Detailed radiosynthesis of [18 F]mG4P027 as a positron emission tomography radiotracer for mGluR4.

We report here the detailed radiosynthesis of [18 F]mG4P027, a metabotropic glutamate receptor 4 (mGluR4) PET radiotracer, which showed superior properties to the currently reported mGluR4 radiotracers. The radiosynthesis in the automated system has been challenging, therefore we disclose here the major limiting factors for the synthesis via step-by-step examination. And we hope this thorough study will help its automation for human use in the future.

Organomediated cleavage of benzoyl group enables an efficient synthesis of 1-(6-nitropyridin-2-yl)thiourea and its application for developing 18F-labeled PET tracers.

A novel organomediated cleavage of benzoyl group using ethane-1,2-diamine and acetic acid under neutral condition enables an efficient synthesis of 1-(6-nitropyridin-2-yl)thiourea, which previously has been challenging to prepare by conventional methods. The successful synthesis of 1-(6-nitropyridin-2-yl)thiourea as a synthon permits development of a variety of 18F labeled heterocycles as PET imaging ligands such as N-(pyridin-2-yl)thiazol-2-amine derivatives. The utility of this synthon is demonstrated with the synthesis of a 18F-labeled PET tracer for studying prion disease. In vitro autoradiography using this PET tracer on sagittal rat brain slices showed highest accumulation of radioactivity in the hippocampus, cortex, and striatum, in accordance with reported immunostaining of PrPc in rat brain.

Improved Synthesis of the Thiophenol Precursor N-(4-Chloro-3-mercaptophenyl)picolinamide for Making the mGluR4 PET Ligand.

Recently [11C]mG4P012 (previously [11C]KALB012 and presently named as [11C]PXT012253 by Prexton Therapeutics) had been used as a biomarker during the preclinical development of a potential therapeutic drug, PXT0002331 (an mGluR4 PAM), for PD and L-dopa-induced dyskinesia. [11C]mG4P012 was shown to be a promising PET radioligand for mGluR4 in the monkey brain and for further development in human subjects. However, the previously reported multi-step synthesis of the thiophenol precursor suffered from low yields and difficult workup procedures. To support the translational research of [11C]mG4P012 and the other potential applications, we have developed a new route for synthesis of the thiophenol precursor and optimized the reaction conditions. The synthesis of N-(4-chloro-3-mercaptophenyl)picolinamide from 1-chloro-4-nitrobenzene has been greatly improved from 8% to 52% total yield with easy handling and in gram scales.

Tau positron emission tomographic imaging in aging and early Alzheimer disease.

OBJECTIVE: Detection of focal brain tau deposition during life could greatly facilitate accurate diagnosis of Alzheimer disease (AD), staging and monitoring of disease progression, and development of disease-modifying therapies. METHODS: We acquired tau positron emission tomography (PET) using (18)F T807 (AV1451), and amyloid-ß PET using (11)C Pittsburgh compound B (PiB) in older clinically normal individuals, and symptomatic patients with mild cognitive impairment or mild AD dementia. RESULTS: We found abnormally high cortical (18)F T807 binding in patients with mild cognitive impairment and AD dementia compared to clinically normal controls. Consistent with the neuropathology literature, the presence of elevated neocortical (18)F T807 binding particularly in the inferior temporal gyrus was associated with clinical impairment. The association of cognitive impairment was stronger with inferior temporal (18)F T807 than with mean cortical (11)C PIB. Regional (18)F T807 was correlated with mean cortical (11)C PiB among both impaired and control subjects. INTERPRETATION: These findings suggest that (18)F T807 PET could have value as a biomarker that reflects both the progression of AD tauopathy and the emergence of clinical impairment.

Single-scan rest/stress imaging: validation in a porcine model with 18F-Flurpiridaz.

