The need for carbon-labeled radiotracers is increasingly higher in drug discovery and development (carbon-14, β–, t 1/2 = 5730 years) as well as in positron emission tomography (PET) for in vivo ...molecular imaging applications (carbon-11, β+, t 1/2 = 20.4 min). However, the structural diversity of radiotracers is still systematically driven by the narrow available labeled sources and methodologies. In this context, the emergence of carbon dioxide radical anion chemistry might set forth potential unexplored opportunities. Based on a dynamic isotopic equilibration between formate salts and 13C, 14C, 11CCO2, C-labeled radical anion CO2 •– could be accessed under extremely mild conditions within seconds. This methodology was successfully applied to hydrocarboxylation and dicarboxylation reactions in late-stage carbon isotope labeling of pharmaceutically relevant compounds. The relevance of the method in applied radiochemistry was showcased by the whole-body PET biodistribution profile of 11Coxaprozin in mice.
Neuroimaging biomarkers are needed to investigate the impact of smoking withdrawal on brain function. NFL-101 is a denicotinized aqueous extract of tobacco leaves currently investigated as an ...immune-based smoking cessation therapy in humans. However, the immune response to NFL-101 and its ability to induce significant changes in brain function remain to be demonstrated. Brain glucose metabolism was investigated using 18Ffluoro-deoxy-glucose (18FFDG) PET imaging in a mouse model of cigarette smoke exposure (CSE, 4-week whole-body inhalation, twice daily). Compared with control animals, the relative uptake of 18FFDG in CSE mice was decreased in the thalamus and brain stem (p < 0.001, n = 14 per group) and increased in the hippocampus, cortex, cerebellum, and olfactory bulb (p < 0.001). NFL-101 induced a humoral immune response (specific IgGs) in mice and activated human natural-killer lymphocytes in vitro. In CSE mice, but not in control mice, single-dose NFL-101 significantly increased 18FFDG uptake in the thalamus (p < 0.01), thus restoring normal brain glucose metabolism after 2-day withdrawal in this nicotinic receptor-rich region. In tobacco research, 18FFDG PET imaging provides a quantitative method to evaluate changes in the brain function associated with the withdrawal phase. This method also showed the CNS effects of NFL-101, with translational perspectives for future clinical evaluation in smokers.
Overcoming the efflux mediated by ATP–binding cassette (ABC) transporters at the blood-brain barrier (BBB) remains a challenge for the delivery of small molecule tyrosine kinase inhibitors (TKIs) ...such as erlotinib to the brain. Inhibition of ABCB1 and ABCG2 at the mouse BBB improved the BBB permeation of erlotinib but could not be achieved in humans. BBB disruption induced by focused ultrasound (FUS) was investigated as a strategy to overcome the efflux transport of erlotinib in vivo.
In rats, FUS combined with microbubbles allowed for a large and spatially controlled disruption of the BBB in the left hemisphere. ABCB1/ABCG2 inhibition was performed using elacridar (10 mg/kg i.v). The brain kinetics of erlotinib was studied using 11C-erlotinib Positron Emission Tomography (PET) imaging in 5 groups (n = 4–5 rats per group) including a baseline group, immediately after sonication (FUS), 48 h after FUS (FUS + 48 h), elacridar (ELA) and their combination (FUS + ELA). BBB integrity was assessed using the Evan's Blue (EB) extravasation test. Brain exposure to 11C-erlotinib was measured as the area under the curve (AUC) of the brain kinetics (% injected dose (%ID) versus time (min)) in volumes corresponding to the disrupted (left) and the intact (right) hemispheres, respectively.
EB extravasation highlighted BBB disruption in the left hemisphere of animals of the FUS and FUS + ELA groups but not in the control and ELA groups. EB extravasation was not observed 48 h after FUS suggesting recovery of BBB integrity. Compared with the control group (AUCBaseline = 1.4 ± 0.5%ID.min), physical BBB disruption did not impact the brain kinetics of 11C-erlotinib in the left hemisphere (p > .05) either immediately (AUCFUS = 1.2 ± 0.1%ID.min) or 48 h after FUS (AUCFUS+48h = 1.1 ± 0.3%ID.min). Elacridar similarly increased 11C-erlotinib brain exposure to the left hemisphere in the absence (AUCELA = 2.2 ± 0.5%ID.min, p < .001) and in the presence of BBB disruption (AUCFUS+ELA = 2.1 ± 0.5%ID.min, p < .001). AUCleft was never significantly different from AUCright (p > .05), in any of the tested conditions.
BBB integrity is not the rate limiting step for erlotinib delivery to the brain which is mainly governed by ABC-mediated efflux. Efflux transport of erlotinib persisted despite BBB disruption.
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•ABC-transporters limit the blood-brain barrier (BBB) permeation of erlotinib.•Ultrasound-induced BBB disruption is used to enhance the brain exposure to drugs.•The brain kinetics of 11C-erlotinib was assessed using in vivo PET imaging in rats.•Erlotinib was able to freely cross the BBB when ABC-transporters were inhibited.•ABC-mediated efflux of erlotinib persisted despite ultrasound-based BBB disruption.
