Automated radiosynthesis of Al[18F]PSMA-11 for large scale routine use.

OBJECTIVES: We report a reproducible automated radiosynthesis for large scale batch production of clinical grade Al[18F]PSMA-11. METHODS: A SynthraFCHOL module was optimized to synthesize Al[18F]PSMA-11 by Al[18F]-chelation. Results Al[18F]PSMA-11 was synthesized within 35min in a yield of 21 ± 3% (24.0 ± 6.0GBq) and a radiochemical purity > 95%. Batches were stable for 4h and conform the European Pharmacopeia guidelines. CONCLUSIONS: The automated synthesis of Al[18F]PSMA-11 allows for large scale production and distribution of Al[18F]PSMA-11.

Correction: Kinetic Modeling and Graphical Analysis of 18F-Fluoromethylcholine (FCho), 18F-Fluoroethyltyrosine (FET) and 18F-Fluorodeoxyglucose (FDG) PET for the Fiscrimination between High-Grade Glioma and Radiation Necrosis in Rats.

[This corrects the article DOI: 10.1371/journal.pone.0161845.].

Kinetic Modeling and Graphical Analysis of 18F-Fluoromethylcholine (FCho), 18F-Fluoroethyltyrosine (FET) and 18F-Fluorodeoxyglucose (FDG) PET for the Fiscrimination between High-Grade Glioma and Radiation Necrosis in Rats.

BACKGROUND: Discrimination between glioblastoma (GB) and radiation necrosis (RN) post-irradiation remains challenging but has a large impact on further treatment and prognosis. In this study, the uptake mechanisms of 18F-fluorodeoxyglucose (18F-FDG), 18F-fluoroethyltyrosine (18F-FET) and 18F-fluoromethylcholine (18F-FCho) positron emission tomography (PET) tracers were investigated in a F98 GB and RN rat model applying kinetic modeling (KM) and graphical analysis (GA) to clarify our previous results. METHODS: Dynamic 18F-FDG (GB n = 6 and RN n = 5), 18F-FET (GB n = 5 and RN n = 5) and 18F-FCho PET (GB n = 5 and RN n = 5) were acquired with continuous arterial blood sampling. Arterial input function (AIF) corrections, KM and GA were performed. RESULTS: The influx rate (Ki) of 18F-FDG uptake described by a 2-compartmental model (CM) or using Patlak GA, showed more trapping (k3) in GB (0.07 min-1) compared to RN (0.04 min-1) (p = 0.017). K1 of 18F-FET was significantly higher in GB (0.06 ml/ccm/min) compared to RN (0.02 ml/ccm/min), quantified using a 1-CM and Logan GA (p = 0.036). 18F-FCho was rapidly oxidized complicating data interpretation. Using a 1-CM and Logan GA no clear differences were found to discriminate GB from RN. CONCLUSIONS: Based on our results we concluded that using KM and GA both 18F-FDG and 18F-FET were able to discriminate GB from RN. Using a 2-CM model more trapping of 18F-FDG was found in GB compared to RN. Secondly, the influx of 18F-FET was higher in GB compared to RN using a 1-CM model. Important correlations were found between SUV and kinetic or graphical measures for 18F-FDG and 18F-FET. 18F-FCho PET did not allow discrimination between GB and RN.

Generation and in vivo characterization of a chimeric αvß5-targeting antibody 14C5 and its derivatives.

BACKGROUND: Previous studies showed that radiolabeled murine monoclonal antibody (mAb) 14C5 and its Fab and F(ab')2 fragments, targeting αvß5 integrin, have promising properties for diagnostic and therapeutic applications in cancer. To diminish the risk of generating a human anti-mouse antibody response in patients, chimeric variants were created. The purpose of this study was to recombinantly produce chimeric antibody (chAb) derivatives of the murine mAb 14C5 and to evaluate the in vitro and in vivo characteristics. METHODS: In vitro stability, specificity, and affinity of radioiodinated chAb and fragments (Iodo-Gen method) were examined on high-expressing αvß5 A549 lung tumor cells. In vivo biodistribution and pharmacokinetic characteristics were studied in A549 lung tumor-bearing Swiss Nu/Nu mice. RESULTS: Saturation binding experiments revealed high in vitro affinity of radioiodinated chAb, F(ab')2, and Fab, with dissociation constants (KD) of 1.19 ± 0.19, 0.68 ± 0.10, and 2.11 ± 0.58 nM, respectively. ChAb 14C5 showed highest tumor uptake (approximately 10%ID/g) at 24 h post injection, corresponding with other high-affinity Abs. ChF(ab')2 and chFab fragments showed faster clearance from the blood compared to the intact Ab. CONCLUSIONS: The chimerization of mAb 14C5 and its fragments has no or negligible effect on the properties of the antibody. In vitro and in vivo properties show that the chAb 14C5 is promising for radioimmunotherapy, due to its high maximum tumor uptake and its long retention in the tumor. The chF(ab')2 fragment shows a similar receptor affinity and a faster blood clearance, causing less non-specific retention than the chAb. Due to their fast blood clearance, the fragments show high potential for radioimmunodiagnosis.