PURPOSE: 18F-labeled myocardial flow agents are becoming available for clinical application but the ∼2 hour half-life of 18F complicates their clinical application for rest-stress measurements. The goal of this work is to evaluate in a pig model a single-scan method which provides quantitative rest-stress blood flow in less than 15 minutes. METHODS: Single-scan rest-stress measurements were made using 18F-Flurpiridaz. Nine scans were performed in healthy pigs and seven scans were performed in injured pigs. A two-injection, single-scan protocol was used in which an adenosine infusion was started 4 minutes after the first injection of 18F-Flurpiridaz and followed either 3 or 6 minutes later by a second radiotracer injection. In two pigs, microsphere flow measurements were made at rest and during stress. Dynamic images were reoriented into the short axis view, and regions of interest (ROIs) for the 17 myocardial segments were defined in bull's eye fashion. PET data were fitted with MGH2, a kinetic model with time varying kinetic parameters, in which blood flow changes abruptly with the introduction of adenosine. Rest and stress myocardial blood flow (MBF) were estimated simultaneously. RESULTS: The first 12-14 minutes of rest-stress PET data were fitted in detail by the MGH2 model, yielding MBF measurement with a mean precision of 0.035 ml/min/cc. Mean myocardial blood flow across pigs was 0.61 ± 0.11 mL/min/cc at rest and 1.06 ± 0.19 mL/min/cc at stress in healthy pigs and 0.36 ± 0.20 mL/min/cc at rest and 0.62 ± 0.24 mL/min/cc at stress in the ischemic area. Good agreement was obtained with microsphere flow measurement (slope = 1.061 ± 0.017, intercept = 0.051 ± 0.017, mean difference 0.096 ± 0.18 ml/min/cc). CONCLUSION: Accurate rest and stress blood flow estimation can be obtained in less than 15 min of PET acquisition. The method is practical and easy to implement suggesting the possibility of clinical translation.

A Food and Drug Administration-approved asthma therapeutic agent impacts amyloid ß in the brain in a transgenic model of Alzheimer disease.

Interfering with the assembly of Amyloid ß (Aß) peptides from monomer to oligomeric species and fibrils or promoting their clearance from the brain are targets of anti-Aß-directed therapies in Alzheimer disease. Here we demonstrate that cromolyn sodium (disodium cromoglycate), a Food and Drug Administration-approved drug already in use for the treatment of asthma, efficiently inhibits the aggregation of Aß monomers into higher-order oligomers and fibrils in vitro without affecting Aß production. In vivo, the levels of soluble Aß are decreased by over 50% after only 1 week of daily intraperitoneally administered cromolyn sodium. Additional in vivo microdialysis studies also show that this compound decreases the half-life of soluble Aß in the brain. These data suggest a clear effect of a peripherally administered, Food and Drug Administration-approved medication on Aß economy, supporting further investigation of the potential long-term efficacy of cromolyn sodium in Alzheimer disease.

First human use of a radiopharmaceutical prepared by continuous-flow microfluidic radiofluorination: proof of concept with the tau imaging agent [18F]T807.

Despite extensive preclinical imaging with radiotracers developed by continuous-flow microfluidics, a positron emission tomographic (PET) radiopharmaceutical has not been reported for human imaging studies by this technology. The goal of this study was to validate the synthesis of the tau radiopharmaceutical 7-(6-fluoropyridin-3-yl)-5H-pyrido[4,3-b]indole ([18F]T807) and perform first-in-human PET scanning enabled by microfluidic flow chemistry. [18F]T807 was synthesized by our modified one-step method and adapted to suit a commercial microfluidic flow chemistry module. For this proof of concept, the flow system was integrated to a GE Tracerlab FXFN unit for high-performance liquid chromatography purification and formulation. Three consecutive productions of [18F]T807 were conducted to validate this radiopharmaceutical. Uncorrected radiochemical yields of 17 ± 1% of crude [18F]T807 (≈ 500 mCi, radiochemical purity 95%) were obtained from the microfluidic device. The crude material was then purified, and > 100 mCi of the final product was obtained in an overall uncorrected radiochemical yield of 5 ± 1% (n  =  3), relative to starting [18F]fluoride (end of bombardment), with high radiochemical purity (≥ 99%) and high specific activities (6 Ci/µmol) in 100 minutes. A clinical research study was carried out with [18F]T807, representing the first reported human imaging study with a radiopharmaceutical prepared by this technology.

