Evaluating immunotherapeutic outcomes in triple-negative breast cancer with a cholesterol radiotracer in mice.

Evaluating the response to immune checkpoint inhibitors (ICIs) remains an unmet challenge in triple-negative breast cancer (TNBC). The requirement for cholesterol in the activation and function of T cells led us to hypothesize that quantifying cellular accumulation of this molecule could distinguish successful from ineffective checkpoint immunotherapy. To analyze accumulation of cholesterol by T cells in the immune microenvironment of breast cancer, we leveraged the PET radiotracer, eFNP-59. eFNP-59 is an analog of cholesterol that our group validated as an imaging biomarker for cholesterol uptake in preclinical models and initial human studies. In immunocompetent mouse models of TNBC, we found that elevated uptake of exogenous labeled cholesterol analogs functions as a marker for T cell activation. When comparing ICI-responsive and -nonresponsive tumors directly, uptake of fluorescent cholesterol and eFNP-59 increased in T cells from ICI-responsive tumors. We discovered that accumulation of cholesterol by T cells increased in ICI-responding tumors that received anti-PD-1 checkpoint immunotherapy. In patients with TNBC, tumors containing cycling T cells had features of cholesterol uptake and trafficking within those populations. These results suggest that uptake of exogenous cholesterol analogs by tumor-infiltrating T cells allows detection of T cell activation and has potential to assess the success of ICI therapy.

Predicting efficacy of immunotherapy in mice with triple negative breast cancer using a cholesterol PET radiotracer.

Predicting the response to cancer immunotherapy remains an unmet challenge in triple-negative breast cancer (TNBC) and other malignancies. T cells, the major target of current checkpoint inhibitor immunotherapies, accumulate cholesterol during activation to support proliferation and signaling. The requirement of cholesterol for anti-tumor functions of T cells led us to hypothesize that quantifying cellular accumulation of this molecule could distinguish successful from ineffective checkpoint immunotherapy. To analyze accumulation of cholesterol by T cells in the immune microenvironment of breast cancer, we leveraged a novel positron emission tomography (PET) radiotracer, FNP-59. FNP-59 is an analog of cholesterol that our group has validated as an imaging biomarker for cholesterol uptake in pre-clinical models and initial human studies. In immunocompetent mouse models of TNBC, we found that elevated uptake of exogenous labeled cholesterol analogs functions as a marker for T cell activation. When comparing immune checkpoint inhibitor (ICI)-responsive EO771 tumors to non-responsive AT-3 tumors, we found significantly higher uptake of a fluorescent cholesterol analog in T cells of the ICI-responsive tumors both in vitro and in vivo. Using the FNP-59 radiotracer, we discovered that accumulation of cholesterol by T cells increased further in ICI-responding tumors that received ant-PD-1 checkpoint immunotherapy. We verified these data by mining single cell sequencing data from patients with TNBC. Patients with tumors containing cycling T cells showed gene expression signatures of cholesterol uptake and trafficking. These results suggest that uptake of exogenous cholesterol analogs by tumor-infiltrating T cells predict T cell activation and success of ICI therapy.

Synthesis and Evaluation of a Fluorine-18 Radioligand for Imaging Huntingtin Aggregates by Positron Emission Tomographic Imaging.

Mutations in the huntingtin gene (HTT) triggers aggregation of huntingtin protein (mHTT), which is the hallmark pathology of neurodegenerative Huntington's disease (HD). Development of a high affinity 18F radiotracer would enable the study of Huntington's disease pathology using a non-invasive imaging modality, positron emission tomography (PET) imaging. Herein, we report the first synthesis of fluorine-18 imaging agent, 6-(5-((5-(2,2-difluoro-2-(fluoro-18F)ethoxy)pyridin-2-yl)methoxy)benzo[d]oxazol-2-yl)-2-methylpyridazin-3(2H)-one ([18F]1), a radioligand for HD and its preclinical evaluation in vitro (autoradiography of post-mortem HD brains) and in vivo (rodent and non-human primate brain PET). [18F]1 was synthesized in a 4.1% RCY (decay corrected) and in an average molar activity of 16.5 ± 12.5 GBq/µmol (445 ± 339 Ci/mmol). [18F]1 penetrated the blood-brain barrier of both rodents and primates, and specific saturable binding in post-mortem brain slices was observed that correlated to mHTT aggregates identified by immunohistochemistry.

