Fast implementation of positron emission tomography (PET) into clinical and preclinical studies highly demands automated synthesis for the preparation of PET radiopharmaceuticals in a safe and reproducible manner. The aim of this study was to develop automated synthesis methods for these six 18F-labeled radiopharmaceuticals produced on a routine basis at the University of Pennsylvania using the AllinOne synthesis module.The development of automated syntheses with varying complexity was accomplished including HPLC purification, SPE procedures and final formulation with sterile filtration. The six radiopharmaceuticals were obtained in high yield and high specific activity with full automation on the AllinOne synthesis module under current good manufacturing practice (cGMP) guidelines.The study demonstrates the versatility of this synthesis module for the preparation of a wide variety of 18F-labeled radiopharmaceuticals for PET imaging studies.

Cancer and neurodegeneration represent two opposite ends of the biological spectrum but contain many common biological mechanisms. Two such mechanisms include the elevated levels of oxidative stress and DNA damage. In this brief review, we describe current approaches for imaging these biological pathways with the molecular imaging technique, Positron Emission Tomography (PET), and the potential of PET imaging studies to measure the efficacy of anticancer drugs and strategies for delaying the progression of neurodegenerative disorders.

G protein-coupled receptors (GPRCs) are a class of proteins that are expressed in high abundance and are responsible for numerous signal transduction pathways in the central nervous system. Consequently, alterations in GPRC function have been associated with a wide variety of neurologic and neuropsychiatric disorders. The development of PET probes for imaging GPRCs has served as a major emphasis of PET radiotracer development and PET imaging studies over the past 30 years. In this review, a basic description of the biology of G proteins and GPRCs is provided. This includes recent evidence of the existence of dimeric and multimeric species of GPRCs that have been termed “receptor mosaics,” with an emphasis on the different GPRCs that form complexes with the dopamine D2 receptor. An overview of the different PET radiotracers for imaging the component GPRC within these different multimeric complexes of the D2 receptor is also provided.

A facile Pd-catalyzed methodology providing an efficient synthetic route to biologically relevant arylpiperazines under aerobic conditions is reported. Electron donating and sterically hindered aryl chlorides were aminated to afford yields up to 97%, with examples using piperazine as solvent, illustrating an ecofriendly, cost-effective synthesis of these privileged structures.

We examined the progesterone receptor membrane component 1 (PGRMC1) protein expression in rat brain cells as the prerequisite step to understand the biology of PGRMC1 in the central nervous system (CNS). We also performed correlation studies between the PGRMC1 protein level and the binding activity of a sigma-2 fluorescent probe, SW120, in order to explore the possibility of using sigma-2 radiotracer of positron emission tomography (PET) to noninvasively image the CNS.Embryonic primary neurons, astrocytes, oligodendrocytes, and microglia cells were cultured. Immunocytochemistry, Western blot, and SW120 staining were performed in these cells.The protein expression of PGRMC1 determined by immunocytochemistry and SW120 staining is prominent in neurons and relatively low in astrocytes, oligodendrocytes, and microglia cells. The PGRMC1 expression level correlates with the binding activity of SW120 in rat brain cells.The sigma-2 receptor PET radiotracer can be potentially used to noninvasively image neuron/synapse densities in the CNS.

A series of 3-(benzylidine)indolin-2-one derivatives were synthesized and evaluated for their in vitro binding to alpha synuclein (α-syn), beta amyloid (Aβ), and tau fibrils. Compounds with a single double bond in the 3-position had only a modest affinity for α-syn and no selectivity for α-syn versus Aβ or tau fibrils. Homologation to the corresponding diene analogues yielded a mixture of Z,E and E,E isomers; substitution of the indoline nitrogen with an N-benzyl group resulted in increased binding to α-syn and reasonable selectivity for α-syn versus Aβ and tau. Introduction of a para-nitro group into the benzene ring of the diene enabled separation of the Z,E and E,E isomers and led to the identification of the Z,E configuration as the more active regioisomer. The data described here provide key structural information in the design of probes which bind preferentially to α-syn versus Aβ or tau fibrils.

A facile method was developed to purify 2-[18F]fluoroethyl azide ([18F]FEA) using a C18 cartridge and an Oasis® HLB cartridge in series, in which [18F]FEA was exclusively trapped on the HLB cartridge. [18F]FEA can be eluted for reactions in solution; alternatively click labeling can be carried out on the HLB cartridge itself by loading an alkyne substrate and copper (I) catalyst dissolved in DMF onto the cartridge. This solid phase extraction methodology for purification and click labeling with [18F]FEA, either in solution or on the cartridge, is safe, simple, reproducible in high yield, and compatible with automated synthesis of 18F-labeled PET tracers.