PET imaging of fatty acid amide hydrolase with [(18)F]DOPP in nonhuman primates.

Fatty acid amide hydrolase (FAAH) regulates endocannabinoid signaling. [(11)C]CURB, an irreversibly binding FAAH inhibitor, has been developed for clinical research imaging with PET. However, no fluorine-18 labeled radiotracer for FAAH has yet advanced to human studies. [(18)F]DOPP ([(18)F]3-(4,5-dihydrooxazol-2-yl)phenyl (5-fluoropentyl)carbamate) has been identified as a promising (18)F-labeled analogue based on rodent studies. The goal of this work is to evaluate [(18)F]DOPP in nonhuman primates to support its clinical translation. High specific activity [(18)F]DOPP (5-6 Ci·µmol(-1)) was administered intravenously (iv) to three baboons (2M/1F, 3-4 years old). The distribution and pharmacokinetics were quantified following a 2 h dynamic imaging session using a simultaneous PET/MR scanner. Pretreatment with the FAAH-selective inhibitor, URB597, was carried out at 200 or 300 µg/kg iv, 10 min prior to [(18)F]DOPP administration. Rapid arterial blood sampling for the first 3 min was followed by interval sampling with metabolite analysis to provide a parent radiotracer plasma input function that indicated ∼95% baseline metabolism at 60 min and a reduced rate of metabolism after pretreatment with URB597. Regional distribution data were analyzed with 1-, 2-, and 3-tissue compartment models (TCMs), with and without irreversible trapping since [(18)F]DOPP covalently links to the active site of FAAH. Consistent with previous findings for [(11)C]CURB, the 2TCM with irreversible binding was found to provide the best fit for modeling the data in all regions. The composite parameter λk3 was therefore used to evaluate whole brain (WB) and regional binding of [(18)F]DOPP. Pretreatment studies showed inhibition of λk3 across all brain regions (WB baseline: 0.112 mL/cm(3)/min; 300 µg/kg URB597: 0.058 mL/cm(3)/min), suggesting that [(18)F]DOPP binding is specific for FAAH, consistent with previous rodent data.

Membrane potential-dependent uptake of 18F-triphenylphosphonium--a new voltage sensor as an imaging agent for detecting burn-induced apoptosis.

BACKGROUND: Mitochondrial dysfunction has been closely related to many pathologic processes, such as cellular apoptosis. Alterations in organelle membrane potential are associated with mitochondrial dysfunction. A fluorine-18 labeled phosphonium compound: (18)F-triphenylphosphonium ((18)F-TPP) was prepared to determine its potential use as a mitochondria-targeting radiopharmaceutical to evaluate cellular apoptosis. METHODS: Studies were conducted in both ex vivo cell lines and in vivo using a burned animal model. Uptake of (18)F-TPP was assessed in PC-3 cells by gamma counting under the following conditions: graded levels of extracellular potassium concentrations, incubation with carbonyl cyanide m-chlorophenylhydrazone and staurosporine. Apoptosis was studied in a burn animal model using terminal deoxynucleotidyl transferase dUTP nick end labeling staining and simultaneous assessment of (18)F-TPP uptake by biodistribution. RESULTS: We found that stepwise membrane depolarization by potassium (K) resulted in a linear decrease in (18)F-TPP uptake, with a slope of 0.62 ± 0.08 and a correlation coefficient of 0.936 ± 0.11. Gradually increased concentrations of m-chlorophenylhydrazone lead to decreased uptake of (18)F-TPP. Staurosporine significantly decreased the uptake of (18)F-TPP in PC-3 cells from 14.2 ± 3.8% to 5.6 ± 1.3% (P < 0.001). Burn-induced significant apoptosis (sham: 4.4 ± 1.8% versus burn: 24.6 ± 6.7 %; P < 0.005) and a reduced uptake of tracer in the spleens of burn-injured animals as compared with sham burn controls (burn: 1.13 ± 0.24% versus sham: 3.28 ± 0.67%; P < 0.005). Biodistribution studies demonstrated that burn-induced significant reduction in (18)F-TPP uptake in spleen, heart, lung, and liver, which were associated with significantly increased apoptosis. CONCLUSIONS: (18)F-TPP is a promising new voltage sensor for detecting mitochondrial dysfunction and apoptosis in various tissues.

