In Vivo Evaluation of Radiofluorinated Caspase-3/7 Inhibitors as Radiotracers for Apoptosis Imaging and Comparison with [18F]ML-10 in a Stroke Model in the Rat.

PURPOSE: The first biological evaluation of two potent fluorine-18 radiolabelled inhibitors of caspase-3/7 was achieved in a cerebral stroke rat model to visualize apoptosis. PROCEDURES: In vivo characteristics of isatins [(18)F]-2 and [(18)F]-3 were studied and compared by µPET to previously described 1-[4-(2-[(18)F]fluoroethyl)benzyl]-5-(2-methoxymethylpyrrolidin-1-ylsulfonyl)isatin ([(18)F]-1) and to 2-(5-[(18)F]fluoropentyl)-2-methyl-malonic acid ([(18)F]ML-10) used as a reference radiotracer in a rat stroke model. RESULTS: [(18)F]-2 and [(18)F]-3 were radiolabelled with high radiochemical purity and high specific radioactivity. Radioactivity uptakes in ischemic and contralateral brain regions were weak for the three radiolabelled isatins and lower for [(18)F]ML-10. In µPET, time activity curves showed significant uptake differences between both regions of interest for [(18)F]-1 after 45 min. No differences were observed for [(18)F]ML-10. CONCLUSIONS: Radiolabelled isatins are more promising radiotracers to image apoptosis than [(18)F]ML-10 in this stroke animal model without craniectomy. In particular, [(18)F]-1 presented significant uptake in apoptotic area 45 min after administration.

Nucleophilic Fluorination and Radiofluorination via Aziridinium Intermediates: N-Substituent Influence, Unexpected Regioselectivity, and Differences between Fluorine-19 and Fluorine-18.

The efficient dehydrofluorination and radiofluorination of N,N-disubstituted-ß-aminoalcohols through an anchimeric-assisted mechanism was developed. An investigation into the influence of N-substituents on the ring opening of the aziridinium intermediate indicated differences in the isomeric ratio and the yields of fluorinated products obtained from N,N-disubstituted-phenylalaninol. This influence was substantial for (18)F-radiofluorination, with yields varying from 0 to 71% at room temperature (RT). Although no significant effects were observed in the fluorine-19 chemistry when the reaction was heated to 90 °C, considerable changes appeared during radiofluorination. In the latter case, the radiochemical yields increased, and degradation of the 2-fluoro-propan-1-amine isomer (b) occurred, leading to a regiospecific reaction in the radiolabeling of [(18)F]-fluorodeprenyl. This method involving nucleophilic radiofluorination at RT was successfully applied to the radiolabeling of [(18)F]-2-fluoroethylamines in which the influence of the N-substituent was also observed.

Radiosynthesis of carbon-11 and fluorine-18 labelled radiotracers to image the ionotropic and metabotropic glutamate receptors.

l-Glutamate is the major neurotransmitter in the central nervous system and activates both ionotropic and metabotropic receptors. Here the radiosynthesis of radiotracers developed for both types of receptors are reviewed with a highlight on the radiopharmaceuticals used or evaluated in humans. At first, radiotracers were developed for ionotropic N-methyl-d-aspartate receptors without any success to obtain radiopharmaceuticals useable for clinical or even preclinical positron emission tomography (PET) imaging purposes. Some compounds were radiolabelled and evaluated for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors without any successful results. The recent development of radiotracers for metabotropic glutamate receptors was more efficient because radiopharmaceuticals are currently evaluated or used in clinical trials to study the mGluR1, mGluR2 or mGluR5 receptors by PET. Although the majority of the radiotracers were classically labelled with carbon-11 by O- or N-[(11) C]-methylation or with fluorine-18 nucleophilic substitution of aromatic nitro or halogeno precursors using krypofix 2.2.2/potassium [(18) F]fluoride complex, some radiosyntheses were performed with recent radiolabelling reactions like the use of iodionium salt for [(18) F]-labelling.

Automated radiosynthesis of [(18)F]ML-10, a PET radiotracer dedicated to apoptosis imaging, on a TRACERLab FX-FN module.

