[18F]FEPPA a TSPO Radioligand: Optimized Radiosynthesis and Evaluation as a PET Radiotracer for Brain Inflammation in a Peripheral LPS-Injected Mouse Model.

[18F]FEPPA is a specific ligand for the translocator protein of 18 kDa (TSPO) used as a positron emission tomography (PET) biomarker for glial activation and neuroinflammation. [18F]FEPPA radiosynthesis was optimized to assess in a mouse model the cerebral inflammation induced by an intraperitoneal injection of Salmonella enterica serovar Typhimurium lipopolysaccharides (LPS; 5 mg/kg) 24 h before PET imaging. [18F]FEPPA was synthesized by nucleophilic substitution (90 °C, 10 min) with tosylated precursor, followed by improved semi-preparative HPLC purification (retention time 14 min). [18F]FEPPA radiosynthesis were carried out in 55 min (from EOB). The non-decay corrected radiochemical yield were 34 ± 2% (n = 17), and the radiochemical purity greater than 99%, with a molar activity of 198 ± 125 GBq/µmol at the end of synthesis. Western blot analysis demonstrated a 2.2-fold increase in TSPO brain expression in the LPS treated mice compared to controls. This was consistent with the significant increase of [18F]FEPPA brain total volume of distribution (VT) estimated with pharmacokinetic modelling. In conclusion, [18F]FEPPA radiosynthesis was implemented with high yields. The new purification/formulation with only class 3 solvents is more suitable for in vivo studies.

Amoxicillin/clavulanic acid-warfarin drug interaction: a randomized controlled trial.

AIMS: To investigate whether an interaction exists between amoxicillin/clavulanic acid (amoxiclav) and warfarin in patients treated with stable oral anticoagulant therapy. METHODS: In a double-blind, cross-over, placebo-controlled study, 12 patients on stable warfarin therapy, received a 7 day amoxiclav regimen or placebo. RESULTS: The mean maximum increase in INR observed was 0.22 ± 0.3 with amoxiclav vs. 0.24 ± 0.6 with placebo (P=0.94). The day 7-day 1 factor II, R(-) and S(-) warfarin plasma concentrations were similar during the amoxiclav and placebo study periods (P=0.81, P=0.45, P=0.75, respectively). CONCLUSION: Amoxiclav did not modify anticoagulation in patients treated with stable warfarin therapy and without infection.

From positron emission tomography to cell analysis of the 18-kDa Translocator Protein in mild traumatic brain injury.

Traumatic brain injury (TBI) leads to a deleterious neuroinflammation, originating from microglial activation. Monitoring microglial activation is an indispensable step to develop therapeutic strategies for TBI. In this study, we evaluated the use of the 18-kDa translocator protein (TSPO) in positron emission tomography (PET) and cellular analysis to monitor microglial activation in a mild TBI mouse model. TBI was induced on male Swiss mice. PET imaging analysis with [18F]FEPPA, a TSPO radiotracer, was performed at 1, 3 and 7 days post-TBI and flow cytometry analysis on brain at 1 and 3 days post-TBI. PET analysis showed no difference in TSPO expression between non-operated, sham-operated and TBI mice. Flow cytometry analysis demonstrated an increase in TSPO expression in ipsilateral brain 3 days post-TBI, especially in microglia, macrophages, lymphocytes and neutrophils. Moreover, microglia represent only 58.3% of TSPO+ cells in the brain. Our results raise the question of the use of TSPO radiotracer to monitor microglial activation after TBI. More broadly, flow cytometry results point the lack of specificity of TSPO for microglia and imply that microglia contribute to the overall increase in TSPO in the brain after TBI, but is not its only contributor.

Astroglial Connexin 43 Deficiency Protects against LPS-Induced Neuroinflammation: A TSPO Brain µPET Study with [18F]FEPPA.