A concise radiosynthesis of the tau radiopharmaceutical, [(18) F]T807.

Fluorine-18 labeled 7-(6-fluoropyridin-3-yl)-5H-pyrido[4,3-b]indole ([(18) F]T807) is a potent and selective agent for imaging paired helical filaments of tau and is among the most promising PET radiopharmaceuticals for this target in early clinical trials. The present study reports a simplified one-step method for the synthesis of [(18) F]T807 that is broadly applicable for routine clinical production using a GE TRACERlab™ FXFN radiosynthesis module. Key facets of our optimized radiosynthesis include development and use of a more soluble protected precursor, tert-butyl 7-(6-nitropyridin-3-yl)-5H-pyrido[4,3-b]indole-5-carboxylate, as well as new HPLC separation conditions that enable a facile one-step synthesis. During the nucleophilic fluorinating reaction with potassium cryptand [(18) F]fluoride (K[(18) F]/K222 ) in DMSO at 130 °C over 10 min the precursor is concurrently deprotected. Formulated [(18) F]T807 was prepared in an uncorrected radiochemical yield of 14 ± 3%, with a specific activity of 216 ± 60 GBq/µmol (5837 ± 1621 mCi/µmol) at the end of synthesis (60 min; n = 3) and validated for human use. This methodology offers the advantage of faster synthesis in fewer steps, with simpler automation that we anticipate will facilitate widespread clinical use of [(18) F]T807.

Mannose-binding lectin binds to amyloid ß protein and modulates inflammation.

Mannose-binding lectin (MBL), a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid ß peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aß are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

PET imaging of mitochondrial function in acute doxorubicin-induced cardiotoxicity: a proof-of-principle study.

Mitochondrial dysfunction plays a key role in doxorubicin-induced cardiotoxicity (DIC). In this proof-of-principle study, we investigated whether PET mapping of cardiac membrane potential, an indicator of mitochondrial function, could detect an acute cardiotoxic effect of doxorubicin (DOX) in a large animal model. Eight Yucatan pigs were imaged dynamically with [18F](4-Fluorophenyl)triphenylphosphonium ([18F]FTPP+) PET/CT. Our experimental protocol included a control saline infusion into the left anterior descending coronary artery (LAD) followed by a DOX test infusion of either 1 mg/kg or 2 mg/kg during PET. We measured the change in total cardiac membrane potential (ΔΨT), a proxy for the mitochondrial membrane potential, ΔΨm, after the saline and DOX infusions. We observed a partial depolarization of the mitochondria following the DOX infusions, which occurred only in myocardial areas distal to the intracoronary catheter, thereby demonstrating a direct association between the exposure of the mitochondria to DOX and a change in ΔΨT. Furthermore, doubling the DOX dose caused a more severe depolarization of myocardium in the LAD territory distal to the infusion catheter. In conclusion, [18F]FTPP+ PET-based ΔΨT mapping can measure partial depolarization of myocardial mitochondria following intracoronary DOX infusion in a large animal model.

Synthesis and Characterization of 5-(2-Fluoro-4-[11C]methoxyphenyl)-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridine-7-carboxamide as a PET Imaging Ligand for Metabotropic Glutamate Receptor 2.

Metabotropic glutamate receptor 2 (mGluR2) is a therapeutic target for several neuropsychiatric disorders. An mGluR2 function in etiology could be unveiled by positron emission tomography (PET). In this regard, 5-(2-fluoro-4-[11C]methoxyphenyl)-2,2-dimethyl-3,4-dihydro-2H-pyrano[2,3-b]pyridine-7-carboxamide ([11C]13, [11C]mG2N001), a potent negative allosteric modulator (NAM), was developed to support this endeavor. [11C]13 was synthesized via the O-[11C]methylation of phenol 24 with a high molar activity of 212 ± 76 GBq/µmol (n = 5) and excellent radiochemical purity (>99%). PET imaging of [11C]13 in rats demonstrated its superior brain heterogeneity and reduced accumulation with pretreatment of mGluR2 NAMs, VU6001966 (9) and MNI-137 (26), the extent of which revealed a time-dependent drug effect of the blocking agents. In a nonhuman primate, [11C]13 selectively accumulated in mGluR2-rich regions and resulted in high-contrast brain images. Therefore, [11C]13 is a potential candidate for translational PET imaging of the mGluR2 function.