PURPOSE: [(18)F]ML-10 is the most advanced radiopharmaceutical for the clinical imaging of the apoptosis phenomenon by PET. The preparation of this radiopharmaceutical on a commercial radiosynthesis module and the requested quality controls for its release are presented herein. PROCEDURES: ML-10 as reference and its mesyloxy derivative as precursor for labelling with fluorine-18 were prepared. [(18)F]ML-10 was synthesized via a [(18)F]fluorine-de-mesyloxy aliphatic nucleophilic substitution via a GE TRACERLab® FX-FN module. Quality controls were performed. RESULTS: The labelling precursor was obtained in a four step synthesis in 28 % overall yield affording ML-10 in two steps (88 % yield). Pure [(18)F]ML-10 was obtained with a decay corrected yield of 39.8 % ± 8.4 % (n = 7) in 70 min and a specific activity of 235 ± 85 GBq/µmol at the end of synthesis. CONCLUSIONS: [(18)F]ML-10 was prepared on a widely available automated module and passed the quality control. A LC/MS method was developed to measure specific radioactivity.

Radiolabelling of 1,4-disubstituted 3-[18F]fluoropiperidines and its application to new radiotracers for NR2B NMDA receptor visualization.

In order to develop a novel and useful building block for the development of radiotracers for positron emission tomography (PET), we studied the radiolabelling of 1,4-disubstituted 3-[(18)F]fluoropiperidines. Indeed, 3-fluoropiperidine became a useful building block in medicinal chemistry for the pharmacomodulation of piperidine-containing compounds. The radiofluorination was studied on substituted piperidines with electron-donating and electron-withdrawing N-substituents. In the instance of electron-donating N-substituents such as benzyl or butyl, configuration retention and satisfactory fluoride-18 incorporation yields up to 80% were observed. In the case of electron-withdrawing N-substituents leading to carbamate or amide functions, the incorporation yields depend on the 4-susbtitutent (2 to 63%). The radiolabelling of this building block was applied to the automated radiosynthesis of NR2B NMDA receptor antagonists and effected by a commercially available radiochemistry module. The in vivo evaluation of three radiotracers demonstrated minimal brain uptakes incompatible with the imaging of NR2B NMDA receptors in the living brain. Nevertheless, moderate radiometabolism was observed and, in particular, no radiodefluorination was observed which demonstrates the stability of the 3-position of the fluorine-18 atom. In conclusion, the 1,4-disubstituted 3-[(18)F]fluoropiperidine moiety could be of value in the development of other radiotracers for PET even if the evaluation of the NR2B NMDA receptor antagonists failed to demonstrate satisfactory properties for PET imaging of this receptor.

Synthesis and in vitro characterization of trans- and cis-[(18)F]-4-methylbenzyl 4-[(pyrimidin-2-ylamino)methyl]-3-fluoropiperidine-1-carboxylates as new potential PET radiotracer candidates for the NR2B subtype N-methyl-D-aspartate receptor.

Diastereoisomeric compounds [(18)F]cis- and [(18)F]trans-4-methylbenzyl 4-[(pyrimidin-2-ylamino)methyl]-3-fluoro-piperidine-1-carboxylates were successfully synthesized as new subtype-selective PET radiotracers for imaging the NR2B subunit containing NMDA receptors. Rat brain section autoradiographies demonstrated a high specific binding in NR2B/NMDA receptor rich regions for both radioligands. The measured logD(7.4) values as well as B(max)/K(d) ratios indicated that both radiotracers possess the adequate properties required for PET radiotracers.

Complementary information from magnetic resonance imaging and (18)F-fluoromisonidazole positron emission tomography in the assessment of the response to an antiangiogenic treatment in a rat brain tumor model.

INTRODUCTION: No direct proof has been brought to light in a link between hypoxic changes in glioma models and the effects of antiangiogenic treatments. Here, we assessed the sensitivity of the detection of hypoxia through the use of (18)F-fluoromisonidazole positron emission tomography ([(18)F]-FMISO PET) in response to the evolution of the tumor and its vasculature. METHODS: Orthotopic glioma tumors were induced in rats after implantation of C6 or 9L cells. Sunitinib was administered from day (D) 17 to D24. At D17 and D24, multiparametric magnetic resonance imaging was performed to characterize tumor growth and vasculature. Hypoxia was assessed by [(18)F]-FMISO PET. RESULTS: We showed that brain hypoxic volumes are related to glioma volume and its vasculature and that an antiangiogenic treatment, leading to an increase in cerebral blood volume and a decrease in vessel permeability, is accompanied by a decrease in the degree of hypoxia. CONCLUSIONS: We propose that [(18)F]-FMISO PET and multiparametric magnetic resonance imaging are pertinent complementary tools in the evaluation of the effects of an antiangiogenic treatment in glioma.

Synthesis, evaluation and metabolic studies of radiotracers containing a 4-(4-[18F]-fluorobenzyl)piperidin-1-yl moiety for the PET imaging of NR2B NMDA receptors.