Astroglial connexin 43 (Cx43) has been recognized as a crucial immunoregulating factor in the brain. Its inactivation leads to a continuous immune recruitment, cytokine expression modification and a specific humoral autoimmune response against the astrocytic extracellular matrix but without brain lesions or cell lysis. To assess the impact of Cx43 deletion on the brain's inflammatory response, TSPO expression was studied by positron emission tomography (PET) imaging with a specific radioligand, [18F]FEPPA, in basal conditions or upon Lipopolysaccharides (LPS)-induced inflammatory challenge. Astroglial Cx43-deleted mice underwent [18F]FEPPA PET/CT dynamic imaging with or without LPS injection (5 mg/kg) 24 h before imaging. Quantification and pharmacokinetic data modelling with a 2TCM-1K compartment model were performed. After collecting the mice brains, TSPO expression was quantified and localized by Western blot and FISH analysis. We found that astroglial Cx43 deficiency does not significantly alter TSPO expression in the basal state as observed with [18F]FEPPA PET imaging, FISH and Western blot analysis. However, deletion of astrocyte Cx43 abolishes the LPS-induced TSPO increase. Autoimmune encephalopathy observed in astroglial Cx43-deleted mice does not involve TSPO overexpression. Consistent with previous studies showing a unique inflammatory status in the absence of astrocyte Cx43, we show that a deficient expression of astrocytic Cx43 protects the animals from LPS-induced neuroinflammation as addressed by TSPO expression.

Changes in dipole membrane potential at the mouse blood-brain barrier enhance the transport of 99mTechnetium Sestamibi more than inhibiting Abcb1, Abcc1, or Abcg2.

Cationic (99m)Tc-agents like (99m)Tc-hexakis-2-methoxyisobutyl isonitrile ((99m)Tc-MIBI) cannot be used for brain imaging because they do not enter the brain as readily as some uncharged (99m)Tc-compounds. The mechanism by which cationic (99m)Tc-agents are transported across the blood-brain barrier (BBB) remains unclear. We explored (99m)Tc-MIBI transport by in situ mouse brain perfusion to determine the influence of BBB features like the ATP-binding cassette transporters (Abcb1/P-glycoprotein (P-gp), Abcc1/Mrp1, and Abcg2/Bcrp), organic cation transporters (Slc22a1-3/Oct1-3), the transmembrane potential and the dipole membrane potential. P-gp reduced (99m)Tc-MIBI transport across the BBB of P-gp-deficient mice 2.2-fold, as confirmed by PSC833 and GF120918 inhibition. Paradoxically verapamil decreased its transport '0.6-fold'. Reducing the BBB dipole membrane potential with tetraphenylborate or phloretin increased (99m)Tc-MIBI transport about 12- and 20-fold, respectively. Guanidine, diphenhydramine, and carnitine significantly decreased (99m)Tc-MIBI transport, but tetraethylammonium did not. (99m)Tc-MIBI transport at the BBB is restricted by P-gp but not by Mrp1 or Bcrp. Some organic cations reduced the influx of (99m)Tc-MIBI into the brain independently of Oct1, 2 and 3, but this could be due to their effect on another cation transporter. The membrane dipole potential of the luminal BBB membrane appeared to be the main factor restricting (99m)Tc-MIBI permeability.

Gefitinib-phenytoin interaction is not correlated with the C-erythromycin breath test in healthy male volunteers.

