activity of transcription factor oncogenes exhibits a shift during prostate cancer progression with distinctive phenotypic effects on metabolism. These epigenetically driven changes in lipid metabolism may serve as novel targets for the development of novel imaging agents and therapeutics., (© 2022. The Author(s).)

High expression of prostate-specific membrane antigen (PSMA) in prostate cancers prompted the development of the PSMA-targeted PET-imaging agent [ 18 F]DCFPyL, which was recently approved by the FDA. Fluorine-18-labeled Lys-Urea-Glu-based oxime derivatives of [ 18 F]DCFPyL were prepared for the comparison of their in vitro and in vivo properties to potentially improve kidney clearance and tumor targeting. The oxime radiotracers were produced by condensation of an aminooxy functionalized PSMA-inhibitor Lys-Urea-Glu scaffold with fluorine-18-labeled aldehydes. The radiochemical yields were between 15-42% (decay uncorrected) in 50-60 min. In vitro saturation and competition binding assays with human prostate cancer cells transfected with PSMA, PC3(+), indicated similar high nM binding affinities to PSMA for all radiotracers. In vivo biodistribution studies with positive control PC3(+) tumor xenografts showed that the kidneys had the highest uptake followed by tumors at 60 min. The PC3(+) tumor uptake was blocked with non-radioactive DCFPyL, and PC3(-) tumor xenograft (negative control) tumor uptake was negligible indicating that PSMA targeting was preserved. The most lipophilic tracer, [ 18 F] 2a , displayed comparable tumor-targeting to [ 18 F]DCFPyL and a desirable alteration in pharmacokinetics and metabolism, resulting in significantly lower kidney uptake with a shift towards hepatobiliary clearance and increased liver uptake.

Purpose: PSMA overexpression has been associated with aggressive prostate cancer (PCa). However, PSMA PET imaging has revealed highly variable changes in PSMA expression in response to ADT treatment ranging from increases to moderate decreases. To better understand these PSMA responses and potential relationship to progressive PCa, the PET imaging agent, [ 18 F]DCFPyL, was used to assess changes in PSMA expression in response to ADT using genomically characterized LuCaP patient-derived xenograft mouse models (LuCaP-PDXs) which were found to be sensitive to ADT (LuCaP73 and LuCaP136;CS) or resistant (LuCaP167;CR)., Methods: [ 18 F]DCFPyL (2-(3-{1-carboxy-5-[(6-[ 18 F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid) was used to assess PSMA in vitro (saturation assays) in LuCaP tumor membrane homogenates and in vivo (imaging/biodistribution) in LuCaP-PDXs. Control and ADT-treated LuCaPs were imaged before ADT (0 days) and 2-, 7-, 14-, and 21-days post-ADT from which tumor:muscle ratios (T:Ms) were determined and concurrently tumor volumes were measured (caliper). After the 21-day imaging, biodistributions and histologic/genomic (PSMA, AR) analysis were done., Results: [ 18 F]DCFPyL exhibited high affinity for PSMA and distinguished different levels of PSMA in LuCaP tumors. Post-ADT CS LuCaP73 and LuCaP136 tumor volumes significantly decreased at day 7 or 14 respectively vs controls, whereas the CR LuCaP167 tumor volumes were minimally changed. [ 18 F]DCFPyL imaging T:Ms were increased 3-5-fold in treated LuCaP73 tumors vs controls, while treated LuCaP136 T:Ms remained unchanged which was confirmed by day 21 biodistribution results. For treated LuCaP167, T:Ms were decreased (~ 45 %) vs controls but due to low T:M values (<2) may not be indicative of PSMA level changes. LuCaP73 tumor PSMA histologic/genomic results were comparable to imaging/biodistribution results, whereas the results for other tumor types varied., Conclusion: Tumor responses to ADT varied from sensitive to resistant among these LuCaP PDXs, while only the high PSMA expressing LuCaP model exhibited an increase in PSMA levels in response to ADT. These models may be useful in understanding the clinical relevance of PSMA PET responses to ADT and potentially the relationship to disease progression as it may relate to the genomic signature., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Introduction: [ 227 Th]Th-3,2-HOPO-MSLN-mAb, a mesothelin (MSLN)-targeted thorium-227 therapeutic conjugate, is currently in phase I clinical trial; however, direct PET imaging using this conjugate is technically challenging. Thus, using the same MSLN antibody, we synthesized 3,2-HOPO and deferoxamine (DFO)-based zirconium-89 antibody conjugates, [ 89 Zr]Zr-3,2-HOPO-MSLN-mAb and [ 89 Zr]Zr-DFO-MSLN-mAb, respectively, and compared them in vitro and in vivo . Methods: [ 89 Zr]Zr-3,2-HOPO-MSLN-mAb and [ 89 Zr]Zr-DFO-MSLN-mAb were evaluated in vitro to determine binding affinity and immunoreactivity in HT29-MSLN and PDX (NCI-Meso16, NCI-Meso21) cells. For both the zirconium-89 conjugates, in vivo studies (biodistribution/imaging) were performed at days 1, 3, and 6, from which tissue uptake was determined. Results: Both the conjugates demonstrated a low nanomolar binding affinity for MSLN and >95% immunoreactivity. In all the three tumor types, biodistribution of [ 89 Zr]Zr-DFO-MSLN-mAb resulted in higher tumor uptake(15.88-28-33%ID/g) at all time points compared with [ 89 Zr]Zr-3,2-HOPO-MSLN-mAb(7-13.07%ID/g). [ 89 Zr]Zr-3,2-HOPO-MSLN-mAb femur uptake was always higher than [ 89 Zr]Zr-DFO-MSLN-mAb, and imaging results concurred with the biodistribution studies. Conclusions: Even though the conjugates exhibited a high binding affinity for MSLN, [ 89 Zr]Zr-DFO-MSLN-mAb showed a higher tumor and lower femur uptake than [ 89 Zr]Zr-3,2-HOPO-MSLN-mAb. Nevertheless, [ 89 Zr]Zr-3,2-HOPO-MSLN-mAb could be used to study organ distribution and lesion uptake with the caveat of detecting MSLN-positive bone lesions. Clinical trial (NCT03507452).

Background: Patients with osteoblastic bone metastases are candidates for radium-223 ( 223 RaCl 2 ) therapy and may undergo sodium fluoride-18 ( 18 F-NaF) positron emission tomography-computed tomography imaging to identify bone lesions. 18 F-NaF has been shown to predict 223 RaCl 2 uptake, but intratumor distributions of these two agents remain unclear. In this study, the authors evaluate the spatial distribution and relative uptakes of 18 F-NaF and 223 RaCl 2 in Hu09-H3 human osteosarcoma mouse xenograft tumors at macroscopic and microscopic levels to better quantify their correlation. Materials and Methods: 18 F-NaF and 223 RaCl 2 were co-injected into Hu09-H3 xenograft tumor severe combined immunodeficient mice. Tumor content was determined from in vivo biodistributions and visualized by PET, single photon emission computed tomography, and CT imaging. Intratumor distributions were visualized by quantitative autoradiography of tumor tissue sections and compared to histology of the same or adjacent sections. Results: 18 F and 223 Ra accumulated in proportional amounts in whole Hu09-H3 tumors ( r 2  = 0.82) and in microcalcified regions within these tumors ( r 2  = 0.87). Intratumor distributions of 18 F and 223 Ra were spatially congruent in these microcalcified regions. Conclusions: 18 F-NaF and 223 RaCl 2 uptake are strongly correlated in heterogeneously distributed microcalcified regions of Hu09-H3 xenograft tumors, and thus, tumor accumulation of 18 F is predictive of 223 Ra accumulation. Hu09-H3 xenograft tumors appear to possess certain histopathological features found in patients with metastatic bone disease and may be useful in clarifying the relationship between administered 223 Ra dose and therapeutic effect.

The C-X-C motif chemokine receptor 4 (CXCR4) is a seven-transmembrane G protein-coupled receptor that is overexpressed in numerous diseases, particularly in various cancers and is a powerful chemokine, attracting cells to the bone marrow niche. Therefore, CXCR4 is an attractive target for imaging and therapeutic purposes. The goal of this study is to develop an efficient, reproducible, and straightforward method to prepare a fluorine-18 labeled CXCR4 ligand. 6-[ 18 F]Fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester (6-[ 18 F]FPy-TFP) and nicotinic acid N-hydroxysuccinimide ester (6-[ 18 F]SFPy) have been prepared using 'fluorination on the Sep-Pak' method. Conjugation of 6-[ 18 F]SFPy or 6-[ 18 F]FPy-TFP with the alpha-amino group at the N terminus of the protected T140 precursor followed by deprotection, yielded the final product 6-[ 18 F]FPy-T140. The overall radiochemical yields were 6-17% ( n = 15, decay-corrected) in a 90-min radiolabeling time with a radiochemical purity >99%. 6-[ 18 F]FPy-T140 exhibited high specific binding and nanomolar affinity for CXCR4 in vitro, indicating that the biological activity of the peptide was preserved. For the first time, [ 18 F]SFPy has been prepared using 'fluorination on the Sep-Pak' method that allows rapid automated synthesis of 6-[ 18 F]FPy-T140. In addition to increased synthetic efficiency, this construct binds with CXCR4 in high affinity and may have potential as an in vivo positron emission tomography (PET) imaging agent. This radiosynthesis method should encourage wider use of this PET agent to quantify CXCR4 in both research and clinical settings.