In this study, novel specific PET radioligands containing the 4-(4-fluorobenzyl)piperidine moiety and selectively antagonistic for the NR2B subunit containing NMDA receptors were developed. Two antagonists, RGH-896 (1a) and 4-(4-fluorobenzyl)piperidinyl-1-methyl-2-benzimidazol-5-ol (2a), belonging to two different structural families, were radiolabeled by an aromatic nucleophilic radiofluorination followed by a reduction of the para-position carbonyl function. Radiotracers [18F]1a, [18F]2a or the pattern 4-(4-[18F]-fluorobenzyl)piperidine ([18F]6) demonstrated an identical in vivo behavior with high accumulation of radioactivity in bone and cartilage which would suggest a radiodefluorination of the radiotracers. The identification of metabolites from 6 by LC-MS-MS confirmed the significant degree of defluorination as a result of the in vivo hydroxylation in the benzyl ring. In conclusion, [18F]1a or [18F]2a are not suitable for imaging the NR2B NMDA receptors due to their poor brain penetration. We also argue for a cautious use of the radiolabeled pattern, 4-(4-[18F]-fluorobenzyl)piperidine, to develop PET radiotracers.

Efficient radiosynthesis of 2-[(18)f]fluoroadenosine: a new route to 2-[(18)f]fluoropurine nucleosides.

An efficient method to incorporate the fluorine-18 radionuclide in 2-nitropurine-based nucleosides was developed. The nucleophilic radiofluorination of the labeling precursor with [(18)F]KF under aminopolyether-mediated conditions (Kryptofix 2.2.2/K2CO3) followed by deprotection was straightforward and, after formulation, gave 2-[(18)F]fluoroadenosine, ready for injection with a radiochemical yield of 45 ± 5%, a radiochemical purity of >98%, and a specific radioactivity up to 148 GBq/µmol. A micropositron emission tomography imaging and biodistribution study on rodents was reported.

Simultaneous tritium labelling of two potent 5-HT4 ligands.

Two potent and selective 5-HT(4) ligands, [(3)H]-5-[(N-propylpiperidin-4-yl)methoxy]-1,2,3,4-tetrahydrobenzo[h][1,6] naphthyridine (1a) and [(3)H]-1-methyl-5-[(N-propylpiperidin-4-yl)methoxy]pyrrolo[1,2-a]thieno[2,3-e]pyrazine (2a) were radiolabelled with tritium. Radioactive labelling was achieved by simultaneous tritium reduction of a mixture of both propargylic precursors (1c-2c). The two tritiated ligands thus obtained were radiochemically pure and possessed high radioactive specific activities. These tritiated 5-HT(4) ligands will allow for binding characterization as an essential tool for their further development.

Radiosynthesis and ex vivo evaluation of [11C]-SIB-1553A as a PET radiotracer for beta4 selective subtype nicotinic acetylcholine receptor.

[11C]-SIB-1553A ((+/-)-4-[2-((N-[11C]-methyl)-2-pyrrolidinyl)ethyl]thiophenol) was labelled with carbon-11 (t1/2=20.4 min) and evaluated in vivo as potential radiotracer for noninvasive assessment of the beta4 subunit nicotinic acetylcholine neurotransmission system with positron emission tomography (PET). The labelling precursor was obtained within five steps from N-Boc-prolinal in 45-56% overall yields. The radiosynthesis of [11C]-SIB-1553A was achieved by a selective N-[11C]-methylation in 32 min with a radiochemical purity greater than 97%, 7.5-30 GBq/micromol of specific radioactivity and 55-65% radiochemical yield (decay corrected, based on [11C]methyl iodide). The ex vivo pharmacological profile of [11C]-SIB-1553A was evaluated in rats with biodistribution studies in organs and in brain structures by autoradiography. The radiotracer uptake in the brain reached 0.49 %ID/g at 10 min and no brain radiometabolite was detected 40 min after intravenous injection. The quantification of radioactivity in various cerebral structures indicated a significantly higher radioactivity level at 15 min than at 30 min. Among the beta4 nAChR subunit-rich structures studied in the rat brain, only the thalamus at 15 and 30 min and the hippocampus at 30 min showed significantly higher uptake. Moreover, competition studies performed with SIB-1553A (15 min before the radiotracer injection) revealed only a low specific binding estimated to 7% of the total binding at 15 min and 13% at 30 min.

Methyl transfer reaction from monomethyltin reagent under palladium(0) catalysis: a versatile method for labelling with carbon-11.