The quantitative relationship between the disruption of the blood-brain barrier (BBB) and the recruitment of glial cells was explored in a mouse model of endotoxemia. 18F2-Fluoro-2-deoxy-sorbitol ...(18FFDS) PET imaging was used as a paracellular marker for quantitative monitoring of BBB permeability after i.v injection of increasing doses of lipopolysaccharide (LPS) or vehicle (saline, n = 5). The brain distribution of 18FFDS (VT, mL.cm−3) was estimated using kinetic modeling. LPS dose-dependently increased the brain VT of 18FFDS after injection of LPS 4 mg/kg (5.2 ± 2.4-fold, n = 4, p < 0.01) or 5 mg/kg (9.0 ± 9.1-fold, n = 4, p < 0.01) but not 3 mg/kg (p > 0.05, n = 7). In 12 individuals belonging to the different groups, changes in BBB permeability were compared with expression of markers of astrocyte (GFAP) and microglial cell (CD11b) using ex vivo immunohistochemistry. Increased expression of CD11b and GFAP expression was observed in mice injected with 3 mg/kg of LPS, which did not increase with higher LPS doses. Quantitative 18FFDS PET imaging can capture different levels of BBB permeability in vivo. A biphasic effect was observed with the lowest dose of LPS that triggered neuroinflammation without disruptive changes in BBB permeability, and higher LPS doses that increased BBB permeability without additional recruitment of glial cells.
Only partial deficiency/inhibition of P-glycoprotein (P-gp, ABCB1) function at the blood-brain barrier (BBB) is likely to occur in pathophysiological situations or drug-drug interactions. This raises ...questions regarding the sensitivity of available PET imaging probes to detect moderate changes in P-gp function at the living BBB. In vitro, the half-maximum inhibitory concentration (IC50) of the potent P-gp inhibitor tariquidar in P-gp-overexpressing cells was significantly different using either 11Cverapamil (44 nM), 11CN-desmethyl-loperamide (19 nM) or 11Cmetoclopramide (4 nM) as substrate probes. In vivo PET imaging in rats showed that the half-maximum inhibition of P-gp-mediated efflux of 11Cmetoclopramide, achieved using 1 mg/kg tariquidar (in vivo IC50 = 82 nM in plasma), increased brain exposure by 2.1-fold for 11Cmetoclopramide (p < 0.05, n = 4) and 2.4-fold for 11Cverapamil (p < 0.05, n = 4), whereby cerebral uptake of the “avid” substrate 11CN-desmethyl-loperamide was unaffected (p > 0.05, n = 4). This comparative study points to differences in the “vulnerability” to P-gp inhibition among radiolabeled substrates, which were apparently unrelated to their “avidity” (maximal response to P-gp inhibition). Herein, we advocate that partial inhibition of transporter function, in addition to complete inhibition, should be a primary criterion of evaluation regarding the sensitivity of radiolabeled substrates to detect moderate but physiologically-relevant changes in transporter function in vivo.
Knowledge of the repeatability of quantitative parameters derived from 18FFDG PET images is essential to define the group size and allow correct interpretation. Here we tested repeatability and ...accuracy of different 18FFDG absolute and relative quantification parameters in a standardized preclinical setup in nonhuman primates (NHP).
Repeated brain 18FFDG scans were performed in 6 healthy NHP under controlled experimental factors likely to account for variability. Regional cerebral metabolic rate of glucose (CMRglu) was calculated using a Patlak plot with blood input function Semi-quantitative approaches measuring standard uptake values (SUV, SUV×glycemia and SUVR (SUV Ratio) using the pons or cerebellum as a reference region) were considered. Test-retest variability of all quantification parameters were compared in different brain regions in terms of absolute variability and intra-and-inter-subject variabilities. In an independent 18FFDG PET experiment, robustness of these parameters was evaluated in 4 naive NHP.
Experimental conditions (injected dose, body weight, animal temperature) were the same at both imaging sessions (p >0.4). No significant difference in the 18FFDG quantification parameters was found between test and retest sessions. Absolute variability of CMRglu, SUV, SUV×glycemia and normalized SUV ranged from 25 to 43%, 16 to 21%, 23 to 28%, and 7 to 14%, respectively. Intra-subject variability largely explained the absolute variability of all quantitative parameters. They were all significantly correlated to each other and they were all robust. Arterial and venous glycemia were highly correlated (r = 0.9691; p<0.0001).
18FFDG test-retest studies in NHP protocols need to be conducted under well-standardized experimental conditions to assess and select the most reliable and reproducible quantification approach. Furthermore, the choice of the quantification parameter has to account for the transversal or follow-up study design. If pons and cerebellum regions are not affected, non-invasive SUVR is the most favorable approach for both designs.