Molecular imaging approaches for metabolic and physiologic imaging of tumors have become important for treatment planning and response monitoring. However, the relationship between the physiologic and metabolic aspects of tumors is not fully understood. Here, we developed new hyperpolarized MRI and electron paramagnetic resonance imaging procedures that allow more direct assessment of tumor glycolysis and oxygenation status quantitatively. We investigated the spatial relationship between hypoxia, glucose uptake, and glycolysis in three human pancreatic ductal adenocarcinoma tumor xenografts with differing physiologic and metabolic characteristics. At the bulk tumor level, there was a strong positive correlation between 18 F-FDG-PET and lactate production, while pO 2 was inversely related to lactate production and 18 F-2-fluoro-2-deoxy-D-glucose ( 18 F-FDG) uptake. However, metabolism was not uniform throughout the tumors, and the whole tumor results masked different localizations that became apparent while imaging. 18 F-FDG uptake negatively correlated with pO 2 in the center of the tumor and positively correlated with pO 2 on the periphery. In contrast to pO 2 and 18 F-FDG uptake, lactate dehydrogenase activity was distributed relatively evenly throughout the tumor. The heterogeneity revealed by each measure suggests a multimodal molecular imaging approach can improve tumor characterization, potentially leading to better prognostics in cancer treatment. SIGNIFICANCE: Novel multimodal molecular imaging techniques reveal the potential of three interrelated imaging biomarkers to profile the tumor microenvironment and interrelationships of hypoxia, glucose uptake, and glycolysis., (©2020 American Association for Cancer Research.)

Background: Prostate-specific membrane antigen (PSMA) has emerged as a promising target for developing radionuclide therapy (RNT) in prostate cancer; however, accumulation of PSMA-RNT in salivary glands can result in irreversible xerostomia. Methods to prevent PSMA-RNT-related xerostomia could be clinically useful; however, little is known about PSMA expression in salivary glands of preclinical animal models. Using [ 18 F]DCFPyL autoradiography/biodistribution, PSMA expression levels were determined in salivary glands of various preclinical monkey and rodent species and compared with humans. Methods: Binding affinities (K d ) and PSMA levels (B max ) were determined by in vitro [ 18 F]DCFPyL autoradiography studies. In vivo rodent tissue uptakes (%ID/g) were determined from [ 18 F]DCFPyL biodistributions. Results: [ 18 F]DCFPyL exhibited low nanomolar K d for submandibular gland (SMG) PSMA across all the species. PSMA levels in human SMG (B max  = 60.91 nM) were approximately two-fold lower compared with baboon SMG but were two- to three-fold higher than SMG PSMA levels of cynomolgus and rhesus. Rodents had the lowest SMG PSMA levels, with the mouse being 10-fold higher than the rat. In vivo rodent biodistribution studies confirmed these results. Conclusions: SMG of monkeys exhibited comparable PSMA expression to human SMG whereas rodents were lower. However, the results suggest that mice are relatively a better small animal preclinical model than rats for PSMA salivary gland studies.

To further explore the scope of our recently developed "fluorination on Sep-Pak" method, we prepared two well-known positron emission tomography (PET) tracers 21-[ 18 F]fluoro-16α,17α-[(R)-(1'-α-furylmethylidene)dioxy]-19-norpregn-4-ene-3,20-dione furanyl norprogesterone ([ 18 F]FFNP) and 16β-[ 18 F]fluoro-5α-dihydrotestosterone ([ 18 F]FDHT). Following the "fluorination on Sep-Pak" method, over 70% elution efficiency was observed with 3 mg of triflate precursor of [ 18 F]FFNP. The overall yield of [ 18 F]FFNP was 64-72% (decay corrected) in 40 min synthesis time with a molar activity of 37-81 GBq/µmol (1000-2200 Ci/mmol). Slightly lower elution efficiency (~55%) was observed with the triflate precursor of [ 18 F]FDHT. Fluorine-18 labeling, reduction, and deprotection to prepare [ 18 F]FDHT were performed on Sep-Pak cartridges (PS-HCO 3 and Sep-Pak plus C-18). The overall yield of [ 18 F]FDHT was 25-32% (decay corrected) in 70 min. The molar activity determined by using mass spectrometry was 63-148 GBq/µmol (1700-4000 Ci/mmol). Applying this quantitative measure of molar activity to in vitro assays [ 18 F]FDHT exhibited high-affinity binding to androgen receptors (K d ~2.5 nM) providing biological validation of this method.

Objective: 18 F-albumin, a vascular imaging agent, may have potential to assess tumor responses to anti-angiogenic therapies. In these studies tumor distribution volume of 18 F-albumin were first determined in various human tumor xenografts from biodistribtuion measurments and then one of the tumor type was used to evaluate changes in 18 F-albumin uptake in anti-angiognic tumor model. Method: 18 F-albumin was synthesized via conjugation of 6-[ 18 F]fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester, [ 18 F]F-Py-TFP, with rat albumin. From the biodistribution of 18 F-albumin in various human tumor xenografts tumor distribution volumes (DVs; tumor %ID/g :blood %ID/g ) were first determined at various time points. Then, the ability of 18 F-albumin to detect tumor angiogenic inhibition in one of these tumor types (U87MG) following treatment with sunitinib was evaluated by position emission tomography (PET) imaging at 0, 7, 14, and 21 days post treatment. Caliper measurements of tumor dimensions were also made at these same times. At Day 21, following imaging, biodistributions, autoradiography of tumor tissues and tumor blood vessel counts (CD31 IHC) were performed. Results: 18 F-albumin retention in various tumors steadily increased over time with U87MG tumor exhibiting the highest uptake (DV) at all times. Significant decreases in 18 F-albumin DVs were observed one week post-treatement (-39%) vs. controls whereas tumor caliper volumes were not significantly decreased until days 14 and 21. At day 21 the significant decrease in DVs in the treatment group (-44%) paralleled biodistribution DV measurements and was consistent with autoradiography and CD31 IHC findings. Conclusion: These data suggest that 18 F-albumin DVs obtained by imaging may serve as an early biomarker of the effectiveness of anti-angiogenic therapy and thus aid in patient management and treatment planning.

Objective: The goal is to evaluate avelumab, an anti-PD-L1 monoclonal immunoglobulin G antibody labeled with zirconium-89 in human PD-L1-expressing cancer cells and mouse xenografts for clinical translation., Methods: [ 89 Zr]Zr-DFO-PD-L1 monoclonal antibody (mAb) was synthesized using avelumab conjugated to desferrioxamine. In vitro binding studies and biodistribution studies were performed with PD-L1+MDA-MB231 cells and MDA-MB231 xenograft mouse models, respectively. Biodistributions were determined at 1, 2, 3, 5, and 7 days post coinjection of [ 89 Zr]Zr-DFO-PD-L1 mAb without or with unlabeled avelumab (10, 20, 40, and 400 µg)., Results: [ 89 Zr]Zr-DFO-PD-L1 mAb exhibited high affinity (K d ∼ 0.3 nM) and detected moderate PD-L1 expression levels in MDA-MB231 cells. The spleen and lymph nodes exhibited the highest [ 89 Zr]Zr-DFO-PD-L1 mAb uptakes in all time points, while MDA-MB231 tumor uptakes were lower but highly retained. In the unlabeled avelumab dose escalation studies, spleen tissue-muscle ratios decreased in a dose-dependent manner indicating specific [ 89 Zr]Zr-DFO-PD-L1 mAb binding to PD-L1. In contrast, lymph node and tumor tissue-muscle ratios increased 4- to 5-fold at 20 and 40 µg avelumab doses., Conclusions: [ 89 Zr]Zr-DFO-PD-L1 mAb exhibited specific and high affinity for PD-L1 in vitro and had target tissue uptakes correlating with PD-L1 expression levels in vivo. [ 89 Zr]Zr-DFO-PD-L1 mAb uptake in PD-L1+tumors increased with escalating doses of avelumab.