Identification of AV-1451 as a Weak, Nonselective Inhibitor of Monoamine Oxidase.

[18F]AV-1451 is one of the most widely used radiotracers for positron emission tomography (PET) imaging of tau protein aggregates in neurodegenerative disorders. While the radiotracer binds with high affinity to tau neurofibrillary tangles, extensive clinical studies have simultaneously revealed off-target tracer accumulation in areas of low tau burden such as the basal ganglia and choroid plexus. Though there are a number of possible reasons for this accumulation, it is often attributed to off-target binding to monoamine oxidase (MAO). In this paper, we investigate the association between [18F]AV-1451 and MAO through (i) enzyme inhibition assays, (ii) autoradiography with postmortem tissue samples, and (iii) nonhuman primate PET imaging. We confirm that [18F]AV-1451 is a weak inhibitor of MAO-A and -B and that MAO inhibitors can alter binding of [18F]AV-1451 in autoradiography and in vivo PET imaging.

Evaluating Cholinergic Receptor Expression in Guinea Pig Primary Auditory and Rostral Belt Cortices After Noise Damage Using [3H]Scopolamine and [18F]Flubatine Autoradiography.

Noise-induced hearing loss leads to anatomic and physiologic changes in primary auditory cortex (A1) and the adjacent dorsal rostral belt (RB). Since acetylcholine is known to modulate plasticity in other cortical areas, changes in A1 and RB following noise damage may be due to changes in cholinergic receptor expression. We used [3H]scopolamine and [18F]flubatine binding to measure muscarinic acetylcholine receptor (mAChR) and nicotinic acetylcholine receptor (nAChR) expression, respectively, in guinea pig A1 and RB 3 weeks following unilateral, left ear noise exposure, and a temporary threshold shift in hearing. [3H]Scopolamine binding decreased in right A1 and RB (contralateral to noise) compared to sham controls across all cortical layers. [18F]Flubatine binding showed a nonsignificant upward trend in right A1 following noise but only significantly increased in right RB and 2 layers of left RB (ipsilateral to noise). This selective response may ultimately influence cortical plasticity and function. The mechanism(s) by which cholinergic receptors are altered following noise exposure remain unknown. However, these data demonstrate noise exposure may differentially influence mAChRs that typically populate interneurons in A1 and RB more than nAChRs that are traditionally located on thalamocortical projections and provide motivation for cholinergic imaging in clinical patient populations of temporary or permanent hearing loss.

Futureproofing [18F]Fludeoxyglucose manufacture at an Academic Medical Center.

BACKGROUND: We recently upgraded our [18F]fludeoxyglucose (FDG) production capabilities with the goal of futureproofing our FDG clinical supply, expanding the number of batches of FDG we can manufacture each day, and improving patient throughput in our nuclear medicine clinic. In this paper we report upgrade of the synthesis modules to the GE FASTLab 2 platform (Phase 1) and cyclotron updates (Phase 2) from both practical and regulatory perspectives. We summarize our experience manufacturing FDG on the FASTLab 2 module with a high-yielding self-shielded niobium (Nb) fluorine-18 target. RESULTS: Following installation of Nb targets for production of fluorine-18, a 55 µA beam for 22 min generated 1330 ± 153 mCi of [18F]fluoride. Using these cyclotron beam parameters in combination with the FASTLab 2, activity yields (AY) of FDG were 957 ± 102 mCi at EOS, corresponding to 72% non-corrected AY (n = 235). Our workflow, inventory management and regulatory compliance have been greatly simplified following the synthesis module and cyclotron upgrades, and patient wait times for FDG PET have been cut in half at our nuclear medicine clinic. CONCLUSIONS: The combination of FASTlab 2 and self-shielded Nb fluorine-18 targets have improved our yield of FDG, and enabled reliable and repeatable manufacture of the radiotracer for clinical use.

Automated synthesis of [68Ga]oxine, improved preparation of 68Ga-labeled erythrocytes for blood-pool imaging, and preclinical evaluation in rodents.