A series of 2-methoxyphenyl piperazine analogues containing a triazole ring were synthesized and their in vitro binding affinities at human dopamine D2 and D3 receptors were evaluated. Compounds 5b, 5c, 5d, and 4g, demonstrate high affinity for dopamine D3 receptors and moderate selectivity for the dopamine D3 versus D2 receptor subtypes. To further examine their potential as therapeutic agents, their intrinsic efficacy at both D2 and D3 receptors was determined using a forskolin-dependent adenylyl cyclase inhibition assay. Affinity at dopamine D4 and serotonin 5-HT1A receptors was also determined. In addition, information from previous molecular modeling studies of the binding of a panel of 163 structurally-related benzamide analogues at dopamine D2 and D3 receptors was applied to this series of compounds. The results of the modeling studies were consistent with our previous experimental data. More importantly, the modeling study results explained why the replacement of the amide linkage with the hetero-aromatic ring leads to a reduction in the affinity of these compounds at D3 receptors.

Two novel classes of compounds targeting the sigma-2 (σ2) receptor were synthesized, and their bioactivities to binding σ1 and σ2 receptors were measured. Four novel triazole carboxamide analogues, 24d, 24e, 24f, and 39c, demonstrated high affinity and selectivity for the σ2 receptor. These data suggest 11C-labeled versions of these compounds may be potential σ2-selective radiotracers for imaging the proliferative status of solid tumors.

Reactive oxygen species (ROS) have been implicated in the pathogenesis of a wide range of human disease states and drug toxicities, but development of imaging tools to study ROS biology in vivo remains a challenge. Here we synthesized and validated a novel PET tracer (12) and its 18F radiolabeled version [18F]12 to allow PET (positron emission tomography) imaging of superoxide in vivo. Initial analysis of ROS reaction kinetics found that compound 12 was rapidly and selectively oxidized by superoxide, but not other ROS. Cell culture studies in EMT6 cells exposed to the cancer chemotherapeutic agent Doxorubicin (DOX), which activates the superoxide-generating enzyme, NADPH oxidase, showed that compound 12 was a sensitive and specific probe for superoxide in cells. The microPET imaging of heart in mice with DOX-induced cardiac inflammation observed 2-fold greater oxidation of [18F]12 in the DOX-treated mice compared to controls (p = 0.02), the results were confirmed by distribution studies on organs subsequently removed from the mice and HPLC analysis of [18F] radioactivity compounds. These data indicate that compound 12 is a useful PET tracer to imaging ROS in vivo.

IntroductionNine novel analogues were synthesized including a 6-carbon spacer analogue of ISO-1 (7). They have moderate binding affinity for sigma-2 (σ2) receptors and high selectivity for σ2 receptors relative to sigma-1 (σ1) receptors.Methods([18F]7) was synthesized and evaluated as a candidate ligand for positron emission (PET) imaging of the σ2 receptor in tumors. Radioligand [18F]7 was radiolabeled with 18F via displacement of the corresponding mesylate precursor with [18F]fluoride. Cellular uptake study of [18F]7 was performed in EMT-6 tumor cell, and in vivo biodistribution study of [18F]7 and microPET imaging study of [18F]3 and [18F]7 carried out in female Balb/c mice bearing EMT-6 tumors.Results[18F]7 had a respectable tumor uptake (1.55%ID/g at 60 min post-injection) and high tumor/muscle ratios at 60 and 120 min post-injection. MicroPET imaging of [18F]7 in tumor-bearing mice as above showed significant tumor localization and a high tumor/muscle ratio as well.ConclusionsThese results are similar to or better than [18F]ISO-1 ([18F]3), which indicates that [18F]7 has potential for imaging the σ2 receptor status of solid tumors.

•We report a cancer-selective drug delivery concept targeting the sigma-2 receptor.•SW IV-134 is a dual-domain drug conjugate with delivery and effector component.•All features of the individual components are well retained in the drug conjugate.•SW IV-134 represents a potent pancreatic cancer drug in vitro and in vivo.•This delivery concept can be expanded to other sigma-2 receptor expressing cancers.Pancreatic adenocarcinoma is highly resistant to conventional therapeutics and has been shown to evade apoptosis by deregulation of the X-linked and cellular inhibitors of apoptosis proteins (XIAP and cIAP). Second mitochondria-derived activator of caspases (Smac) induces and amplifies cell death by reversing the anti-apoptotic activity of IAPs. Thus, Smac-derived peptide analogues (peptidomimetics) have been developed and shown to represent promising cancer therapeutics. Sigma-2 receptors are overexpressed in many proliferating tumor cells including pancreatic cancer. Selected ligands to this receptor are rapidly internalized by cancer cells. These characteristics have made the sigma-2 receptor an attractive target for drug delivery because selective delivery to cancer cells has the potential to increase therapeutic efficacy while minimizing toxicity to normal tissues.Here, we describe the initial characterization of SW IV-134, a chemically linked drug conjugate between the sigma-2 ligand SW43 and the Smac mimetic SW IV-52 as a novel treatment option for pancreatic adenocarcinoma. The tumor killing characteristics of our dual-domain therapeutic SW IV-134 was far greater than either component in isolation or in an equimolar mix and suggests enhanced cellular delivery when chemically linked to the sigma-2 ligand. One of the key findings was that SW IV-134 retained target selectivity of the Smac cargo with the involvement of the NF-κB/TNFα signaling pathway. Importantly, SW IV-134 slowed tumor growth and improved survival in murine models of pancreatic cancer. Our data support further study of this novel therapeutic and this drug delivery strategy because it may eventually benefit patients with pancreatic cancer.