Cromolyn platform suppresses fibrosis and inflammation, promotes microglial phagocytosis and neurite outgrowth.

Neurodegenerative diseases are characterized by chronic neuroinflammation and may perpetuate ongoing fibrotic reactions within the central nervous system. Unfortunately, there is no therapeutic available that treats neurodegenerative inflammation and its sequelae. Here we utilize cromolyn, a mast cell inhibitor with anti-inflammatory capabilities, and its fluorinated analogue F-cromolyn to study fibrosis-related protein regulation and secretion downstream of neuroinflammation and their ability to promote microglial phagocytosis and neurite outgrowth. In this report, RNA-seq analysis shows that administration of the pro-inflammatory cytokine TNF-α to HMC3 human microglia results in a robust upregulation of fibrosis-associated genes. Subsequent treatment with cromolyn and F-cromolyn resulted in reduced secretion of collagen XVIII, fibronectin, and tenascin-c. Additionally, we show that cromolyn and F-cromolyn reduce pro-inflammatory proteins PLP1, PELP1, HSP90, IL-2, GRO-α, Eotaxin, and VEGF-Α, while promoting secretion of anti-inflammatory IL-4 in HMC3 microglia. Furthermore, cromolyn and F-cromolyn augment neurite outgrowth in PC12 neuronal cells in concert with nerve growth factor. Treatment also differentially altered secretion of neurogenesis-related proteins TTL, PROX1, Rab35, and CSDE1 in HMC3 microglia. Finally, iPSC-derived human microglia more readily phagocytose Aß42 with cromolyn and F-cromolyn relative to controls. We propose the cromolyn platform targets multiple proteins upstream of PI3K/Akt/mTOR, NF-κB, and GSK-3ß signaling pathways to affect cytokine, chemokine, and fibrosis-related protein expression.

Synthesis and Characterization of [18F]JNJ-46356479 as the First 18F-Labeled PET Imaging Ligand for Metabotropic Glutamate Receptor 2.

PURPOSE: Metabotropic glutamate receptor 2 (mGluR2) has been implicated in various psychiatric and neurological disorders, such as schizophrenia and Alzheimer's disease. We have previously developed [11C]7 as a PET radioligand for imaging mGluR2. Herein, [18F]JNJ-46356479 ([18F]8) was synthesized and characterized as the first 18F-labeled mGluR2 imaging ligand to enhance diagnostic approaches for mGluR2-related disorders. PROCEDURES: JNJ-46356479 (8) was radiolabeled via the copper (I)-mediated radiofluorination of organoborane 9. In vivo PET imaging experiments with [18F]8 were conducted first in C57BL/6 J mice and Sprague-Dawley rats to obtain whole body biodistribution and brain uptake profile. Subsequent PET studies were done in a cynomolgus monkey (Macaca fascicularis) to investigate the uptake of [18F]8 in the brain, its metabolic stability, as well as pharmacokinetic properties. RESULTS: JNJ-46356479 (8) exhibited excellent selectivity against other mGluRs. In vivo PET imaging studies showed reversible and specific binding characteristic of [18F]8 in rodents. In the non-human primate, [18F]8 displayed good in vivo metabolic stability, excellent brain permeability, fast and reversible kinetics with moderate heterogeneity across brain regions. Pre-treatment studies with compound 7 revealed time-dependent decrease of [18F]8 accumulation in mGluR2 rich regions based on SUV values with the highest decrease in the nucleus accumbens (18.7 ± 5.9%) followed by the cerebellum (18.0 ± 7.9%), the parietal cortex (16.9 ± 7.8%), and the hippocampus (16.8 ± 6.9%), similar to results obtained in the rat studies. However, the volume of distribution (VT) results derived from 2T4k model showed enhanced VT from a blocking study with compound 7. This is probably because of the potentiating effect of compound 7 as an mGluR2 PAM as well as related non-specific binding in the tissue data. CONCLUSIONS: [18F]8 readily crosses the blood-brain barrier and demonstrates fast and reversible kinetics both in rodents and in a non-human primate. Further investigation of [18F]8 on its binding specificity would warrant translational study in human.