Radiolabeled erythrocytes have multiple applications in nuclear medicine, including blood pool imaging. Historically they have been labeled with SPECT radionuclides. A PET blood pool imaging agent is highly desirable as it would improve clinical applications with better image quality and resolution, higher sensitivity, and dynamic scanning capabilities. With the coming of age of modern 68Ge/68Ga generator systems, gallium-68 is now widely accessible. In this paper we describe an updated method for the preparation of 68Ga-labeled erythrocytes and their preliminary use in rodent blood pool imaging. A novel automated synthesis of [68Ga]oxine using a 68Ga/68Ge generator and automated synthesis module is reported. [68Ga]Oxine was synthesized in 50 ± 5% (n = 3) non-decay corrected radiochemical yield and >99% radiochemical purity. Rat and human erythrocytes were successfully labeled with the complex in 42% RCY, and the 68Ga-labeled erythrocytes have been shown to clearly image the blood pool in a healthy rat. Human erythrocytes labelled with [68Ga]oxine were shown to be viable up to 2 hours post-labelling, and washout of the radiolabel was minimal up to 1 hour post-labelling. Further optimization of the labeling method to translate for use in human cardiac and oncologic blood pool PET imaging studies, is underway.

Synthesis and pre-clinical evaluation of a potential radiotracer for PET imaging of the dopamine D3 receptor.

There is considerable interest in using positron emission tomography (PET) imaging to understand the function of dopamine D3 receptors. Due to high sequence homology with D2 receptors, development of D3-selective PET radiotracers has been challenging. In an effort to overcome this issue, we report the radiosynthesis of a new selective D3 ligand with carbon-11 ([11C]1 ), and its initial preclincial evaluation as a potential PET radiotracer for in vivo imaging of D3 receptors. [11C]1 was prepared via [11C]CO2 fixation in 0.1% non-corrected radiochemical yield, good radiochemical purity (>95%) and high specific activity (>2000 Ci mmol-1). [11C]1 exhibited specific binding to D3 receptors using ex vivo autoradiography experiments with rat brain, but only 14-fold selectivity over D2 receptors which is lower than the 1400-fold value reported previously for cell studies. Rodent PET imaging revealed reasonable uptake of the radiotracer in areas of the brain known to be rich in D3 receptors.

Investigation of Proposed Activity of Clarithromycin at GABAA Receptors Using [(11)C]Flumazenil PET.

Clarithromycin is a potential treatment for hypersomnia acting through proposed negative allosteric modulation of GABAA receptors. We were interested whether this therapeutic benefit might extend to Parkinson's disease (PD) patients because GABAergic neurotransmission is implicated in postural control. Prior to initiating clinical studies in PD patients, we wished to better understand clarithromycin's mechanism of action. In this work we investigated whether the proposed activity of clarithromycin at the GABAA receptor is associated with the benzodiazepine binding site using in vivo [(11)C]flumazenil positron emission tomography (PET) in primates and ex vivo [(3)H]flumazenil autoradiography in rat brain. While the studies demonstrate that clarithromycin does not change the K d of FMZ, nor does it competitively displace FMZ, there is preliminary evidence from the primate PET imaging studies that clarithromycin delays dissociation and washout of flumazenil from the primate brain in a dose-dependent fashion. These findings would be consistent with the proposed GABAA allosteric modulator function of clarithromycin. While the results are only preliminary, further investigation of the interaction of clarithromycin with GABA receptors and/or GABAergic medications is warranted, and therapeutic applications of clarithromycin alone or in combination with flumazenil, to treat hyper-GABAergic status in PD at minimally effective doses, should also be pursued.

Synthesis and Evaluation of [(18)F]RAGER: A First Generation Small-Molecule PET Radioligand Targeting the Receptor for Advanced Glycation Endproducts.

The receptor for advanced glycation endproducts (RAGE) is a 35 kDa transmembrane receptor that belongs to the immunoglobulin superfamily of cell surface molecules. Its role in Alzheimer's disease (AD) is complex, but it is thought to mediate influx of circulating amyloid-ß into the brain as well as amplify Aß-induced pathogenic responses. RAGE is therefore of considerable interest as both a diagnostic and a therapeutic target in AD. Herein we report the synthesis and preliminary preclinical evaluation of [(18)F]RAGER, the first small molecule PET radiotracer for RAGE (Kd = 15 nM). Docking studies proposed a likely binding interaction between RAGE and RAGER, [(18)F]RAGER autoradiography showed colocalization with RAGE identified by immunohistochemistry in AD brain samples, and [(18)F]RAGER microPET confirmed CNS penetration and increased uptake in areas of the brain known to express RAGE. This first generation radiotracer represents initial proof-of-concept and a promising first step toward quantifying CNS RAGE activity using PET. However, there were high levels of nonspecific [(18)F]RAGER binding in vitro, likely due to its high log P (experimental log P = 3.5), and rapid metabolism of [(18)F]RAGER in rat liver microsome studies. Therefore, development of second generation ligands with improved imaging properties would be advantageous prior to anticipated translation into clinical PET imaging studies.