Focused ultrasound in combination with microbubbles (FUS) provides an effective means to locally enhance the delivery of therapeutics to the brain. Translational and quantitative imaging techniques ...are needed to noninvasively monitor and optimize the impact of FUS on blood-brain barrier (BBB) permeability in vivo. Positron-emission tomography (PET) imaging using 18F2-fluoro-2-deoxy-sorbitol (18FFDS) was evaluated as a small-molecule (paracellular) marker of blood-brain barrier (BBB) integrity. 18FFDS was straightforwardly produced from chemical reduction of commercial 18F2-deoxy-2-fluoro-D-glucose. 18FFDS and the invasive BBB integrity marker Evan’s blue (EB) were i.v. injected in mice after an optimized FUS protocol designed to generate controlled hemispheric BBB disruption. Quantitative determination of the impact of FUS on the BBB permeability was determined using kinetic modeling. A 2.2 ± 0.5-fold higher PET signal (n = 5; p < 0.01) was obtained in the sonicated hemisphere and colocalized with EB staining observed post mortem. FUS significantly increased the blood-to-brain distribution of 18FFDS by 2.4 ± 0.8-fold (VT; p < 0.01). Low variability (=10.1%) of VT values in the sonicated hemisphere suggests reproducibility of the estimation of BBB permeability and FUS method. 18FFDS PET provides a readily available, sensitive and reproducible marker of BBB permeability to noninvasively monitor the extent of BBB disruption induced by FUS in vivo.
Duchenne muscular dystrophy (DMD) is a neurodevelopmental disorder primarily caused by the loss of the full-length Dp427 dystrophin in both muscle and brain. The basis of the central comorbidities in ...DMD is unclear. Brain dystrophin plays a role in the clustering of central gamma-aminobutyric acid A receptors (GABAARs), and its loss in the mdx mouse alters the clustering of some synaptic subunits in central inhibitory synapses. However, the diversity of GABAergic alterations in this model is still fragmentary. In this study, the analysis of in vivo PET imaging of a benzodiazepine-binding site radioligand revealed that the global density of central GABAARs is unaffected in mdx compared with WT mice. In contrast, semi-quantitative immunoblots and immunofluorescence confocal imaging in tissue sections revealed complex and differential patterns of alterations of the expression levels and/or clustered distribution of a variety of synaptic and extrasynaptic GABAAR subunits in the hippocampus, cerebellum, cortex, and spinal cord. Hence, dystrophin loss not only affects the stabilization of synaptic GABAARs but also influences the subunit composition of GABAARs subtypes at both synaptic and extrasynaptic sites. This study provides new molecular outcome measures and new routes to evaluate the impact of treatments aimed at compensating alterations of the nervous system in DMD.
(18)FDPA-714 N,N-diethyl-2-(2-(4-(2(18)F-fluoroethoxy)phenyl)5,7dimethylpyrazolo1,5apyrimidin-3-yl)acetamide is a new radioligand currently used for imaging the 18-kDa translocator protein in animal ...models of neuroinflammation and recently in humans. The biodistribution by positron emission tomography (PET) in baboons and the in vitro and in vivo metabolism of (18)FDPA-714 were investigated in rats, baboons, and humans. Whole-body PET experiments showed a high uptake of radioactivity in the kidneys, heart, liver, and gallbladder. The liver was a major route of elimination of (18)FDPA-714, and urine was a route of excretion for radiometabolites. In rat and baboon plasma, high-performance liquid chromatography (HPLC) metabolic profiles showed three major radiometabolites accounting for 85% and 89% of total radioactivity at 120 minutes after injection, respectively. Rat microsomal incubations and analyses by liquid chromatography-mass spectrometry (LC-MS) identified seven metabolites, characterized as O-deethyl, hydroxyl, and N-deethyl derivatives of nonradioactive DPA-714, two of them having the same retention times than those detected in rat and baboon plasma. The third plasma radiometabolite was suggested to be a carboxylic acid compound that accounted for 15% of the rat brain radioactivity. O-deethylation led to a nonradioactive compound and (18)Ffluoroacetic acid. Human CYP3A4 and CYP2D6 were shown to be involved in the oxidation of the radioligand. Finally an easy, rapid, and accurate method--indispensable for PET quantitative clinical studies--for quantifying (18)FDPA-714 by solid-phase extraction was developed. In vivo, an extensive metabolism of (18)FDPA-714 was observed in rats and baboons, identified as (18)Fdeethyl, (18)Fhydroxyl, and (18)Fcarboxylic acid derivatives of (18)FDPA-714. The main route of excretion of the unchanged radioligand in baboons was hepatobiliary while that of radiometabolites was the urinary system.
Sulfonylurea receptor 1 (SUR1) overexpression in the central nervous system is a potential biomarker for positron emission tomography (PET) imaging of brain damage and recovery. VU0071063, a ...selective ligand of SUR1 able to cross the blood–brain barrier, was isotopically radiolabeled with carbon‐11 from a desmethyl precursor obtained quantitatively in one step. Ready‐to‐inject 11CVU0071063 was obtained in 18 ± 2% radiochemical yield and 103 ± 22 GBq/μmol molar activity. PET imaging in healthy rats demonstrated a significant brain penetration and rapid elimination of the tracer in vivo, encouraging further investigation in animal models of SUR1 overexpression.
Automated isotopic carbon‐11 radiolabeling of SURI ligand VU0071063 was successfully developed to demonstrate in vivo the brain penetration of this tracer in healthy rats using PET imaging.
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