The 11C-monomethylstannate prepared from [11C]-methyl iodide and Lappert's stannylene, was subject to a palladium-mediated cross-coupling reaction with an aryl halide under ligand-free conditions, to afford easily purified 11C-methyl(hetero)arenes in high radiochemical yields.

Efficiency of 18F-FDG and 99mTc-depreotide SPECT in the diagnosis of malignancy of solitary pulmonary nodules.

PURPOSE: The purpose of the study was to compare ( 18)F-fluorodeoxyglucose (FDG) positron emission tomography (PET) and ( 99m)Tc-depreotide single-photon emission computed tomography (SPECT) in the diagnosis of malignancy of solitary pulmonary nodules (SPNs). METHODS: Twenty-eight patients without any history of cancer and presenting an SPN (0.8-3 cm in size) underwent FDG PET and depreotide SPECT. Depreotide SPECT and FDG PET were performed on a double-head gamma camera and a dedicated PET scanner respectively. Twenty-five out of 28 lesions were removed by thoracotomy or assessed by biopsy (n=1) and histologically examined. A strategy of serial CT scanning was adopted in the three remaining patients. RESULTS: Histological findings revealed 18 malignant nodules and seven benign lesions. Stability over a 2-year period indicated a benign process in the remaining three cases. Both techniques yielded true positive results in 15 of the 18 cancers. FDG PET identified two additional adenocarcinomas not detected by depreotide SPECT. A carcinoid tumour not visualised on FDG PET was identified by depreotide SPECT. Seven of the ten benign lesions did not reveal tracer uptake on either depreotide SPECT or FDG PET. Both techniques showed false positive results for the same two lesions. One more false positive was seen on FDG PET. FDG PET and depreotide SPECT had a sensitivity of 94.4% and 88.9% respectively; this difference was not significant. In our experience, depreotide SPECT and FDG PET are equally sensitive (92.3%) for large (>1.5 cm) and equally specific (85.7%) for small (up to 1.5 cm) SPNs suspicious for malignancy. CONCLUSION: This study showed( 18)F-FDG PET to be more sensitive than ( 99m)Tc-depreotide SPECT in the diagnosis of malignancy of SPNs. However, the combination of both techniques may provide additional accuracy.

Evaluation in rats and primates of [11C]-mecamylamine, a potential nicotinic acetylcholine receptor radioligand for positron emission tomography.

Mecamylamine is a well-described non specific antagonist of nicotinic acetylcholine receptors (nAChRs), used in therapy and in psychopharmacological studies. [(11)C]-Mecamylamine was prepared and evaluated as a putative radioligand for positron emission tomography to study nicotinic acetylcholine receptors. The radiosynthesis consisted in the [(11)C]-methylation of the desmethyl precursor within 40 min with 30-40% radiochemical yield decay corrected. Biodistribution studies in rats showed that radioligand crossed the blood-brain barrier (0.39% ID at 30 min) and only unmetabolized tracer was recovered from brain at 45 min. Ex vivo autoradiography studies in rats did not indicate preferential uptake, and pre-treatment mecamylamine or with chlorisondamine, an nicotinic receptor inhibitor, did not demonstrate a significant specific binding. To investigate possible specie differences and effects of anesthesia, in vivo positron emission tomography (PET) studies were carried out on anaesthetized baboons and conscious macaques. The regional brain distribution of [(11)C]-mecamylamine in the two species of primates exhibited similar kinetics as did the rat with steady state reached about 45-50 min after radiotracer administration. Uptake values were two-fold higher in brain of conscious macaque than in anaesthetized baboon (thalamus: 0.258% ID/(kg mL) in conscious macaques and 0.129% ID/(kg mL) in baboons). PET images showed a radioactivity distribution which was quite homogeneous throughout the brain but with somewhat higher uptake in grey matter than in white. Brain distribution was unaltered by saturation or displacement studies. Possible explanation for the failure to establish specific binding in vivo could be long-lived structural modifications of the ionotropic channel by the unlabeled ligand administered before the tracer. In conclusion, [(11)C]-mecamylamine did not satisfy the requirements for a PET tracer of nicotinic acetylcholine receptors.

Debenzylation of tertiary amines using phosgene or triphosgene: an efficient and rapid procedure for the preparation of carbamoyl chlorides and unsymmetrical ureas. Application in carbon-11 chemistry.