Introduction: Equilibrium single-photon radionuclide imaging methods for assessing cardiac function and the integrity of the vascular system have long been in use for both clinical and research purposes. However, positron-emitting blood pool agents that could provide PET equivalents to these (and other) clinical procedures have not yet been adopted despite technical imaging advantages offered by PET. Our goal was to develop a PET blood pool tracer that not only meets necessary in vivo biological requirements but can be produced with an uncomplicated and rapid synthesis method which would facilitate clinical translation. Herein, albumin labeled with fluorine-18 was synthesized using a one-pot method and evaluated in vitro and in vivo in rats., Methods: A ligand (NODA-Bz-TFPE), containing NODA attached to a tetrafluorophenylester (TFPE) via a phenyl linker (Bz), was labeled with aluminum fluoride (Al[ 18 F]F). Conjugation of the serum albumin with the ligand (Al[ 18 F]F-NODA-Bz-TFPE), followed by purification (size exclusion chromatography), yielded the final product (Al[ 18 F]F-NODA-Bz-RSA/HSA). In vitro stability was evaluated in human serum albumin by HPLC. Rat biodistributions and whole-body PET imaging over a 4 h time course were used for the in vivo evaluation., Results: This synthesis exhibited an overall radiochemical yield of 45 ± 10% (n = 30), a 50-min radiolabeling time, a radiochemical purity >99% and apparent stability up to 4 h in human serum. Blood had the highest retention of Al[ 18 F]F-NODA-Bz-RSA at all times with a blood half-life of 5.2 h in rats. Al[ 18 F]F-NODA-Bz-RSA distribution in most rat tissues remained relatively constant for up to 1 h, indicating that the tissue radioactivity content represents the respective tissue plasma volume. Dynamic whole-body PET images were in agreement with these findings., Conclusions: A new ligand has been developed and radiolabeled with Al[ 18 F]F that allows rapid (50-min) preparation of fluorine-18 serum albumin in one-pot. In addition to increased synthetic efficiency, the construct appears to be metabolically stable in rats. This method could encourage wider use of PET to quantify cardiac function and tissue vascular integrity in both research and clinical settings., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Following our recently published fluorine-18 labeling method, "Radio-fluorination on the Sep-Pak", we have successfully synthesized 6-[ 18 F]fluoronicotinaldehyde by passing a solution (1:4 acetonitrile: t-butanol) of its quaternary ammonium salt precursor, 6-(N,N,N-trimethylamino)nicotinaldehyde trifluoromethanesulfonate (2), through a fluorine-18 containing anion exchange cartridge (PS-HCO 3 ). Over 80% radiochemical conversion was observed using 10 mg of precursor within 1 minute. The [ 18 F]fluoronicotinaldehyde ([ 18 F]5) was then conjugated with 1-(6-(aminooxy)hexyl)-1H-pyrrole-2,5-dione to prepare the fluorine-18 labeled maleimide functionalized prosthetic group, 6-[ 18 F]fluoronicotinaldehyde O-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl) oxime, 6-[ 18 F]FPyMHO ([ 18 F]6). The current Sep-Pak method not only improves the overall radiochemical yield (50 ± 9%, decay-corrected, n = 9) but also significantly reduces the synthesis time (from 60-90 minutes to 30 minutes) when compared with literature methods for the synthesis of similar prosthetic groups., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

Introduction: We describe a simple fixture that can be added to the imaging bed of a small-animal PET scanner that allows for automated counting of multiple organ or tissue samples from mouse-sized animals and counting of injection syringes prior to administration of the radiotracer. The combination of imaging and counting capabilities in the same machine offers advantages in certain experimental settings., Methods: A polyethylene block of plastic, sculpted to mate with the animal imaging bed of a small-animal PET scanner, is machined to receive twelve 5-ml containers, each capable of holding an entire organ from a mouse-sized animal. In addition, a triangular cross-section slot is machined down the centerline of the block to secure injection syringes from 1-ml to 3-ml in size. The sample holder is scanned in PET whole-body mode to image all samples or in one bed position to image a filled injection syringe. Total radioactivity in each sample or syringe is determined from the reconstructed images of these objects using volume re-projection of the coronal images and a single region-of-interest for each. We tested the accuracy of this method by comparing PET estimates of sample and syringe activity with well counter and dose calibrator estimates of these same activities., Results: PET and well counting of the same samples gave near identical results (in MBq, R 2 =0.99, slope=0.99, intercept=0.00-MBq). PET syringe and dose calibrator measurements of syringe activity in MBq were also similar (R 2 =0.99, slope=0.99, intercept=- 0.22-MBq)., Conclusion: A small-animal PET scanner can be easily converted into a multi-sample and syringe counting device by the addition of a sample block constructed for that purpose. This capability, combined with live animal imaging, can improve efficiency and flexibility in certain experimental settings., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Introduction: Quantitative small animal radionuclide imaging studies are often carried out with the intention of estimating the total radioactivity content of various tissues such as the radioactivity content of mouse xenograft tumors exposed to putative diagnostic or therapeutic agents. We show that for at least one specific application, positron projection imaging (PPI) and PET yield comparable estimates of absolute total tumor activity and that both of these estimates are highly correlated with direct well-counting of these same tumors. These findings further suggest that in this particular application, PPI is a far more efficient data acquisition and processing methodology than PET., Methods: Forty-one athymic mice were implanted with PC3 human prostate cancer cells transfected with prostate-specific membrane antigen (PSMA (+)) and one additional animal (for a total of 42) with a control blank vector (PSMA (-)). All animals were injected with [ 18 F] DCFPyl, a ligand for PSMA, and imaged for total tumor radioactivity with PET and PPI. The tumors were then removed, assayed by well counting for total radioactivity and the values between these methods intercompared., Results: PET, PPI and well-counter estimates of total tumor radioactivity were highly correlated (R 2 >0.98) with regression line slopes near unity (0.95

Fluorine-18 labeling of biomolecules is mostly performed by an indirect labeling method using a prosthetic group. Fluorine-18 labeled 6-fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester is a useful prosthetic group to radiolabel a protein. Recently, we reported an improved preparation of this prosthetic group. To test the conjugation efficiency of the labeled ester prepared by this method, we have performed conjugation reactions with a peptide, a protein, and a small molecule. Prostate-specific membrane antigen targeting small molecule [ 18 F]DCFPyL, αvβ3 integrin receptors targeting peptide [ 18 F]c(RGDfK) and [ 18 F]albumin were prepared in good radiochemical yields. The conjugation reactions were completed at 40°C to 50°C in 10 minutes. The overall radiochemical yield was 25% to 43% in 30 to 45 minutes., (Published 2016. This article is a U.S. Government work and is in the public domain in the USA.)

Fluorine-18 labeled fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester has been successfully synthesized in an unprecedented way by flowing an acetonitrile solution of its quaternary ammonium salt precursor (N,N,N-trimethyl-5-((2,3,5,6-tetrafluorophenoxy)carbonyl)pyridin-2-aminium trifluoromethanesulfonate, 1) through an anion exchange cartridge. The fluorination reaction proceeded at room temperature without azeotropic drying of the fluoride. Over 75% conversion was observed with 10mg of precursor in 2:8, acetonitrile: t-butanol in 1min. The total synthesis time was 5min which is ~30min shorter than the current literature method., (Published by Elsevier Inc.)

Background: HIV-associated neuroinflammation is believed to be a major contributing factor in the development of HIV-associated neurocognitive disorders (HAND). In this study, we used micropositron emission tomography (PET) imaging to quantify neuroinflammation in HIV-1 transgenic rat (Tg), a small animal model of HIV, known to develop neurological and behavioral problems., Methods: Dynamic [(18)F]DPA-714 PET imaging was performed in Tg and age-matched wild-type (WT) rats in three age groups: 3-, 9-, and 16-month-old animals. As a positive control for neuroinflammation, we performed unilateral intrastriatal injection of quinolinic acid (QA) in a separate group of WT rats. To confirm our findings, we performed multiplex immunofluorescent staining for Iba1 and we measured cytokine/chemokine levels in brain lysates of Tg and WT rats at different ages., Results: [(18)F]DPA-714 uptake in HIV-1 Tg rat brains was generally higher than in age-matched WT rats but this was not statistically significant in any age group. [(18)F]DPA-714 uptake in the QA-lesioned rats was significantly higher ipsilateral to the lesion compared to contralateral side indicating neuroinflammatory changes. Iba1 immunofluorescence showed no significant differences in microglial activation between the Tg and WT rats, while the QA-lesioned rats showed significant activation. Finally, cytokine/chemokine levels in brain lysates of the Tg rats and WT rats were not significantly different., Conclusion: Microglial activation might not be the primary mechanism for neuropathology in the HIV-1 Tg rats. Although [(18)F]DPA-714 is a good biomarker of neuroinflammation, it cannot be reliably used as an in vivo biomarker of neurodegeneration in the HIV-1 Tg rat.

We sought to develop a practical, reproducible and clinically translatable method of radiolabeling serum albumins with fluorine-18 for use as a PET blood pool imaging agent in animals and man. Fluorine-18 radiolabeled fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester, [(18)F]F-Py-TFP was prepared first by the reaction of its quaternary ammonium triflate precursor with [(18)F]tetrabutylammonium fluoride ([(18)F]TBAF) according to a previously published method for peptides, with minor modifications. The incubation of [(18)F]F-Py-TFP with rat serum albumin (RSA) in phosphate buffer (pH9) for 15 min at 37-40 °C produced fluorine-18-radiolabeled RSA and the product was purified using a mini-PD MiniTrap G-25 column. The overall radiochemical yield of the reaction was 18-35% (n=30, uncorrected) in a 90-min synthesis. This procedure, repeated with human serum albumin (HSA), yielded similar results. Fluorine-18-radiolabeled RSA demonstrated prolonged blood retention (biological half-life of 4.8 hours) in healthy awake rats. The distribution of major organ radioactivity remained relatively unchanged during the 4 hour observation periods either by direct tissue counting or by dynamic PET whole-body imaging except for a gradual accumulation of labeled metabolic products in the bladder. This manual method for synthesizing radiolabeled serum albumins uses fluorine-18, a widely available PET radionuclide, and natural protein available in both pure and recombinant forms which could be scaled up for widespread clinical applications. These preclinical biodistribution and PET imaging results indicate that [(18)F]RSA is an effective blood pool imaging agent in rats and might, as [(18)F]HSA, prove similarly useful as a clinical imaging agent., (Published by Elsevier Inc.)