Targeting Metal-Aß Aggregates with Bifunctional Radioligand [11C]L2-b and a Fluorine-18 Analogue [18F]FL2-b.

Interest in quantifying metal-Aß species in vivo led to the synthesis and evaluation of [11C]L2-b and [18F]FL2-b as radiopharmaceuticals for studying the metallobiology of Alzheimer's disease (AD) using positron emission tomography (PET) imaging. [11C]L2-b was synthesized in 3.6% radiochemical yield (nondecay corrected, n = 3), >95% radiochemical purity, from the corresponding desmethyl precursor. [18F]FL2-b was synthesized in 1.0% radiochemical yield (nondecay corrected, n = 3), >99% radiochemical purity, from a 6-chloro pyridine precursor. Autoradiography experiments with AD positive and healthy control brain samples were used to determine the specificity of binding for the radioligands compared to [11C]PiB, a known imaging agent for ß-amyloid (Aß) aggregates. The Kd for [11C]L2-b and [18F]FL2-b were found to be 3.5 and 9.4 nM, respectively, from those tissue studies. Displacement studies of [11C]L2-b and [18F]FL2-b with PiB and AV-45 determined that L2-b binds to Aß aggregates differently from known radiopharmaceuticals. Finally, brain uptake of [11C]L2-b was examined through microPET imaging in healthy rhesus macaque, which revealed a maximum uptake at 2.5 min (peak SUV = 2.0) followed by rapid egress (n = 2).

High affinity radiopharmaceuticals based upon lansoprazole for PET imaging of aggregated tau in Alzheimer's disease and progressive supranuclear palsy: synthesis, preclinical evaluation, and lead selection.

Abnormally aggregated tau is the hallmark pathology of tauopathy neurodegenerative disorders and is a target for development of both diagnostic tools and therapeutic strategies across the tauopathy disease spectrum. Development of carbon-11- or fluorine-18-labeled radiotracers with appropriate affinity and specificity for tau would allow noninvasive quantification of tau burden using positron emission tomography (PET) imaging. We have synthesized [(18)F]lansoprazole, [(11)C]N-methyl lansoprazole, and [(18)F]N-methyl lansoprazole and identified them as high affinity radiotracers for tau with low to subnanomolar binding affinities. Herein, we report radiosyntheses and extensive preclinical evaluation with the aim of selecting a lead radiotracer for translation into human PET imaging trials. We demonstrate that [(18)F]N-methyl lansoprazole, on account of the favorable half-life of fluorine-18 and its rapid brain entry in nonhuman primates, favorable kinetics, low white matter binding, and selectivity for binding to tau over amyloid, is the lead compound for progression into clinical trials.

Synthesis and evaluation of [(11)C]PyrATP-1, a novel radiotracer for PET imaging of glycogen synthase kinase-3ß (GSK-3ß).

INTRODUCTION: The dysfunction of glycogen synthase kinase-3ß (GSK-3ß) has been implicated in a number of diseases, including Alzheimer's disease. The ability to non-invasively quantify GSK-3ß activity in vivo is therefore of critical importance, and this work is focused upon development of inhibitors of GSK-3ß radiolabeled with carbon-11 to examine quantification of the enzyme using positron emission tomography (PET) imaging. METHODS: (11)C PyrATP-1 was prepared from the corresponding desmethyl-piperazine precursor in an automated synthesis module. In vivo rodent and primate imaging studies were conducted on a Concorde MicroPET P4 scanner to evaluate imaging properties and in vitro autoradiography studies with rat brain samples were carried out to examine specific binding. RESULTS: 2035±518MBq (55±14mCi) of [(11)C]PyrATP-1 was obtained (1%-2% non-corrected radiochemical yield at end-of-synthesis based upon [(11)C]CO2) with high chemical (>95%) and radiochemical (>99%) purities, and good specific activities (143±52GBq/µmol (3874±1424Ci/mmol)), n=5. In vivo microPET imaging studies revealed poor brain uptake in rodents and non-human primates. Pretreatment of rodents with cyclosporin A resulted in moderately increased brain uptake suggesting Pgp transporter involvement. Autoradiography demonstrated high levels of specific binding in areas of the rodent brain known to be rich in GSK-3ß. CONCLUSION: (11)C PyrATP-1 is readily synthesized using standard carbon-11 radiochemistry. However the poor brain uptake in rodents and non-human primates indicates that the radiotracer is not suitable for the purposes of quantifying GSK-3ß in neurological and psychiatric disorders.