Evaluation of Fluorinated Cromolyn Derivatives as Potential Therapeutics for Alzheimer's Disease.

BACKGROUND: Cromolyn is an anti-neuroinflammatory modulator with a multifactorial mechanism of action that has been shown to inhibit amyloid-ß (Aß) aggregation and enhance microglial uptake and clearance of Aß. OBJECTIVE: We report the effects of fluoro-cromolyn derivatives on microglial cell toxicity and microglial clearance of Aß42. METHODS: Microglial cell toxicity for cromolyn derivatives were determined in naive BV2 microglial cells. Microglial clearance assays were performed with Aß42 in naive BV2 microglial cell line and single cell clone BV2 line expressing CD33WT. PET imaging was performed for three F-18 analogs in a rhesus macaque. RESULTS: All compounds but derivative 8 exhibited low microglial cell toxicity. Cromolyn 1 and derivatives 2, 4, and 7 displayed an increased uptake on Aß42 in naïve BV2 microglial cells. Derivative 4 increased Aß42 uptake in a dose-dependent manner and at 75µM resulted in a one-fold increase in Aß42 uptake in BV2-CD33WT. PET imaging for three [18F]cromolyn analogs revealed the order of brain tracer penetration to be 4a > 10 > 2a. Tracer 4a exhibited enhanced uptake in areas of high perfusion (putamen, grey matter, and cerebellum) and lower signal in areas of lower perfusion (caudate, thalamus, and white matter). CONCLUSION: Substantial uptake of Aß42 in both naïve BV2 and BV2-CD33WT cells observed with 4 indicate conversion of microglial cells from a pro-inflammatory to an activation state favoring Aß phagocytosis/clearance. These findings suggest that a fluoro-cromolyn analog could reduce fibril-prone Aß42in vivo and thereby serve as a therapeutic for the treatment and prevention of AD.

A concise method for fully automated radiosyntheses of [18F]JNJ-46356479 and [18F]FITM via Cu-mediated 18F-fluorination of organoboranes.

A modified alcohol-enhanced 18F-fluorodeboronation has been developed for the radiosyntheses of [18F]JNJ-46356479 and [18F]FITM. Unlike the [18F]KF/K222 approach, this method tolerates the presence of sensitive heterocycles in Bpin precursors 4 and 8 allowing a one-step 18F-fluorodeboronation on the fully automated TRACERlab™ FXFN platform.

Synthesis and Characterization of Fluorine-18-Labeled N-(4-Chloro-3-((fluoromethyl-d2)thio)phenyl)picolinamide for Imaging of mGluR4 in Brain.

We have synthesized and characterized [18F]-N-(4-chloro-3-((fluoromethyl-d2)thio)phenyl)-picolinamide ([18F]15) as a potential ligand for the positron emission tomography (PET) imaging of mGluR4 in the brain. Radioligand [18F]15 displays central nervous system drug-like properties, including mGluR4 affinity, potent mGluR4 PAM activity, and selectivity against other mGluRs, as well as sufficient metabolic stability. Radiosynthesis was carried out in two steps. The radiochemical yield of [18F]15 was 11.6 ± 2.9% (n = 7, decay corrected) with a purity of 99% and a molar activity of 84.1 ± 11.8 GBq/µmol. Ex vivo biodistribution studies showed reversible binding of [18F]15 in all investigated tissues including the brain, liver, heart, lungs, and kidneys. PET imaging studies in male Sprague Dawley rats showed that [18F]15 accumulates in the brain regions known to express mGluR4. Pretreatment with the unlabeled mGluR4 PAM compounds 13 (methylthio analogue) and 15 showed significant dose-dependent blocking effects. These results suggest that [18F]15 is a promising radioligand for PET imaging mGluR4 in the brain.