Developing an imaging agent targeting the hepatocyte growth factor receptor protein (Met) status of cancerous lesions would aid in the diagnosis and monitoring of Met-targeted tyrosine kinase inhibitors (TKIs). A peptide targeting Met labeled with [(99m)Tc] had high affinity in vitro (Kd = 3.3 nM) and detected relative changes in Met in human cancer cell lines. In vivo [(99m)Tc]-Met peptide (AH-113018) was retained in Met-expressing tumors, and high-expressing Met tumors (MKN-45) were easily visualized and quantitated using single-photon emission computed tomography or optical imaging. In further studies, MKN-45 mouse xenografts treated with PHA 665752 (Met TKI) or vehicle were monitored weekly for tumor responses by [(99m)Tc]-Met peptide imaging and measurement of tumor volumes. Tumor uptake of [(99m)Tc]-Met peptide was significantly decreased as early as 1 week after PHA 665752 treatment, corresponding to decreases in tumor volumes. These results were comparable to Cy5**-Met peptide (AH-112543) fluorescence imaging using the same treatment model. [(99m)Tc] or Cy5**-Met peptide tumor uptake was further validated by histologic (necrosis, apoptosis) and immunoassay (total Met, p Met, and plasma shed Met) assessments in imaged and nonimaged cohorts. These data suggest that [(99m)Tc] or Cy5**-Met peptide imaging may have clinical diagnostic, prognostic, and therapeutic monitoring applications.

Tumor endothelial marker 8 (TEM8) is a cell surface receptor that is highly expressed in a variety of human tumors and promotes tumor angiogenesis and cell growth. Antibodies targeting TEM8 block tumor angiogenesis in a manner distinct from the VEGF receptor pathway. Development of a TEM8 imaging agent could aid in patient selection for specific antiangiogenic therapies and for response monitoring. In these studies, L2, a therapeutic anti-TEM8 monoclonal IgG antibody (L2mAb), was labeled with (89)Zr and evaluated in vitro and in vivo in TEM8 expressing cells and mouse xenografts (NCI-H460, DLD-1) as a potential TEM8 immuno-PET imaging agent. (89)Zr-df-L2mAb was synthesized using a desferioxamine-L2mAb conjugate (df-L2mAb); (125)I-L2mAb was labeled directly. In vitro binding studies were performed using human derived cell lines with high, moderate, and low/undetectable TEM8 expression. (89)Zr-df-L2mAb in vitro autoradiography studies and CD31 IHC staining were performed with cryosections from human tumor xenografts (NCI-H460, DLD-1, MKN-45, U87-MG, T-47D, and A-431). Confirmatory TEM8 Western blots were performed with the same tumor types and cells. (89)Zr-df-L2mAb biodistribution and PET imaging studies were performed in NCI-H460 and DLD-1 xenografts in nude mice. (125)I-L2mAb and (89)Zr-df-L2mAb exhibited specific and high affinity binding to TEM8 that was consistent with TEM8 expression levels. In NCI-H460 and DLD-1 mouse xenografts nontarget tissue uptake of (89)Zr-df-L2mAb was similar; the liver and spleen exhibited the highest uptake at all time points. (89)Zr-L2mAb was highly retained in NCI-H460 tumors with <10% losses from day 1 to day 3 with the highest tumor to muscle ratios (T:M) occurring at day 3. DLD-1 tumors exhibited similar pharmacokinetics, but tumor uptake and T:M ratios were reduced ∼2-fold in comparison to NCI-H460 at all time points. NCI-H460 and DLD-1 tumors were easily visualized in PET imaging studies despite low in vitro TEM8 expression in DLD-1 cells indicating that in vivo expression might be higher in DLD-1 tumors. From in vitro autoradiography studies (89)Zr-df-L2mAb specific binding was found in 6 tumor types (U87-MG, NCI-H460, T-47D MKN-45, A-431, and DLD-1) which highly correlated to vessel density (CD31 IHC). Westerns blots confirmed the presence of TEM8 in the 6 tumor types but found undetectable TEM8 levels in DLD-1 and MKN-45 cells. This data would indicate that TEM8 is associated with the tumor vasculature rather than the tumor tissue, thus explaining the increased TEM8 expression in DLD-1 tumors compared to DLD-1 cell cultures. (89)Zr-df-L2mAb specifically targeted TEM8 in vitro and in vivo although the in vitro expression was not necessarily predictive of in vivo expression which seemed to be associated with the tumor vasculature. In mouse models, (89)Zr-df-L2mAb tumor uptakes and T:M ratios were sufficient for visualization during PET imaging. These results would suggest that a TEM8 targeted PET imaging agent, such as (89)Zr-df-L2mAb, may have potential clinical, diagnostic, and prognostic applications by providing a quantitative measure of tumor angiogenesis and patient selection for future TEM8 directed therapies.

Unlabelled: Interleukin-13 Pseudomonas exotoxin (IL-13PE), a targeted agent for interleukin-13 receptor α2 (IL-13Rα2)-expressing tumors, has been administered intracranially by convection-enhanced delivery (CED) for glioma therapy in several clinical trials including a randomized phase 3 clinical trial. However, its intracranial distribution was not optimally evaluated. We investigated the intracranial distribution of radiolabeled IL-13PE after CED in a murine model of glioblastoma multiforme., Methods: IL-13PE was radiolabeled with Na(125)I and evaluated for its activity in vitro in receptor-positive U251 or -negative T98G human glioma cell lines. Gliomas were grown in nude mice after intracranial implantation with U251 cells, and (125)I-IL-13PE was stereotactically administered by bolus or CED for 3 d, followed by micro-SPECT/CT imaging. SPECT images were evaluated quantitatively and compared with histology and autoradiography results., Results: The radioiodination technique resulted in a specific and biologically active (125)I-IL-13PE, which bound and was cytotoxic to IL-13Rα2-positive but not to IL-13Rα2-negative tumor cells. Both the binding and the cytotoxic activities were blocked by a 100-fold excess of IL-13, which indicated the specificity of binding and cytotoxicity. SPECT/CT imaging revealed retention of (125)I-IL-13PE administered by CED in U251 tumors and showed significantly higher volumes of distribution and maintained detectable drug levels for a longer period of time than the bolus route. These results were confirmed by autoradiography., Conclusion: IL-13PE can be radioiodinated without the loss of specificity, binding, or cytotoxic activity. Intracranial CED administration produces a higher volume of distribution for a longer period of time than the bolus route. Thus, CED of IL-13PE is superior to bolus injection in delivering the drug to the entire tumor., (© 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Introduction: We describe and illustrate a method for creating ECG-gated PET images of the heart for each of several mice imaged at the same time. The method is intended to increase "throughput" in PET research studies of cardiac dynamics or to obtain information derived from such studies, e.g. tracer concentration in end-diastolic left ventricular blood., Methods: An imaging bed with provisions for warming, anesthetic delivery, etc., was fabricated by 3D printing to allow simultaneous PET imaging of two side-by-side mice. After electrode attachment, tracer injection and placement of the animals in the scanner field of view, ECG signals from each animal were continuously analyzed and independent trigger markers generated whenever an R-wave was detected in each signal. PET image data were acquired in "list" mode and these trigger markers were inserted into this list along with the image data. Since each mouse is in a different spatial location in the FOV, sorting of these data using trigger markers first from one animal and then the other yields two independent and correctly formed ECG-gated image sequences that reflect the dynamical properties of the heart during an "average" cardiac cycle., Results: The described method yields two independent ECG-gated image sequences that exhibit the expected properties in each animal, e.g. variation of the ventricular cavity volumes from maximum to minimum and back during the cardiac cycle in the processed animal with little or no variation in these volumes during the cardiac cycle in the unprocessed animal., Conclusion: ECG-gated image sequences for each of several animals can be created from a single list mode data collection using the described method. In principle, this method can be extended to more than two mice (or other animals) and to other forms of physiological gating, e.g. respiratory gating, when several subjects are imaged at the same time., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The dopaminergic system is especially vulnerable to the effects of human immunodeficiency virus (HIV) infection, rendering dopaminergic deficits early surrogate markers of HIV-associated neuropathology. We quantified dopamine D2/3 receptors in young HIV-1 transgenic (Tg) (n  =  6) and age-matched control rats (n  =  7) and adult Tg (n  =  5) and age-matched control rats (n  =  5) using [18F]fallypride positron emission tomography (PET). Regional uptake was quantified as binding potential (BPND) using the two-tissue reference model with the cerebellum as the reference. Time-activity curves were generated for the ventral striatum, dorsal striatum, thalamus, and cerebellum. Whereas BPND values were significantly lower in the ventral striatum (p < .001) and dorsal striatum (p  =  .001) in the adult Tg rats compared to controls rats, they were significantly lower only in the dorsal striatum (p < .05) in the young rats. Tg rats had smaller striatal volumes on magnetic resonance imaging. We also found lower expression levels of tyrosine hydroxylase on immunohistochemistry in the Tg animals. Our findings suggest that progressive striatal D2/3 receptor deficits occur in Tg rats as they age and can be detected using small-animal PET imaging. The effectiveness of various approaches in preventing or halting this dopaminergic loss in the Tg rat can thus be measured preclinically using [18F]fallypride PET as a molecular imaging biomarker of HIV-associated neuropathology.