In vivo visualization and quantification of (Disturbed) Oatp-mediated hepatic uptake and Mrp2-mediated biliary excretion of 99mTc-mebrofenin in mice.

UNLABELLED: Hepatic transport of (99m)Tc-mebrofenin through organic anion transport protein 1a and 1b (Oatp1a/1b) and multidrug resistance protein 2 (Mrp2) was investigated by small-animal SPECT. On the basis of the results, a noninvasive method to visualize and quantify disturbances in hepatic transport is proposed. METHODS: Friend virus B wild-type mice (untreated, bile duct-ligated, vehicle- or rifampicin-treated) and strain-matched knockout mice unable to express the uptake transporters Oatp1a/1b (Slco1a/1b(-/-)/(-/-)) or the efflux transporter Mrp2 (Abcc2(-/-)) were intravenously injected with (99m)Tc-mebrofenin (n = 3 per group). After dynamic small-animal SPECT and short CT acquisitions, time-activity curves of the liver and of the gallbladder and intestines were obtained and correlated with direct blood samples. RESULTS: Normal hepatobiliary clearance of (99m)Tc-mebrofenin was severely impaired in the bile duct-ligated animal, as evidenced by elevated hepatic tracer levels. In Slco1a/1b(-/-)/(-/-) mice, a lower area under the curve (AUC) for the liver (P = 0.014) was obtained and no activity was detected in the gallbladder and intestines. Renal rerouting was observed, along with an increase in the blood AUC (P = 0.01). Abcc2(-/-) mice had a higher liver AUC (P = 0.009), a delayed emergence time of (99m)Tc-mebrofenin in the gallbladder (P = 0.009), and a lower AUC for the gallbladder and intestines (P = 0.001). The blood curve was similar to that of wild-type mice. (99m)Tc-mebrofenin disposition was altered after rifampicin treatments. We observed a dose-dependent delayed time point at which tracer maximized in liver, an increased AUC for liver, and a lower AUC for gallbladder and intestines (P = 0.042, 0.034, and 0.001, respectively, highest dose). Emergence in the gallbladder occurred later (P = 0.009, highest dose), and blood AUC was higher (P = 0.006). CONCLUSION: The current study visualized and quantified hepatic uptake and biliary efflux of (99m)Tc-mebrofenin. Our results demonstrated the possibility of discriminating, on a quantitative level, between lack of functional activity of sinusoidal uptake versus that of biliary efflux transporters.

The influence of mass of [11C]-laniquidar and [11C]-N-desmethyl-loperamide on P-glycoprotein blockage at the blood-brain barrier.

INTRODUCTION: An earlier report suggested that mass amount of PET tracers could be an important factor in brain uptake mediated by P-glycoprotein. Thereby, this study investigated the influence of mass dose of laniquidar, desmethyl-loperamide and loperamide on the P-glycoprotein-mediated brain uptake of, respectively, [(11)C]-laniquidar and [(11)C]-N-desmethyl-loperamide ([(11)C]-dLop). METHODS: Wild-type (WT) mice were injected intravenously with solutions of 5.6 MBq [(11)C]-laniquidar (either no carrier added or 60 mg/kg laniquidar added) or with 5.0-7.4 MBq [(11)C]-dLop (either no carrier added or 3 mg/kg desmethyl loperamide). Mice were killed, and brain and blood were collected, weighted and counted for radioactivity. Mdr1a(-/-) knockout mice were incorporated as the control group. RESULTS: Injection of (11)C-laniquidar (no carrier added) in WT mice resulted in a statistical significant lower brain uptake (0.7±0.2 %ID/g) compared to the carrier-added formulation (60 mg/kg laniquidar) (3.1±0.3 %ID/g) (P=.004), while no statistical difference could be observed between formulations of [(11)C]-dLop. The [(11)C]-laniquidar and [(11)C]-dLop blood concentrations were not significantly different between the tested formulations in WT mice. In control animals, no effect of mass amount on brain uptake of both tracers could be demonstrated. CONCLUSIONS: These results demonstrate the bivalent character of laniquidar, acting as a substrate at low doses and as a blocking agent for P-glycoprotein transport in the brain at higher doses. In comparison, no difference was observed in [(11)C]-dLop uptake between carrier- and no-carrier-added formulations, which confirms that desmethyl-loperamide is a substrate of P-glycoprotein at the blood-brain barrier.