Quantitative in vivo mapping of myocardial mitochondrial membrane potential.

BACKGROUND: Mitochondrial membrane potential (ΔΨm) arises from normal function of the electron transport chain. Maintenance of ΔΨm within a narrow range is essential for mitochondrial function. Methods for in vivo measurement of ΔΨm do not exist. We use 18F-labeled tetraphenylphosphonium (18F-TPP+) to measure and map the total membrane potential, ΔΨT, as the sum of ΔΨm and cellular (ΔΨc) electrical potentials. METHODS: Eight pigs, five controls and three with a scar-like injury, were studied. Pigs were studied with a dynamic PET scanning protocol to measure 18F-TPP+ volume of distribution, VT. Fractional extracellular space (fECS) was measured in 3 pigs. We derived equations expressing ΔΨT as a function of VT and the volume-fractions of mitochondria and fECS. Seventeen segment polar maps and parametric images of ΔΨT were calculated in millivolts (mV). RESULTS: In controls, mean segmental ΔΨT = -129.4±1.4 mV (SEM). In pigs with segmental tissue injury, ΔΨT was clearly separated from control segments but variable, in the range -100 to 0 mV. The quality of ΔΨT maps was excellent, with low noise and good resolution. Measurements of ΔΨT in the left ventricle of pigs agree with previous in in-vitro measurements. CONCLUSIONS: We have analyzed the factors affecting the uptake of voltage sensing tracers and developed a minimally invasive method for mapping ΔΨT in left ventricular myocardium of pigs. ΔΨT is computed in absolute units, allowing for visual and statistical comparison of individual values with normative data. These studies demonstrate the first in vivo application of quantitative mapping of total tissue membrane potential, ΔΨT.

[18F]Fluorocholine and [18F]Fluoroacetate PET as Imaging Biomarkers to Assess Phosphatidylcholine and Mitochondrial Metabolism in Preclinical Models of TSC and LAM.

PURPOSE: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by inactivating mutations of the TSC1 or TSC2 gene, characterized by neurocognitive impairment and benign tumors of the brain, skin, heart, and kidneys. Lymphangioleiomyomatosis (LAM) is a diffuse proliferation of α-smooth muscle actin-positive cells associated with cystic destruction of the lung. LAM occurs almost exclusively in women, as a TSC manifestation or a sporadic disorder (TSC1/TSC2 somatic mutations). Biomarkers of whole-body tumor burden/activity and response to rapalogs or other therapies remain needed in TSC/LAM. EXPERIMENTAL DESIGN: These preclinical studies aimed to assess feasibility of [18F]fluorocholine (FCH) and [18F]fluoroacetate (FACE) as TSC/LAM metabolic imaging biomarkers. RESULTS: We previously reported that TSC2-deficient cells enhance phosphatidylcholine synthesis via the Kennedy pathway. Here, we show that TSC2-deficient cells exhibit rapid uptake of [18F]FCH in vivo and can be visualized by PET imaging in preclinical models of TSC/LAM, including subcutaneous tumors and pulmonary nodules. Treatment with rapamycin (72 hours) suppressed [18F]FCH standardized uptake value (SUV) by >50% in tumors. Interestingly, [18F]FCH-PET imaging of TSC2-deficient xenografts in ovariectomized mice also showed a significant decrease in tumor SUV. Finally, we found rapamycin-insensitive uptake of FACE by TSC2-deficient cells in vitro and in vivo, reflecting its mitochondrial accumulation via inhibition of aconitase, a TCA cycle enzyme. CONCLUSIONS: Preclinical models of TSC2 deficiency represent informative platforms to identify tracers of potential clinical interest. Our findings provide mechanistic evidence for testing the potential of [18F]FCH and [18F]FACE as metabolic imaging biomarkers for TSC and LAM proliferative lesions, and novel insights into the metabolic reprogramming of TSC tumors.