Introduction: We describe a prototype positron projection imager (PPI) for visualizing the whole-body biodistribution of positron-emitting compounds in mouse-size animals. The final version of the PPI will be integrated into the MONICA portable dual-gamma camera system to allow the user to interchangeably image either single photon or positron-emitting compounds in a shared software and hardware environment., Methods: A mouse is placed in the mid-plane between two identical, opposed, pixelated LYSO arrays separated by 21.8-cm and in time coincidence. An image of the distribution of positron decays in the animal is formed on this mid-plane by coincidence events that fall within a small cone angle perpendicular to the two detectors and within a user-specified energy window. We measured the imaging performance of this device with phantoms and in tests performed in mice injected with various compounds labeled with positron-emitting isotopes., Results: Representative performance measurements yielded the following results (energy window 250-650keV, cone angle 3.5°): resolution in the image mid-plane, 1.66-mm (FWHM), resolution ±1.5-cm above and below the image plane, 2.2-mm (FWHM), sensitivity: 0.237-cps/kBq (8.76-cps/μCi) (18)F (0.024% absolute). Energy resolution was 15.9% with a linear-count-rate operating range of 0-14.8MBq (0-400μCi) and a corrected sensitivity variation across the field-of-view of <3%. Whole-body distributions of [(18)F] FDG and [(18)F] fluoride were well visualized in mice of typical size., Conclusion: Performance measurements and field studies indicate that the PPI is well suited to whole-body positron projection imaging of mice. When integrated into the MONICA gamma camera system, the PPI may be particularly useful early in the drug development cycle where, like MONICA, basic whole-body biodistribution data can direct future development of the agent under study and where logistical factors (e.g., available imaging space, non-portability, and cost) may be limitations., (Published by Elsevier Inc.)

Unlabelled: The overexpression and overactivation of hepatocyte growth factor receptor (Met) in various cancers has been linked to increased proliferation, progression to metastatic disease, and drug resistance. Developing a PET agent to assess Met expression would aid in the diagnosis and monitoring of responses to Met-targeted therapies. In these studies, onartuzumab, the experimental therapeutic 1-armed monoclonal antibody, was radiolabeled with (76)Br or (89)Zr and evaluated as an imaging agent in Met-expressing cell lines and mouse xenografts., Methods: (89)Zr-desferrioxamine (df)-onartuzumab was synthesized using a df-conjugate; (76)Br-onartuzumab was labeled directly. Met-binding studies were performed using the human tumor-derived cell lines MKN-45, SNU-16, and U87-MG, which have relatively high, moderate, and low levels of Met, respectively. Biodistribution and small-animal PET studies were performed in MKN-45 and U87-MG xenografts., Results: (76)Br-onartuzumab and (89)Zr-df-onartuzumab exhibited specific, high-affinity Met binding (in the nanomolar range) that was concordant with established Met expression levels. In MKN-45 (gastric carcinoma) xenografts, both tracers cleared slowly from nontarget tissues, with the highest uptake in tumor, blood, kidneys, and lungs. (76)Br-onartuzumab MKN-45 tumor uptake remained relatively constant from 18 h (5 percentage injected dose per gram of tissue [%ID/g]) to 48 h (3 %ID/g) and exhibited tumor-to-muscle ratios ranging from 4:1 to 6:1. In contrast, (89)Zr-df-onartuzumab MKN-45 tumor uptake continued to accumulate from 18 h (10 %ID/g) to 120 h (23 %ID/g), attaining tumor-to-muscle ratios ranging from 20:1 to 27:1. MKN-45 tumors were easily visualized in imaging studies with both tracers at 18 h, but after 48 h (89)Zr-df-onartuzumab image quality improved, with at least 2-fold-greater tumor uptake than nontarget tissues. MKN-45 tumor uptake for both tracers correlated significantly with tumor mass and Met expression and was not affected by the presence of plasma shed Met., Conclusion: (89)Zr-df-onartuzumab and (76)Br-onartuzumab specifically targeted Met in vitro and in vivo; (89)Zr-df-onartuzumab achieved higher tumor uptake and tumor-to-muscle ratios than (76)Br-onartuzumab at later times, suggesting that (89)Zr-df-onartuzumab would be better suited to image Met for diagnostic and prognostic purposes.

Corticotropin-releasing factor (CRF), a neuropeptide, regulates endocrine and autonomic responses to stress through G-protein coupled receptors, CRF(1) or CRF(2) . A PET ligand able to monitor changes in CRF(1) receptor occupancy in vivo would aid in understanding the pathophysiology of stress-related diseases as well as in the clinical development of nonpeptide antagonists with therapeutic value. We have radiolabeled the CRF(1) receptor ligand, [8-(4-bromo-2,6-dimethoxyphenyl)-2,7-dimethylpyrazolo[1,5-α][1,3,5]triazin-4-yl]-N,N-bis-(2-methoxyethyl)amine (BMK-152) (ClogP = 2.6), at both the 3 and 4 position with [(76) Br]. Using in vitro autoradiography saturation studies the 4-[(76) Br]BMK-152 exhibited high affinity binding to both rat (K(d) = 0.23 ± 0.07 nM; n = 3) and monkey frontal cortex (K(d) = 0.31 ± 0.08 nM; n = 3) consistent with CRF(1) receptor regional distribution whereas with the 3-[(76) Br]BMK-152, the K(d) s could not be determined due to high nonspecific binding. In vitro autoradiography competition studies using [(125) I]Tyr(0) -o-CRF confirmed that 3-Br-BMK-152 (K(i) = 24.4 ± 4.9 nM; n = 3) had lower affinity (70-fold) than 4-Br-BMK-152 (K(i) = 0.35 ± 0.07 nM; n = 3) in monkey frontal cortex and similiar studies using [(125) I]Sauvagine confirmed CRF(1) receptor selectivity. In vivo studies with P-glycoprotein (PGP) knockout mice (KO) and their wild-type littermates (WT) showed that the brain uptake of 3-[(76) Br]BMK/4-[(76) Br]BMK was increased less than twofold in KO versus WT indicating that 3-[(76) Br]BMK-152/4-[(76) Br]BMK was not a Pgp substrate. Rat brain uptakes of 4-[(76) Br] BMK-152 from ex vivo autoradiography studies showed regional localization consistent with known published CRF(1) receptor distribution and potential as a PET ligand for in vivo imaging of CRF(1) receptors., (Copyright © 2011 Wiley-Liss, Inc.)

High-sensitivity and high-resolution LC/MS instrumentation has been applied in positron emission tomography (PET) radiopharmaceutical development to provide quantitative measurement of the mass of radiotracers extracted from tissues of rats. We employed the highly sensitive Waters Q-TOF premier MS coupled with an Acquity UPLC system to demonstrate that LC-MS can generate ex vivo biodistribution data for PET 5-HT(1A) ligand FPWAY without the need to radiolabel. For the biodistribution studies, we injected rats with [(18)F]FPWAY containing various amounts of nonradioactive FPWAY. At the end of the allotted time, the animals were killed and six regions of brain and plasma from each animal were processed for quantitative measurement of parent compound concentration by LC-MS. These data were then converted to the differential uptake ratio DUR (%ID/g*body weight/100) and the brain tissue-specific binding ratio to allow direct comparison with data obtained by gamma counting of the coinjected radioactive [(18)F]FPWAY. The DUR and the brain tissue-specific binding ratio calculated using the LC-MS method were highly correlated to the values obtained by standard radioactivity measurements of [(18)F]FPWAY. In conclusion, there was significant concordance between the LC/MS and radioactivity method in determination of DUR and the specific binding ratio in the rat brain. This concordance indicated that high-sensitivity LC/MS is an indispensable tool in evaluating the quantity of administered chemical in tissue as part of the development of new molecular imaging probes.