Imaging of carrageenan-induced local inflammation and adjuvant-induced systemic arthritis with [(11)C]PBR28 PET.

INTRODUCTION: [(11)C] PBR28 binding to translocator protein (TSPO) was evaluated for imaging of acute and chronic inflammation using two established rat models. METHODS: Acute inflammation was induced by local carrageenan injection into the paw of Fisher 344 rats (model A). T-cell mediated adjuvant arthritis was induced by heat-inactivated Mycobacterium butyricum injection in Lewis rats (model B). Micro-PET scan was performed after injection of approximately 35 MBq [(11)C]PBR28. In model A, volumes of interest (VOIs) were defined in the paw of Fisher 344 rats (n=6) with contralateral sham treatment as control. For model B, VOIs were defined in the tail, sacroiliac joints, hips, knees and thigh muscles of M. butyricum treated animals (n=8) and compared with sham-treated controls (n=4). The peak (11)C-PBR28 SUV (SUVpeak) and area under the curve (AUCSUV) of 60-minute time-activity data were calculated. Immunohistochemistry for CD68, a macrophage stain, was performed from paw tissues. In addition, the [(11)C]PBR28 cell uptake was measured in lipopolysaccharide (LPS)-stimulated and non-stimulated macrophage cultures. RESULTS: LPS-stimulated macrophages displayed dose-dependent increased [(11)C]PBR28 uptake, which was blocked by non-labeled PBR28. In both models, radiotracer uptake of treated lesions increased rapidly within minutes and displayed overall accumulative kinetics. The SUVpeak and AUCSUV of carrageenan-treated paws was significantly increased compared to controls. Also, the [(11)C]PBR28 uptake ratio of carrageenan-treated vs. sham-treated paw correlated significantly with CD68 staining ratios of the same animals. In adjuvant arthritis, significantly increased [(11)C]PBR28 SUVpeak and AUCSUV values were identified at the tail, knees, and sacroiliac joints, while no significant differences were identified in the lumbar spine and hips. CONCLUSIONS: Based on our initial data, [(11)C]PBR28 PET appears to have potential for imaging of various inflammatory processes involving macrophage activation.

Evaluation of [(11)C]N-Methyl Lansoprazole as a Radiopharmaceutical for PET Imaging of Tau Neurofibrillary Tangles.

[(11)C]N-Methyl lansoprazole ([(11)C]NML, 3) was synthesized and evaluated as a radiopharmaceutical for quantifying tau neurofibrillary tangle (NFT) burden using positron emission tomography (PET) imaging. [(11)C]NML was synthesized from commercially available lansoprazole in 4.6% radiochemical yield (noncorrected RCY, based upon [(11)C]MeI), 99% radiochemical purity, and 16095 Ci/mmol specific activity (n = 5). Log P was determined to be 2.18. A lack of brain uptake in rodent microPET imaging revealed [(11)C]NML to be a substrate for the rodent permeability-glycoprotein 1 (PGP) transporter, but this could be overcome by pretreating with cyclosporin A to block the PGP. Contrastingly, [(11)C]NML was not found to be a substrate for the primate PGP, and microPET imaging in rhesus revealed [(11)C]NML uptake in the healthy primate brain of ∼1600 nCi/cc maximum at 3 min followed by rapid egress to 500 nCi/cc. Comparative autoradiography between wild-type rats and transgenic rats expressing human tau (hTau +/+) revealed 12% higher uptake of [(11)C]NML in the cortex of brains expressing human tau. Further autoradiography with tau positive brain samples from progressive supranuclear palsy (PSP) patients revealed colocalization of [(11)C]NML with tau NFTs identified using modified Bielschowsky staining. Finally, saturation binding experiments with heparin-induced tau confirmed K d and Bmax values of [(11)C]NML as 700 pM and 0.214 fmol/µg, respectively.