Antiepileptic drugs modulate P-glycoproteins in the brain: a mice study with (11)C-desmethylloperamide.

P-glycoprotein transporters (P-gp) located at the blood-brain barrier (BBB) are likely to play a role in refractory epilepsy. In vitro studies already pointed out that several antiepileptic drugs (AEDs) are substrate of P-gp. This study proposes a new in vivo approach to investigate the interaction between some AEDs and P-gp located at the BBB. (11)C-desmethylloperamide ((11)C-dLop), a radiolabelled substrate of P-gp, was intravenously administrated after pretreatment with saline or AEDs (sodium valproate, levetiracetam, topiramate and phenytoin) at their human therapeutic and four times their therapeutic dose. The effect of the different pretreatment on the intracerebral concentration of (11)C-dLop was determined to indirectly investigate possible in vivo interactions between AEDs and P-gp. Pretreatment with levetiracetam, topiramate and phenytoin at therapeutic doses significantly decreased intracerebral concentration of (11)C-dLop. Pretreatment with a therapeutic dose of sodium valproate did not influence brain uptake of (11)C-dLop. In case of pretreatment with supratherapeutic doses of AED, (11)C-dLop brain uptake was not different compared to pretreatment with saline. The metabolisation rate of (11)C-dLop in plasma was unaltered, indicating that observed differences in brain uptake of the tracer were not due to pharmacokinetic changes. The following conclusion can be made: levetiracetam, topiramate and phenytoin demonstrate biphasic modulation of the BBB P-gp. At therapeutic doses they act as inducers of efflux, at supratherapeutic doses they have no effect on the efflux rate. Sodium valproate does not interact with P-gp at therapeutic nor at higher doses.

P-glycoprotein at the blood-brain barrier: kinetic modeling of 11C-desmethylloperamide in mice using a 18F-FDG µPET scan to determine the input function.

PURPOSE: The objective of this study is the implementation of a kinetic model for 11C-desmethylloperamide (11C-dLop) and the determination of a typical parameter for P-glycoprotein (P-gp) functionality in mice. Since arterial blood sampling in mice is difficult, an alternative method to obtain the arterial plasma input curve used in the kinetic model is proposed. METHODS: Wild-type (WT) mice (pre-injected with saline or cyclosporine) and P-gp knock-out (KO) mice were injected with 20 MBq of 11C-dLop, and a dynamic µPET scan was initiated. Afterwards, 18.5 MBq of 18F-FDG was injected, and a static µPET scan was started. An arterial input and brain tissue curve was obtained by delineation of an ROI on the left heart ventricle and the brain, respectively based on the 18F-FDG scan. RESULTS: A comparison between the arterial input curves obtained by the alternative and the blood sampling method showed an acceptable agreement. The one-tissue compartment model gives the best results for the brain. In WT mice, the K1/k2 ratio was 0.4 ± 0.1, while in KO mice and cyclosporine-pretreated mice the ratio was much higher (2.0 ± 0.4 and 1.9 ± 0.2, respectively). K1 can be considered as a pseudo value K1, representing a combination of passive influx of 11C-desmethylloperamide and a rapid washout by P-glycoprotein, while k2 corresponds to slow passive efflux out of the brain. CONCLUSIONS: An easy to implement kinetic modeling for imaging P-glycoprotein function is presented in mice without arterial blood sampling. The ratio of K1/k2 obtained from a one-tissue compartment model can be considered as a good value for P-glycoprotein functionality.

Radiosynthesis and in vivo evaluation of [(11)C]MC80 for P-glycoprotein imaging.

P-glycoprotein (P-gp) is an ATP-dependent efflux pump protecting the body against xenobiotics. The in vitro characterized modulator 6,7-dimethoxy-2-(6-methoxy-naphthalen-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (MC80) of the P-gp pump was labelled with (11)C and evaluated in vivo for its potential to image P-gp function and expression. Radiochemical pure (>98%) [(11)C]MC80 was obtained within 25 min starting from [(11)C]methyl iodide with radiochemical yield of 26%. Biodistribution studies in FVB mice demonstrated a high baseline brain uptake (7.66 + or - 1.38%ID/g at 1 min pi). Cerebral uptake was increased in mdr1a knock-out mice as well as after CsA pretreatment. Pre-administration of an excess of non-radioactive MC80 caused a reduced uptake in several target organs including brain, pancreas and intestines. The results indicate that [(11)C]MC80 kinetics are modulated by P-gp. Reversed phase-HPLC analysis of brain revealed an excellent metabolic profile (>90% intact [(11)C]MC80).