Unlabelled: This study aimed to determine the magnitude and distribution of tissue angiotensin-converting enzyme (ACE), mast-cell chymase, and angiotensin II, type 1, plasma membrane receptor (AT1R), in relation to collagen replacement in infarcted and noninfarcted left ventricular myocardial segments. A new radiotracer, 18F-fluorobenzoyl-lisinopril (FBL), was synthesized without compromising its affinity for tissue ACE., Methods: Five- to 10-microm contiguous short-axis slices of explanted hearts from 3 patients with ischemic cardiomyopathy were incubated in vitro with FBL, with and without 10(-6) M lisinopril. Tissue radioactivity was recorded as a function of position in photostimulating luminescence units (PSL). Immunohistochemistry studies were performed with mouse monoclonal antibody against ACE, anti-mast cell chymase, and polyclonal antibody against the human AT1R., Results: There was specific binding of FBL to ACE; mean FBL binding was 6.6 +/- 5.2 PSL/mm2, compared with 3.4 +/- 2.5 PSL/mm2 in segments incubated in solution containing cold, 10(-6) M lisinopril (P < 0.0001). Mean FBL binding was 6.3 +/- 4.5 PSL/mm2 in infarcted, 7.6 +/- 4.7 PSL/mm2 in periinfarcted, and 5.0 +/- 1.0 PSL/mm2 in remote, noninfarcted (P < 0.02 vs. periinfarcted) segments. The autoradiographic observations concerning FBL binding were confirmed by ACE and AT1R immunoreactivity. Distribution of mast cell chymase differed from ACE, as a higher number of mast cells was present in the remote, noninfarcted myocardium than in the periinfarcted myocardium (5.1 +/- 3.2 vs. 3.2 +/- 2.2 mast cells per field, P < 0.001). The number of mast cells in ischemic hearts exceeded that in normal hearts (4.2 +/- 2.7 vs. 1.5 +/- 1.2 mast cells per field, x200, P < 0.001)., Conclusion: FBL binds specifically to ACE. The binding is nonuniform in infarcted, periinfarcted, and remote, noninfarcted segments, and there is apparently increased ACE activity in the juxtaposed areas of replacement fibrosis. On the other hand, the distribution of mast cell chymase appears nonuniform and disparate from ACE.

Introduction: We set out to develop a muscarinic M1-selective agonist (based on the structure of the functionally M1-selective xanomeline) that could be radiolabeled with fluorine-18 for use as an imaging agent for positron emission tomography., Methods: The radiochemical synthesis was achieved, employing the arts of organic and radiochemical syntheses. Binding selectivity studies employed biodistribution studies, using autoradiography and/or tissue dissection, in wild-type or muscarinic receptor knockout mice., Results: [(18)F]Fluoroxanomeline shows rather uniform uptake in all mouse brain regions and high specific binding, with a brain-to-blood ratio of 32 at 60 min postinjection. In addition, the specific binding is demonstrated by a 58% to 75% decrease in brain uptake upon coinjection with 5 nmol of unlabeled fluoroxanomeline or xanomeline. Brain uptake studies with [(3)H]xanomeline in muscarinic knockout mice show decreased uptake in M1 (17-34%) and M2 (2-20%) knockout mice compared with control. However, statistical significance was observed in only a few regions. Comparison of [(18)F]fluoroxanomeline in knockout mice showed no difference in M1 or M4 knockout mice but a general decrease in M2 (2-24%) knockout mice. The decrease of [(18)F]fluoroxanomeline uptake in M2 knockout mice reached statistical significance in brain stem, cerebellum, frontal cortex, hippocampus, inferior colliculus and superior colliculus., Conclusion: Although xanomeline displays highly selective M1 agonist activity in functional assays, little selectivity for muscarinic subtype binding was observed for xanomeline or its fluorine-containing analogue, fluoroxanomeline. This emphasizes the lack of correlation between functional selectivity and binding selectivity.

[(18)F]FCWAY and [(18)F]FPWAY, analogues of the high affinity 5-HT(1A) receptor (5-HT(1A)R) antagonist WAY100635, were evaluated in rodents as potential radiopharmaceuticals for determining 5-HT(1A)R density and changes in receptor occupancy due to changes in endogenous serotonin (5-HT) levels. The in vivo hippocampus specific binding ratio [(hippocampus(uptake)/cerebellum(uptake))-1] of [(18)F]FPWAY was decreased to 32% of the ratio of [(18)F]FCWAY, indicating that [(18)F]FPWAY has lower affinity than [(18)F]FCWAY. The 5-HT(1A)R selectivity of [(18)F]FPWAY was confirmed using ex vivo autoradiography studies with 5-HT(1A)R knockout, heterozygous, and wildtype mice.Pre- or post-treatment of awake rodents in tissue dissection studies with paroxetine had no effect on hippocampal binding of [(18)F]FCWAY or [(18)F]FPWAY compared to controls, indicating neither tracer was sensitive to changes in endogenous 5-HT. In mouse ex vivo autoradiography studies in which awake mice were treated with fenfluramine following the [(18)F]FPWAY, a significant decrease was not observed in the hippocampus specific binding ratios. In rat dissection studies with fenfluramine administered following [(18)F]FPWAY or [(18)F]FBWAY ([(18)F]-MPPF) in awake or urethane-anesthetized rats, no significant differences in the specific binding ratios of the hippocampus were observed compared to their respective controls. [(18)F]FPWAY and [(18)F]FBWAY uptakes in all brain regions were increased variably in the anesthetized group (with the greatest increase in the hippocampus) vs. the awake group, but were decreased in the fenfluramine-treated anesthetized group vs. the anesthetized group. These data are best explained by changes in blood flow caused by urethane and fenfluramine, which varies from region to region in the brain.

The influence of the renin-angiotensin system (RAS) is recognized in cardiac and vascular injury. An extrinsic RAS has been known for decades, and an equally important intrinsic RAS has been discovered recently. The latter leads to pathologic tissue alterations in the absence of systemic stimuli and may be the main source of local tissue effects of RAS. A new radiotracer fluorobenzoyl-lisinopril was synthesized by radiolabeling benzoic acid active ester with 18F and reacting that with the epsilon-amino group of lisinopril. The presence of angiotensin-converting enzyme (ACE) activity and angiotensin II receptors was examined in relation to myocardial fibrosis. This tissue-specific radioligand represents the first study of ACE in the human heart. This article presents preliminary data on imaging the RAS in the human cardiac tissue and discusses the potential for clinical application of these imaging techniques to human patients.

In vivo imaging using positron emission tomography (PET) is important in the development of new radiopharmaceuticals in rodent animal models for use as biochemical probes, diagnostic agents, or in drug development. We have shown mathematically that, if small animal imaging studies in rodents are to have the same "quality" as human PET studies, the same number of coincidence events must be detected from a typical rodent imaging "voxel" as from the human imaging voxel. To achieve this using the same specific activity preparation, we show that roughly the same total amount of radiopharmaceutical must be given to a rodent as to a human subject. At high specific activities, the mass associated with human doses, when administered to a rodent, may not decrease the uptake of radioactivity at non saturable sites or sites where an enzyme has a high capacity for a substrate. However, in the case of binding sites of low density such as receptors, the increased mass injected could saturate the receptor and lead to physiologic effects and non-linear kinetics. Because of the importance of the mass injected for small animal PET imaging, we experimentally compared high and low mass preparations using ex vivo biodistribution and phosphorimaging of three compounds: 2-fluoro-2-deoxyglucose (FDG), 6-fluoro-L-metatyrosine (FMT) and one receptor-directed compound, the serotonin 5HT1A receptor ligand, trans-4-fluoro-N-[2-[4-(2-methoxylphenyl) piperazino]ethyl]-N-(2-pyridyl) cyclohexane- carboxamide (FCWAY). Changes in the mass injected per rat did not affect the distribution of FDG, FMT, and FCWAY in the range of 0.6-1.9 nmol per rat. Changes in the target to nontarget ratio were observed for injected masses of FCWAY in the range of approximately 5-50 nmol per rat. If the specific activity of such compounds and/or the sensitivity of small animal scanners are not increased relative to human studies, small animal PET imaging will not correctly portray the "true" tracer distribution. These difficulties will only be exacerbated in animals smaller than the rat, e.g., mice.

[(18)F][3-(3-(3-Fluoropropyl)thio)-1,2,5-thiadiazol-4-yl]-1,2,5,6-tetrahydro-1-methylpyridine ([(18)F]FP-TZTP) is an M2 selective muscarinic agonist that may allow noninvasive studies of Alzheimer's disease with PET. 3-(3-(Propylthio)-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (P-TZTP), a nonfluorinated analog of FP-TZTP, and unlabeled FP-TZTP inhibited [(18)F]FP-TZTP binding in vivo. Because muscarinic action of the loading dose of P-TZTP administered might have had pharmacological effects, the apparent inhibition might have resulted from reduced delivery rather than competition with receptor-binding. Therefore, we examined the effects of P-TZTP or FP-TZTP administration on cerebral blood flow (CBF) measured by the [(14)C]iodoantipyrine method and laser-Doppler flowmetry in rats. Statistically significant synchronous decreases in both CBF and mean arterial blood pressure (MABP) were observed within the first minute following administration. The decreases in both CBF and MABP were prevented by pretreatment with atropine methyl bromide (M-At), a peripheral muscarinic antagonist, and coadministration of M-At with either FP-TZTP or P-TZTP resulted in the same degree of inhibition of cerebral [(18)F]FP-TZTP-uptake 30 min after administration as observed without M-At. Also, with programmed infusions designed to produce constant arterial concentrations of [(18)F]FP-TZTP and FP-TZTP, which avoid changes in CBF, significant inhibition of [(18)F]FP-TZTP-binding by FP-TZTP was observed. These results indicate that inhibition of [(18)F]FP-TZTP-binding in the brain by P-TZTP or FP-TZTP in vivo occurs independently of their effects on CBF. The methods employed here may also be of interest to evaluate physiological effects of blocking agents utilized to validate other radiopharmaceuticals., (Published 2003 Wiley-Liss, Inc.)