Investigation on tumor hypoxia in resectable primary prostate cancer as demonstrated by 18F-FAZA PET/CT utilizing multimodality fusion techniques.

PURPOSE: As hypoxia is believed to play an important role in the development and progression of prostate cancer, we evaluated whether 18F-labeled fluoroazomycin arabinoside (18F-FAZA) would be useful to identify tumor hypoxia in resectable prostate cancer. METHODS: Positron emission tomography (PET)/CT was performed on 14 patients with untreated localized primary prostate cancer 3 h post-injection of approximately 390 MBq of 18F-FAZA using forced diuresis to decrease radioactivity in the urinary bladder. Anatomical trans-pelvic coil and pre- and post-contrast 1.5 T MRI with endorectal coil were performed on the same day. Patients underwent radical prostatectomy and ex vivo 3 T MRI of the prostatectomy specimen within 14 days following in vivo imaging. Imaging results were verified by whole mount histopathology plus tissue microarray (TMA) immunohistochemical (IHC) analysis for carbonic anhydrase IX (CAIX) and hypoxia-inducible factor 1α (HIF-1α). Registration of in vivo imaging with histology was achieved using mutual information software and performing ex vivo MRI of the prostatectomy specimen and whole mount sectioning with block face photography as intermediate steps. RESULTS: Whole mount histology identified 43 tumor nodules, 19 of them larger than 1 ml as determined on coregistered volumes featuring 18F-FAZA, MRI, and histological 3-D image information. None of these lesions was found to be positive for CAIX or visualized by 18F-FAZA PET/CT while IHC for HIF-1α showed variable staining of tumor tissues. Accordingly, no correlation was found between 18F-FAZA uptake and Gleason scores. CONCLUSION: Our data based on 18F-FAZA PET/CT and CAIX IHC do not support the presence of clinically relevant hypoxia in localized primary prostate cancer including high-grade disease. Activation of HIF-1α may be independent of tissue hypoxia in primary prostate cancer.

In vivo [11C]dihydrotetrabenazine binding in rat striatum: sensitivity to dopamine concentrations.

INTRODUCTION: The sensitivity of the in vivo binding of [(11)C]dihydrotetrabenazine ([(11)C]DTBZ) and [(11)C]methylphenidate ([(11)C]MPH) to their respective targets - vesicular monoamine transporter type 2 (VMAT2) and neuronal membrane dopamine transporter - after alterations in endogenous levels of dopamine was examined in the rat brain. METHODS: In vivo binding of [(11)C]DTBZ and [(11)C]MPH was determined using a bolus+infusion protocol. The in vitro number of VMAT2 binding sites was determined by autoradiography. RESULTS: Repeated dosing with alpha-methyl-p-tyrosine (AMPT) at doses that significantly (-75%) depleted brain tissue dopamine levels resulted in increased (+36%) in vivo [(11)C]DTBZ binding to VMAT2 in the striatum. The increase in binding could be completely reversed via treatment with L-DOPA/benserazide to restore dopamine levels. There were no changes in the total number of VMAT2 binding sites, as measured using in vitro autoradiography. No changes were observed for in vivo [(11)C]MPH binding to the dopamine transporter in the striatum following AMPT pretreatment. CONCLUSION: These results indicate that large reductions in dopamine concentrations in the rat brain can produce modest but significant changes in the binding of radioligands to VMAT2, which can be reversed by replenishment of dopamine using exogenous L-DOPA.

5-tert-Butyl-2-(4'-[18F]fluoropropynylphenyl)-1,3-dithiane oxides: potential new GABA A receptor radioligands.