Reduced dimethylaminoethanol in [(18)F]fluoromethylcholine: an important step towards enhanced tumour visualization.

PURPOSE: [(18)F]Fluoromethylcholine ([(18)F]FCho) is a radiotracer generally used for tumour visualization in patients. Due to high levels of dimethylaminoethanol (DMAE) remaining in [(18)F]FCho solutions synthesized by currently available methods, tumour visualization might be compromised. METHODS: An improved purification method involving an optimized purification step for reducing the levels of DMAE was conceived. The physiological explanation for the interference of residual DMAE in [(18)F]FCho pharmacokinetics was further elaborated in a xenograft mouse model. RESULTS: The use of a series of polymer solid-phase extraction cartridges (Oasis HLB/WCX), instead of the commonly used combination of tC18 and Accell CM cartridges, reduced DMAE levels from 402.2±49.6 ppm to 3.0±0.5 ppm. Subsequent in vitro tests proved that (1) [(18)F]FCho uptake was reduced in the presence of DMAE at concentrations above 0.5 µM and (2) DMAE is a competitive inhibitor of [(18)F]FCho transport. In vivo experiments in xenograft mouse models corroborated reduced tumour uptake at DMAE plasma levels of about 2.5 µM as found in patients injected with contaminated [(18)F]FCho. CONCLUSION: Residual DMAE, even at levels below choline plasma concentrations found during fasting, compromises [(18)F]FCho uptake in vivo and care should be taken to avoid its interference in molecular imaging with [(18)F]FCho.

Synthesis, in vitro and in vivo evaluation, and radiolabeling of aryl anandamide analogues as candidate radioligands for in vivo imaging of fatty acid amide hydrolase in the brain.

Fatty acid amide hydrolyase (FAAH) is one of the main enzymes responsible for terminating the signaling of endocannabinoids in the brain. Imaging FAAH in vivo using PET or SPECT is important to deeper understanding of its role in neuropsychiatric disorders. However, at present, no radioligand is available for mapping the enzyme in vivo. Here, we synthesized 18 aryl analogues of anandamide, FAAH's endogenous substrate, and in vitro evaluated their potential as metabolic trapping tracers. Interaction studies with recombinant FAAH revealed good to very good interaction of the methoxy substituted aryl anandamide analogues 17, 18, 19, and 20 with FAAH and they were identified as competing substrates. Compounds 17 and 18 did not display significant binding to CB1 and CB2 cannabinoid receptors and stand out as potential candidate metabolic trapping tracers. They were successfully labeled with 11C in good yields and high radiochemical purity and displayed brain uptake in C57BL/6J mice. Radioligands [11C]-17 and [11C]-18 merit further investigation in vivo.

In vivo evaluation in rodents of [(123)I]-3-I-CO as a potential SPECT tracer for the serotonin 5-HT(2A) receptor.

INTRODUCTION: [(123)I]-(4-fluorophenyl)[1-(3-iodophenethyl)piperidin-4-yl]methanone ([(123)I]-3-I-CO) is a potential single photon emission computed tomography tracer with high affinity for the serotonin 5-HT(2A) receptor (K(i)=0.51 nM) and good selectivity over other receptor (sub)types. To determine the potential of the radioligand as a 5-HT(2A) tracer, regional brain biodistribution and displacement studies will be performed. The influence of P-glycoprotein blocking on the brain uptake of the radioligand will also be investigated. METHODS: A regional brain biodistribution study and a displacement study with ketanserin were performed with [(123)I]-3-I-CO. Also, the influence of cyclosporin A (50 mg/kg) on the brain distribution of the radioligand was investigated. For the displacement study, ketanserin (1 mg/kg) was administered 30 min after injection of [(123)I]-3-I-CO. RESULTS: The initial brain uptake of [(123)I]-3-I-CO was quite high, but a rapid wash-out of radioactivity was observed. Cortex-to-cerebellum binding index ratios were low (1.1 - 1.7), indicating considerable aspecific binding and a low specific 'signal' of the radioligand. Tracer uptake was reduced to the levels in cerebellum (a 60% reduction) after ketanserin displacement. Administration of cyclosporin A resulted in a doubling of the brain radioactivity concentration. CONCLUSIONS: Although [(123)I]-3-I-CO showed adequate brain uptake and could be displaced by ketanserin, high aspecific binding to brain tissue was responsible for very low cortex-to-cerebellum binding index ratios, possibly limiting the potential of the radioligand as a serotonin 5-HT(2A) receptor tracer. We also demonstrated that [(123)I]-3-I-CO is probably a weak substrate for the P-glycoprotein efflux transporter.