AIMS: We aimed to describe the pharmacokinetic interaction between phenytoin, a potent CYP3A4 and P-glycoprotein (P-gp) (ABCB1) inducer, and gefitinib, a CYP3A4, CYP2D6 and P-gp substrate. METHODS: An open-label, randomized, two-phase crossover study was conducted. Eighteen healthy male volunteers (nine homozygous CC and nine homozygous TT as determined by their ABCB1 C3435T polymorphism in exon 26) received a single oral dose of 250 mg gefitinib alone or after 5 days treatment with phenytoin (5 mg kg(-1) daily). Gefitinib plasma concentrations were determined by high-performance liquid chromatography. Hepatic CYP3A4 activity was evaluated by the (14)C-erythromycin breath test (ERMBT) and the ABCB1 and CYP2D6 genetic polymorphisms were determined by the TaqMan allelic discrimination assay and long polymerase chain reaction, respectively. RESULTS: Following treatment with phenytoin, mean gefitinib C(max) and AUC(0-infinity) decreased by 26 +/- 44% [95% confidence interval (CI) for the difference 5-48%, P= 0.005] and 47 +/- 26% (95% CI for the difference 34-60%, P= 0.001), respectively, and apparent oral clearance increased by 126 +/- 93% (95% CI for the difference 80-172%, P= 0.004). Concomitantly, phenytoin increased the mean ERMBT by 91 +/- 44% (95% CI 75-105%, P < 0.001) from baseline, but the extent of liver CYP3A4 induction was not correlated to the extent of interaction. Furthermore, this interaction was independent of ABCB1 genetic polymorphism. The CYP2D6 genotype was slightly but significantly related to gefitinib clearance (P= 0.04) during the control phase. CONCLUSIONS: The significant interaction between gefitinib and phenytoin was not correlated with the erythromycin breath test and was independent of ABCB1 polymorphism, but may involve presystemic CYP3A-mediated intestinal first-pass.

Evaluation of planar bioluminescence imaging and microPET/CT for therapy monitoring in a mouse model of pigmented metastatic melanoma.

Bioluminescence imaging (BLI) is widely used for in-vivo monitoring of anti-cancer therapy in mice. [18F]MEL050 is a Positron Emission Tomography (PET) radiotracer which specifically targets melanin. We evaluated planar BLI and [18F]MEL050-PET/CT for therapy (pro-apoptotic peptide LZDP) monitoring in a mouse model of metastatic pigmented melanoma. Twelve B6-albino mice were intravenously injected with B16-F10-luc2 cells on day 0 (D0). The mice received daily from D2 to D17 either an inactive peptide (G1, n=6), or LZDP (G2, n=6). They underwent both BLI and [18F]MEL050-PET/CT imaging on D2, D8 and D17. The number of visible tumors was determined on BLI and PET/CT. [18F]MEL050 uptake in tumor sites was quantified on PET/CT. After sacrifice (D17), the number of black tumors was counted ex-vivo. On D2, BLI and PET/CT images were visually negative. On D8, BLI detected 8 tumor sites in 4/6 mice of G1 vs 5 in 3/6 mice of G2 (NS); PET/CT was visually negative. On D17, BLI detected 17 tumor sites in 5/6 mice of G1 vs 10 in 4/6 mice of G2 (NS). PET/CT detected 18 tumor sites in 4/4 mice of G1 vs 14 in 3/4 mice of G2 (NS). Mean %ID/g of [18F]MEL050 in tumor sites was lower in G2 than in G1 on D17 (P<0.001), whereas bioluminescence intensity was not different between the 2 groups. Ex-vivo examination confirmed lower number of tumors in G2 (P<0.03). In the small number of animals tested in this study, [18F]MEL050-PET/CT and ex-vivo examination could affirm anti-tumoral effect of LZDP, but not BLI.

[18F]MEL050 as a melanin-targeted PET tracer: Fully automated radiosynthesis and comparison to 18F-FDG for the detection of pigmented melanoma in mice primary subcutaneous tumors and pulmonary metastases.