As potential new ligands targeting the binding site of gamma-aminobutyric acid (GABA) receptor ionophore, trans-5-tert-butyl-2-(4'-fluoropropynylphenyl)-2-methyl-1,1-dioxo-1,3-dithiane (1) and cis/trans-5-tert-butyl-2-(4'-fluoropropynylphenyl)-2-methyl-1,1,3,3-tetroxo-1,3-dithiane (2) were selected for radiolabeling and initial evaluation as in vivo imaging agents for positron emission tomography (PET). Both compounds exhibited identical high in vitro binding affinities (K(i)=6.5 nM). Appropriate tosylate-substituted ethynyl precursors were prepared by multistep syntheses involving stepwise sulfur oxidation and chromatographic isolation of desired trans isomers. Radiolabeling was accomplished in one step using nucleophilic [(18)F]fluorination. In vivo biodistribution studies with trans-[(18)F]1 and trans-[(18)F]2 showed significant initial uptake into mouse brain and gradual washout, with heterogeneous regional brain distributions and higher retention in the cerebral cortex and cerebellum and lower retention in the striatum and pons-medulla. These regional distributions of the new radioligands correlated with in vitro and ex vivo measures of standard radioligands binding to the ionophore- and benzodiazepine-binding sites of GABA(A) receptor in rodent brain. A comparison of these results with previously prepared radiotracers for other neurochemical targets, including successes and failures as in vivo radioligands, suggests that higher-affinity compounds with increased retention in target brain tissues will likely be needed before a successful radiopharmaceutical for human PET imaging can be identified.

Hippocampal inactivation disrupts the acquisition and contextual encoding of fear extinction.

In recent studies, inactivation of the dorsal hippocampus before the retrieval of extinguished fear memories disrupted the context-dependent expression of these memories. In the present experiments, we examined the role of the dorsal hippocampus in the acquisition of extinction. After pairing an auditory conditional stimulus (CS) with an aversive footshock [unconditional stimulus (US)], rats received an extinction session in which the CS was presented without the US. In experiment 1, infusion of muscimol, a GABAA receptor agonist, into the dorsal hippocampus before the extinction training session decreased the rate of extinction. Moreover, when later tested for fear to the extinguished CS, all rats that had received hippocampal inactivation before extinction training demonstrated renewed fear regardless of the context in which testing took place. This suggests a role for the dorsal hippocampus in both acquiring the extinction memory and encoding the CS-context relationship that yields the context dependence of extinction. In experiment 2, inactivation of the dorsal hippocampus before testing also disrupted the context dependence of fear to the extinguished CS. In experiment 3, quantitative autoradiography revealed the boundaries of muscimol diffusion after infusion into the dorsal hippocampus. Together, these results reveal that the dorsal hippocampus is involved in the acquisition, contextual encoding, and context-dependent retrieval of fear extinction. Learning and remembering when and where aversive events occur is essential for adaptive emotional regulation.

Intratumoral injection of BCNU in ethanol (DTI-015) results in enhanced delivery to tumor--a pharmacokinetic study.

Solvent facilitated perfusion (SFP) has been proposed as a technique to increase the delivery of chemotherapeutic agents to tumors. SFP entails direct injection of the agent into the tumor in a water-miscible organic solvent, and because the solvent moves easily through both aqueous solutions and cellular membranes it drives the penetration of the solubilized anticancer agent throughout the tumor. To test this hypothesis, we compared the pharmacokinetics (PK) of 14C-labeled 1,3-bis-chlorethyl-1-nitrosourea (BCNU) in intra-cerebral 9L rat gliomas after intravenous (IV) infusion in 90% saline--10% ethanol or direct intratumoral (IT) injection of 14C-BCNU in 100% ethanol (DTI-015). Treatment with DTI-015 yielded a peak radioactive count (Cmax) for the 14C label that was 100-1000 fold higher in the tumor than in all other tissues in addition to a concentration in the tumor that was 100-fold higher than that achieved following IV infusion of 14C-BCNU. Pathologic and auto-radiographic analysis of tissue sections following IT injection of 14C-BCNU in ethanol into either tumor or normal rat brain revealed both an enhanced local volume of distribution and an increased concentration of BCNU delivered to tumor compared to non-tumor bearing brain. To investigate the mechanism behind the SFP of BCNU to the tumor both dynamic contrast and perfusion MRI were performed on 9L tumors before and after treatment and demonstrated a decrease in tumor perfusion following IT injection of DTI-015. Finally, initial PK of patient blood samples following administration of DTI-015 into relapsed high-grade glioma indicated a 20-fold decrease in systemic exposure to BCNU compared to IV infusion of BCNU providing further evidence for the enhanced therapeutic ratio observed for DTI-015.