BackgroundPARP inhibitors (PARPi) have the potential to impact cancer therapy in a selective patient population; however, despite current patient selection methods clinical trials have shown mixed response rates. It is therefore clinically useful to determine which patients will respond prior to receiving PARPi therapy. One essential biomarker is to measure the level of PARP enzyme expression in tumors. Small molecule radiotracers have been developed to accurately quantify PARP-1 expression in vitro and in vivo. [125I]KX-02-019 is the first report of a radioiodinated analogue of the benzimidazole class of PARPi. Herein, we studied the pharmacological properties of [125I]KX-02-019 as well as the in vivo biodistribution.Methods[125I]KX-02-019 was evaluated in both cancer and non-cancer cell lines. We evaluated the pharmacologic properties of [125I]KX-02-019 in live cells by measuring enzyme association and dissociation kinetics, saturation, and specificity. In addition, competitive inhibition experiments were carried out with commercially available PARPi. Protein expression was analyzed by Western blot to compare PARP-1 and PARP-2 expression across cell lines studied. The biodistribution was studied in a mouse EMT6 tumor model at time points of 0.5, 1, 2, 4 and 6 h.Results[125I]KX-02-019 showed subtle differences in pharmacological properties in the absence of PARP-2. In addition, [125I]KX-02-019 was competitively displaced by clinical PARPi. In vivo biodistribution studies showed an increasing tumor to muscle ratio over 6 h as well as fast clearance from healthy tissues.Conclusion[125I]KX-02-019 has binding sites in both PARP1 KO cells as well as PARP2 KO cells showing higher affinity for PARP-2. This observation is supported by a decrease in binding affinity in PARP2 KO cells compared to PARP1 KO cells. The pharmacologic and biological properties of [125I]KX-02-019 studied in vitro and in vivo showed that this analogue may be useful in determining pharmacokinetic and pharmacodynamic properties of clinical PARPi.

There is a need for improved methods to image genetically engineered cells, including immune cells used for cell-based therapy. Given the genetic manipulation inherent to gene therapy, the use of a reporter protein is a logical solution and positron emission tomography (PET) can provide the desired sensitivity and spatial localization. We developed a broadly applicable PET imaging strategy based on the small bacterial protein E. coli dihydrofolate reductase (Ec dhfr) and its highly specific small molecule inhibitor, trimethoprim (TMP). The difference in TMP affinity for bacterial compared to mammalian DHFR suggests that a TMP radioligand would have a low background in unmodified mammalian tissues and high retention in Ec dhfr engineered cells, providing high contrast imaging. Here, we describe the in vitro properties of [11C]TMP and show over 10-fold increased signal in transgenic Ec dhfr cells compared to control. In a mouse xenograft model, [11C]TMP rapidly accumulated in Ec dhfr carrying cells within minutes of intravenous administration. Moreover, [11C]TMP can identify less than a million xenografted cells in a small volume in tissues other than the abdominal compartment. This limit of detection is a clinically relevant number and bodes well for clinical translation especially given that [11C]TMP is an isotopologue of clinically approved antibiotic.Download high-res image (88KB)Download full-size image

Reactive oxygen species (ROS) have been implicated in the pathogenesis of a wide range of human disease states and drug toxicities, but development of imaging tools to study ROS biology in vivo remains a challenge. Here we synthesized and validated a novel PET tracer (12) and its 18F radiolabeled version [18F]12 to allow PET (positron emission tomography) imaging of superoxide in vivo. Initial analysis of ROS reaction kinetics found that compound 12 was rapidly and selectively oxidized by superoxide, but not other ROS. Cell culture studies in EMT6 cells exposed to the cancer chemotherapeutic agent Doxorubicin (DOX), which activates the superoxide-generating enzyme, NADPH oxidase, showed that compound 12 was a sensitive and specific probe for superoxide in cells. The microPET imaging of heart in mice with DOX-induced cardiac inflammation observed 2-fold greater oxidation of [18F]12 in the DOX-treated mice compared to controls (p = 0.02), the results were confirmed by distribution studies on organs subsequently removed from the mice and HPLC analysis of [18F] radioactivity compounds. These data indicate that compound 12 is a useful PET tracer to imaging ROS in vivo.