A muscarinic receptor radioligand, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (fP-TZTP) radiolabeled with the positron emitting radionuclide (18)F ([(18)F]FP-TZTP) displayed regional brain distribution consistent with M2 receptor densities in rat brain. The purpose of the present study is to further elucidate the subtype selectivity of [(18)F]FP-TZTP using genetically engineered mice which lacked functional M1, M2, M3, or M4 muscarinic receptors. Using ex vivo autoradiography, the regional brain localization of [(18)F]FP-TZTP in M2 knockout (M2 KO) was significantly decreased (51.3 to 61.4%; P<0.01) when compared to the wild-type (WT) mice in amygdala, brain stem, caudate putamen, cerebellum, cortex, hippocampus, hypothalamus, superior colliculus, and thalamus. In similar studies with M1KO, M3KO and M4KO compared to their WT mice, [(18)F]FP-TZTP uptakes in the same brain regions were not significantly decreased at P<0.01. However, in amygdala and hippocampus small decreases of 19.5% and 22.7%, respectively, were observed for M1KO vs WT mice at P<0.05. Given the fact that large decreases in [(18)F]FP-TZTP brain uptakes were seen only in M2 KO vs. WT mice, we conclude that [(18)F]FP-TZTP preferentially labels M2 receptors in vivo.

Paclitaxel (Taxol) is a clinically important chemotherapeutic agent. We describe the synthesis of fluoro-, bromo-, and iodopaclitaxel and their [(18)F]fluoro-, [(76)Br]bromo-, and [(124)I]iodo- analogues. [(18)F]Fluoropaclitaxel shows high uptake and rapid clearance from tissues in rats. Preadministration of paclitaxel in normal rats significantly increases (p < 0.005) retention of [(18)F]fluoropaclitaxel and [(76)Br]bromopaclitaxel in blood (33.0%), heart (32.0%), lung (37.6%) kidney (142.4%); and blood (33.4%), lung (42.3%), kidney (62.4%), respectively. [(18)F]Fluoropaclitaxel uptake in the brain of mdr1a/1b(-/-) mice is increased 1400% (p < 1.3e-07) relative to wild-type controls. Preadministration of paclitaxel or XR9576, a modulator, had little effect on the biodistribution in these mdr1a/1b(-/-) mice. As a result, [(18)F]fluoropaclitaxel will be a useful radiopharmaceutical for the study of multidrug resistant tumors.

Purpose: A 18fluorine-labeled derivative of vasoactive intestinal peptide [18F- Arg,Arg VIP(18F-dVIP)] was evaluated as a potential imaging agent for breast cancer by comparison with 2-deoxy-2-[18F]fluoro-D-glucose (FDG) using standard ex vivo determinations and small animal position emission tomography (PET) imaging., Procedures: Human breast carcinomas, T-47D and MDA-MB231, tumor-bearing nude mice were injected intravenously with 18F-dVIP or FDG for imaging and/or biodistribution (ex vivo) determined by gamma counting., Results: FDG had two- to three-fold greater tumor accumulation and target-to-non target contrast relative to 18F-dVIP. VIP receptors were detected in both tumor types but in low concentrations (<15,000 receptors/cell) consistent with lower uptakes. FDG was cleared rapidly from non-target tissues while 18F-dVIP cleared into the kidneys., Conclusions: 18F-dVIP uptake in mice T-47D tumors and kidneys determined by imaging correlated with values determined by ex vivo counting suggesting that tumor and other tissue uptakes can be quantified by in vivo positron projection imaging.

Monitoring gene therapy of glycogen storage disease type 1a in a mouse model was achieved using [(18)F]FDG and a dedicated animal scanner. The G6Pase knockout (KO) mice were compared to the same mice after infusion with a recombinant adenovirus containing the murine G6Pase gene (Ad-mG6Pase). Serial images of the same mouse before and after therapy were obtained and compared with wild-type (WT) mice of the same strain to determine the uptake and retention of [(18)F]FDG in the liver. Image data were acquired from heart, blood pool and liver for twenty minutes after injection of [(18)F]FDG. The retention of [(18)F]FDG was lower for the WT mice compared to the KO mice. The mice treated with adenovirus-mediated gene therapy had retention similar to that found in age-matched WT mice. These studies show that FDG can be used to monitor the G6Pase concentration in liver of WT mice as compared to G6Pase KO mice. In these mice, gene therapy returned the liver function to that found in age matched WT controls as measured by the FDG kinetics in the liver compared to that found in age matched wild type controls.

[76Br]FBAU is a potential PET tracer for assessing proliferation. This study proposes that [76Br]FBAU 3',5'-dibenzoate has higher blood-brain-barrier permeability than [76Br]FBAU itself and thus might be better suited for applications in the brain. The brain uptake indexes of the two compounds measured after carotid injection (29.6 +/- 13.9 for [76Br]FBAU 3',5'-dibenzoate, versus 10.0 +/- 8.7 for [76Br]FBAU) support this claim. Biodistribution study also showed that the brain accumulation of activity was higher in rats injected with [76Br]FBAU 3',5'-dibenzoate than with [76Br]FBAU (0.119+/-0.023 DUR at 1 h, versus 0.061 +/- 0.006). [76Br]FBAU 3',5'-dibenzoate was relatively stable in rat plasma, gradually being hydrolyzed to [76Br]FBAU exponentially with a calculated half-life of 0.8 h. DNA incorporation of [76Br]FBAU was also confirmed. The results presented support the hypothesis that the 3',5'-dibenzoate can act as a prodrug for FBAU and deliver more radiolabeled nucleoside to the brain.

An accurate, rapid method for the determination of unmetabolized 3-(3-[(3-[18F]fluoropropyl)thio]-1,2,5-thiadiazol-4-yl)-1.2,5,6-tetrahydro-1-methylpyridine (FP-TZTP), a selective M2 muscarinic agonist, is necessary in order to obtain quantitative information from positron emission tomography (PET) imaging. Using LC-MS-MS to analyze products from cultured human and rat hepatocytes, we identified metabolites resulting from oxidation of the nitrogen in the tetrahydropyridine ring, sulfur-oxidation, demethylation of the tertiary amine, and oxidation of the tetrahydropyridine ring. From the knowledge of the structure of the metabolites, we have developed a two-step extraction sequence that allows rapid determination of the parent fraction in plasma without time-consuming chromatographic analysis.

The racemate 1, ((+/-)-(5-Chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)- 2H-indol-2-one), is a potent, specific and novel opener of cloned large-conductance, calcium-activated (maxi-K) potassium channels. One of its enantiomers, BMS-204352 (MaxiPost), is undergoing clinical evaluation for efficacy in patients with suspected acute stroke. In the current study, we have prepared [(18)F]-labeled 1 using a silver assisted nucleophilic substitution to examine its distribution and disposition in the rat, with particular emphasis on the brain. Biodistribution studies in rats confirm that brain uptake is rapid and occurs at high levels, and indicate that a major fraction of the compound in the brain does not accumulate by a specific, saturable mechanism.

Use of the [(18)F]-fluoromethyl phenyl group is an attractive alternative to direct fluorination of phenyl groups because the fluorination of the methyl group takes place under milder reaction conditions. However, we have found that 4-FMeBWAY showed femur uptake equal to that of fluoride up to 30 min in rat whereas 4-FMeQNB had a significantly lower percent injected dose per gram in femur up to 120 min. For these and other benzylfluoride derivatives, there was no clear in vivo structure-defluorination relationship. Because benzylchlorides (BzCls) are known alkylating agents, benzylfluorides may be alkylating agents as well, which may be the mechanism of defluorination. On this basis, the effects of substitution on chemical stability were evaluated by the 4-(4-nitro-benzyl)-pyridine (NBP) test, which is used to estimate alkylating activity with NBP. The effect of substitution on the alkylating activity was evaluated for nine BzCl derivatives: BzCl; 3- or 4-methoxy (electron donation) substituted BzCl; 2-, 3-, or 4-nitro (electron withdrawing) substituted BzCl; and 2-, 3-, or 4-chloro (electron withdrawing) substituted BzCl. Taken together, the alkylating reactivity of 3-chloro-BzCl was the weakest. This result was then applied to [(18)F]-benzylfluoride derivatives and in vivo and in vitro stability were evaluated. Consequently, 3-chloro-[(18)F]-benzylfluoride showed a 70-80% decrease of defluorination in both experiments in comparison with [(18)F]-benzylfluoride, as expected. Moreover, a good linear relationship between in vivo femur uptake and in vitro hepatocyte metabolism was observed with seven (18)F-labeled radiopharmaceuticals, which were benzylfluorides, alkylfluorides, and arylfluorides. Apparently, the [(18)F]-fluoride ion is released by metabolism in the liver in vivo. In conclusion, 3-chloro substituted BzCls are the most stable, which suggests that 3-chloro benzylfluorides will be the most chemically stable compound. This result should be important in future design of radioligands labeled with a benzylfluoride moiety.