INTRODUCTION: Melanoma is a highly malignant cutaneous tumor of melanin-producing cells. MEL050 is a synthetic benzamide-derived molecule that specifically binds to melanin with high affinity. Our aim was to implement a fully automated radiosynthesis of [18F]MEL050, using for the first time, the AllInOne™ synthesis module (Trasis), and to evaluate the potential of [18F]MEL050 for the detection of pigmented melanoma in mice primary subcutaneous tumors and pulmonary metastases, and to compare it with that of [18F]FDG. METHODS: Automated radiosynthesis of [18F]MEL050, including HPLC purification and formulation, were performed on an AllInOne™ synthesis module. [18F]MEL050 was synthesized using a one-step bromine-for-fluorine nucleophilic heteroaromatic substitution. Melanoma models were induced by subcutaneous (primary tumor) or intravenous (pulmonary metastases) injection of B16-F10-luc2 cells in NMRI mice. The maximum percentage of [18F]MEL050 Injected Dose per g of lung tissue (%ID/g Max) was determined on PET images, compared to [18F]FDG and correlated to in vivo bioluminescence imaging. RESULTS: The automated radiosynthesis of [18F]MEL050 required an overall radiosynthesis time of 48min, with a yield of 13-18% (not-decay corrected) and radiochemical purity higher than 99%. [18F]MEL050 PET/CT images were concordant with bioluminescence imaging, showing increased radiotracer uptake in all primary subcutaneous tumors and pulmonary metastases of mice. PET quantification of radiotracers uptake in tumors and muscles demonstrated similar tumor-to-background ratio (TBR) with [18F]MEL050 and [18F]FDG in subcutaneous tumors and higher TBR with [18F]MEL050 than with [18F]FDG in pulmonary metastases. CONCLUSION: We successfully implemented the radiosynthesis of [18F]MEL050 using the AllInOne™ module, including HPLC purification and formulation. In vivo PET/CT validation of [18F]MEL050 was obtained in mouse models of pigmented melanoma, where higher [18F]MEL050 uptake was observed in sub-millimetric pulmonary metastases, comparatively to [18F]FDG.

The [14C-N-methyl]-erythromycin breath test dosimetry complies with the French regulations for radiation safety.

The [14C-N-methyl]-erythromycin breath test (14C-ERMBT) is one of the most valuable probes for liver cytochrome P450-3A4 activity in humans. In order to extend the use of this test in France, we herein provide safety data regarding either patient dosimetry or worker exposure to [14C-N-methyl]-erythromycin. In order to determine the maximum radiation exposure for patient and nuclear medicine technician following one intravenous 14C-ERMBT [111 kiloBequerel (kBq)], we have used the dosimetric data gathered in animal studies and extrapolated to humans using a weight-based method, approximate data provided by the French Society of Radioprotection and erythromycin pharmacokinetics in humans, considering always the worst conditions for the patient and worker exposure determination. The radioactivity administered to a patient after one 14C-ERMBT was equal to 108.8 kBq (i.e. 98% of the total radioactivity in the 14C-erythromycin vial) leading to a patient effective dose of 20 microsievert (microSv) and a maximum effective dose after 14CO2 inhalation by the exposed worker of 16 microSv compared with a mean individual annual effective dose from natural and artificial radioactivity exposure of 3500 microSv in France. The 14C-ERMBT is safe and complies with the European regulations regarding the administration of 14C-labelled compounds in humans. It can therefore be used in clinical research in France without any particular safety requirement.

An optimised radiolabel procedure to prepare (99m)Tc-colloidal rhenium sulphide to improve radiochemical purity.

BACKGROUND: More than 25% of 99mTc colloidal rhenium sulphide preparations have been reported to have a radiochemical purity of <95% in 11 radiopharmacies. OBJECTIVES: To identify the key parameters involved in radiochemical purity, different preparation procedures were analysed to develop an optimised preparation method. METHODS: In the first part of this study, various data such as the Nanocis kit batch number, the eluate volume, the time between the two final elutions, the temperature and duration of heating were collected and analysed to determine the critical parameters that significantly decrease radiochemical purity. In the second part, a new procedure was applied and then the same parameters and radiochemical purity values were collected and compared with the results before the new procedure. RESULTS: Among 184 preparations, 137 (75%) had a radiochemical purity exceeding 95%, 25 (13.6%) were between 90 and 95% pure and 22 (12%) were below 90%. Significantly higher radiochemical purity was observed after the implementation of the new preparation procedure (89.5% of 374 preparations had radiochemical purities of > 95%). This new procedure consists in lowering the 99mTc eluate volume and time of heating. CONCLUSIONS: The implementation of a new method for the preparation of (99m)Tc colloidal rhenium sulphide based on a comparison of practices in various radiopharmacies resulted in: i) a determination of the critical points of this preparation, ii) an optimised labelling technique to harmonise different practices, and iii) a significant improvement in the preparations radiochemical purity and the quality of the lymphoscintigraphy in the location of sentinel node.