Based on encouraging in vitro data indicating M2 subtype selectivity, we synthesized, radiolabeled with 18F, and evaluated 3-(3-(2-fluoroethylthio)-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetr ahydro-1-methylpyridine [FE-TZTP], and 3-(3-(3-fluoropropylthio)-1,2,5-thiadiazol-4-yl)-1,2,5,6-tet rahydro-1-methylpyridine [FP-TZTP] for muscarinic subtype selectivity in vivo. [18F]FE-TZTP displays high uptake in vivo but is inhibited only weakly by coinjecting unlabeled P-TZTP. Contrarily, [18F]FP-TZTP shows significant inhibition of uptake by coinjecting unlabeled P-TZTP or the muscarinic agonist L-687,306 (3-(3-cyclopropyl-1,2,4-oxadiazol-5-yl)-1-azabicyclo[2.2.1]heptane ). Using in vivo autoradiography, [18F]FP-TZTP displays regional distribution consistent with M2 subtype distribution. In addition, [18F]FP-TZTP shows specific uptake in the heart at 5 min. Analysis of metabolites in the awake rat brain revealed that the parent compound represents >95% of the extractable activity at 30 min. In vivo studies in rhesus monkeys revealed rapid brain uptake of [18F]FP-TZTP, with clearance sustained over 2 h. Administration of P-TZTP or FP-TZTP (80 nmol/kg) at 60 min after injection of [18F]FP-TZTP results in a significant displacement of brain activity in all regions. Metabolite analysis in monkey plasma shows that parent compound represents 20% of the extractable radioactivity at 40 min postinjection. One metabolite, which increases with time, has similar lipophilicity to the parent. However, based on metabolism in rat we believe metabolites are not in the brain to any significant extent in monkeys during the time of imaging studies. Regional uptake, autoradiographic distribution, and clearance rates in the brain are consistent with the hypothesis that [18F]FP-TZTP is M2 selective in vivo.

Two [18F] labeled ligands for the mAChR were prepared and evaluated in rodents and nonhuman primates. The properties of both compounds, one an agonist and the other an antagonist, were consistent with M2 subtype specificity.

We have developed a multistep radiochemical synthesis of two diastereomers of quinuclidinyl-4-[18F]-fluoromethyl-benzilate ([18F]-FMeQNB), a high-affinity ligand for muscarinic acetylcholine receptors. Previously, we have shown that the nonradioactive (R,R)-diastereomer displays an eightfold selectivity for M1 over M2 while the nonradioactive (R,S)-diastereomer displays a sevenfold selectivity for M2 over M1 in vitro. This paper reports the results of in vivo comparison studies. In the rat, uptake of (R,S)-[18F]-FMeQNB was nearly uniform in all brain regions following the concentration of M2 subtype. The uptake was reduced by 36-54% in all brain regions on coinjection with 50 nmol of unlabeled ligand. An injection of (R,S)-[18F]-FMeQNB followed at 60 min by injection of unlabeled ligand and subsequent sacrifice at 120 min displaced 30-50% of radioactivity in the pons, medulla, and cerebellum, which contain a high proportion of M2 subtype. The most dramatic displacement and inhibition of uptake on coinjection of (R,S)-[18F]-FMeQNB was observed in the heart. In rhesus monkey, the compound showed prolonged uptake and retention in the brain. In the blood, the parent compound degraded rapidly to a single radiolabeled polar metabolite believed to be fluoride. Within 30 min the parent compound represented less than 5% of the plasma activity. Displacement with (R)-QNB was generally slow, but was more rapid from those tissues which contain a higher proportion of M2 subtype. The results are consistent with the hypothesis that (R,S)-[18F]-FMeQNB is M2 selective in vivo. On the other hand, (R,R)-[18F]-FMeQNB showed higher uptake in those brain regions containing a higher concentration of M1 subtype. Uptake in the heart at 60 min was much lower than that observed with the (R,S)-diastereomer. Inhibition of uptake on coinjection with unlabeled (R,S)-FMeQNB is only significant in the heart, thalamus, and pons. Inhibition of uptake on coinjection with unlabeled (R,R)-FMeQNB is quite uniform in all brain regions. Displacement with (R)-QNB shows a more varying amount displaced. These results are consistent with (R,R)-[18F]-FMeQNB being M1 selective in vivo.

An affinity column method was developed to determine the immunoreactivity of 131I-ChiL6 (chimeric L6 monoclonal antibody), a candidate for radioimmunotherapy. This method involved assessing the binding of the radiolabeled antibody to antigen containing membranes bound to a Reacti-gel agarose matrix. The immunoreactivity determined by the affinity column method correlated to other in vitro binding assays including the Lindmo infinite antigen excess method. In tumor-bearing mice which had been injected with 131I-ChiL6, which possessed high immunoreactivities (90-82%), a high tumor uptake (13.5-10.5% ID/g) was observed. A decrease in tumor uptake (5.2-4.8% ID/g) was observed with 131I-ChiL6 samples of low immunoreactivity (42% and 31%, respectively). While a moderate loss of immunoreactivity (4-18%) of the 131I-ChiL6 samples could be detected by the affinity column method, the loss of tumor uptake in vivo observed was not as significant. This method was found to be an efficient and sensitive method for detecting damage to the antibody during radiolabeling and applicable as a quality control method for clinical trials. This rapid method, compared to the other in vitro binding assays (including the Lindmo infinite antigen excess method) has distinct advantages as a quality control method since it requires less manipulation and can be semi-automated.

Gadoteridol, a nonionic gadolinium chelate, is currently being evaluated for contrast-enhanced magnetic resonance imaging. A radioimmunoassay (RIA) has been developed for the measurement of gadoteridol in biological fluids. The RIA has a range of 0 to 25 micrograms/mL and has the sensitivity to detect 0.05 microgram/mL of gadoteridol. Satisfactory zero binding and sensitivity were obtained after an overnight incubation at 4 degrees C. Separation of the antibody-bound and free radiolabel was achieved with 12.5% polyethylene glycol. A quantitative recovery of the exogenous analyte was obtained at all concentrations of gadoteridol tested. Linearity in both serum and urine was satisfactory. Intraassay coefficients of variation were 6.4 and 2.8% for the low and medium controls, respectively. Interassay coefficients of variation were 5.4, 3.8, and 12.2% for the low, medium, and high controls, respectively. Cross reactivities of the ligand 5 and the calcium salt 6 were 37 and 29%, respectively. Clinical samples from the ascending dosage studies were analyzed by the gadoteridol RIA. The results obtained from the serum specimens demonstrated an excellent linear proportionality between drug concentration in blood and administered dosage of gadoteridol. Cumulative urinary excretion data showed that 94% of the drug was excreted in the urine within 24 h.

We have determined the kinetics of dissociation of (R)-3-Quinuclidinyl (R)-4-[125I]Iodobenzilate ((R,R)-[125I]4IQNB) from muscarinic acetylcholine receptor preparations from the cortex, hippocampus, caudate/putamen, thalamus, pons and colliculate bodies. The dissociation curves are well described by a biexponential function and are consistent with subtype selectivity favoring slow dissociation from the M1, M3, and M4 receptors with a 20-fold faster dissociation rate for the M2 receptor. Following intravenous injection, (R,R)-[125I]4IQNB binds to receptor in the rat brain in concentrations which reflect the receptor concentration present in a structure. We determined the extent of radioligand present at two times, 2 and 24 hrs, as an indication of the relative proportions of m-AChR which exhibits rapid vs. slow dissociation of (R,R)-[125I]4IQNB. A good correlation between in vitro and in vivo results suggests that the relative populations of receptor subtypes can be imaged using in vivo pharmacokinetics of (R,R)-[125I]4IQNB.

Among ligands that bind to the alpha- and beta-adrenoceptors and to the muscarinic acetylcholine receptor (m-AChR), those that bind to the latter have the best properties for external detection of receptor sites by gamma-camera imaging. To develop the optimal radiotracer, nonradioactive analogues of 3-quinuclidinyl benzilate (I) were tested in in vivo displacement studies with (-)-[3H]I to determine their ability to compete with (-)-[3H]I for the muscarinic acetylcholine receptor. There is a linear correlation between the ability to compete with (-)-[3H]I for the m-AChR and the affinity constant of the analogue as determined by in vitro assay, suggesting that the test is a valid indicator of in vivo distribution. One radioiodinated analogue, 3-quinuclidinyl p- iodobenzilate , bound to m-AChR in the heart and brain of rats.