Ibogaine labeling with 99mTc-tricarbonyl: synthesis and transport at the mouse blood-brain barrier.

The (99m)Tc-tricarbonyl core may be used as an ideal tool for gamma-labeling ligands in noninvasive SPECT imaging. However, most (99m)Tc-tricarbonyl-labeled agents have difficulty crossing the blood-brain barrier (BBB). We radiolabeled the neuroactive indole ibogaine with (99m)Tc-tricarbonyl and measured its transport into the mouse brain by in situ brain perfusion. We measured the interactions of [(99m)Tc(CO)(3)-ibogaine](+) and (99m)Tc-tricarbonyl with the main BBB efflux transporters P-gp and BCRP in vitro and in vivo. Ibogaine was radiolabeled (yield: over 95%). [(99m)Tc(CO)(3)-ibogaine](+) entered the brain (K(in)) poorly (0.18 microL/g/s), at about the same rate as (99m)Tc-tricarbonyl (0.16 microL/g/s) and [(99m)Tc-sestamibi](+) (0.10 microL/g/s). The CNS tracer [(99m)Tc-HMPAO](0) entered the brain approximately 70-times higher than [(99m)Tc(CO)(3)-ibogaine](+). In vitro studies revealed that neither [(99m)Tc(CO)(3)-ibogaine](+) nor (99m)Tc-tricarbonyl ion were substrates for P-gp or BCRP. But lowering the membrane dipole potential barrier with phloretin enhanced the brain transport of [(99m)Tc(OH(2))(3)(CO)(3)](+) approximately 3-fold. Thus, ibogaine directly labeled with (99m)Tc-tricarbonyl is not suitable for CNS imaging because of its poor uptake. Brain transport is not restricted by efflux transporters but is reduced by its lipophilicity and interaction with the membrane-positive dipole potential.

Effect of widely used combinations of antiretroviral therapy on liver CYP3A4 activity in HIV-infected patients.

AIMS: To evaluate the effects of combined antiretroviral drugs (HAART) on liver CYP3A4 activity using the [(14)C-N-methyl]-erythromycin breath test (ERMBT). METHODS: HIV-infected patients (31 women, 30 men) with mean (+/- SD) age of 38 +/- 9 years were enrolled and underwent complete clinical and laboratory evaluation. Patients were divided into five groups and were treated with two nucleoside analogues (NAs) and one of the following: nelfinavir alone (n = 13), any ritonavir-boosted protease inhibitor with (n = 8) or without (n = 13) nevirapine, nevirapine alone (n = 15), or a third NA (n = 12). Three or four ERMBTs were performed 7 days prior to (D-7) and at the beginning of treatment (D0), D14 (only for patients taking nevirapine) and on D28. RESULTS: Mean baseline liver CYP3A4 activity displayed high interindividual variability (47%) but low intraindividual variability (15%). Women had 30% higher ERMBT values than men [2.7 +/- 1.3 vs. 1.9 +/- 0.7; 95% confidence interval (CI) 20.5, 49.5; P = 0.003]. The ERMBT data correlated with body weight, alpha- and beta-globulins and alanin aminotransferases (0.10 < r(s) < 0.20; P < 0.01). Whereas nevirapine had no effect on liver CYP3A4 activity, nelfinavir-based and ritonavir-boosted drug regimens inhibited it by 69% (95% CI 64.7, 72.9; P = 0.005) and by 95% (95% CI 93.3, 96.7; P = 0.001), respectively. CONCLUSION: Evaluation of the effect of HAART on liver CYP3A4 activity may aid in preventing inappropriate treatment regimens in HIV-infected patients.