Efficient and rapid preparations of carbamoyl chlorides and unsymmetrical ureas from tertiary amines and phosgene or its safe equivalent triphosgene [bis(trichloromethyl)carbonate, BTC] are described. First, the reaction of stoichiometric amounts of phosgene with secondary amines was revisited, and it was shown that the formation of carbamoyl chlorides in high yields required careful adjustments of experimental conditions and the use of pyridine as an HCl scavenger. A phosgene-mediated dealkylation of triethylamine was observed when this base was used instead of pyridine. Taking advantage of this observation, a strategy of synthesis of carbamoyl chlorides from tertiary amines and phosgene has been developed. N-Alkyl-N-benzyl(substituted)tetrahydroisoquinolines, -piperazines, -piperidines, or -anilines were treated with stoichiometric amounts of phosgene (or BTC) in CH(2)Cl(2). Tertiary amines bearing electron-enriched benzyl group(s) afforded carbamoyl chlorides in excellent yields and without any contamination by symmetrical ureas. Subsequent additions of primary or secondary amines to these carbamoyl chlorides produced unsymmetrical ureas in single-pot and high-yielding operations. This methodology was applied in (11)C-chemistry. From [(11)C]phosgene, a common precursor used in the preparation of radiotracers for positron emission tomography, a rapid and efficient synthesis of (11)C-carbamoyl chlorides and (11)C-unsymmetrical ureas derived from tetrahydroisoquinoline and piperazine is described. The first example of (11)C-amide formation from the reaction of a (11)C-carbamoyl chloride and an organometallic (cyanocuprate or a Grignard reagent in the presence of a nickel catalyst) is also presented.

Efficiency of [(18)F]FDG PET in characterising renal cancer and detecting distant metastases: a comparison with CT.

The purpose of this study was to assess the efficiency of fluorine-18 fluoro-2-deoxyglucose (FDG) positron emission tomography (PET) in the characterisation and primary staging of suspicious renal masses, in comparison with computed tomography, the current standard imaging modality. Fifty-three FDG PET studies were performed within the framework of a prospective study: 35 for both characterisation and staging of a suspicious mass, and 18 for staging early after surgical removal of a renal cancer. In the characterisation of renal masses, a high rate of false negative results was observed, leading to a sensitivity, specificity and accuracy of 47%, 80% and 51% respectively, versus 97%, 0/5 and 83% respectively for CT. FDG PET detected all the sites of distant metastasis revealed by CT, as well as eight additional metastatic sites, leading to an accuracy of 94% versus 89% for CT. However, 36/53 patients (68%) did not have any distant metastasis on either CT or on PET. All but one of these patients had a low Fuhrman histological grade and a limited local stage (< or =pT2). We conclude that FDG PET does not offer any advantage over CT for the characterisation of renal masses but that it appears to be an efficient tool for the detection of distant metastasis in renal cancer. However, our data suggest that a selection process could be implemented to determine which patients should undergo PET. FDG PET could be performed in the event of a solitary metastasis or doubtful images on CT. Selection could also be based on adverse histological findings from nephrectomy specimens in order to perform staging early after nephrectomy.

Early response to chemotherapy in hypopharyngeal cancer: assessment with (11)C-methionine PET, correlation with morphologic response, and clinical outcome.

UNLABELLED: Neoadjuvant chemotherapy in hypopharyngeal cancer globally improves survival, but some patients do not respond to chemotherapy and adjuvant therapy is delayed. Prediction of response to chemotherapy may allow physicians to optimize planned treatment. The aim of this study was to compare treatment response assessed early with (11)C-methionine PET and morphologic response assessed after treatment completion with MRI. METHODS: Thirteen patients with previously untreated squamous cell carcinoma of the hypopharynx, T3 or T4, were included. All patients received 3 courses of chemotherapy comprising cisplatin and 5-fluorouracil. (11)C-Methionine PET was performed before and after the first course of chemotherapy. PET estimation of response was expressed in relative variation of mean standardized uptake value (SUVmean), maximal standardized uptake value (SUVmax), volume of (11)C-methionine tumor uptake, and total tumor uptake. Posttreatment response was assessed with MRI, which was performed before the first course and after treatment completion, and expressed in relative variation of tumor volume. Patients were considered responders if their tumor volume was reduced by more than 50%. RESULTS: The relative decrease in all PET parameters correlated significantly with the relative decrease in MRI volume. The larger area under the receiver operating characteristic curve was obtained for SUVmean (0.883), but that area was close to the area of SUVmax (0.857). For methodologic considerations, SUVmax was more reproducible. The optimal threshold of response for SUVmax was -25%, leading to a mean of 83% (range, 36%-93%) sensitivity and 86% (range, 42%-100%) specificity. Using this threshold, survival at 2 y was improved for responders (83%), compared with nonresponders (57%), but the difference was not statistically significant. CONCLUSION: (11)C-Methionine PET provides early useful information about changes in tumor metabolism induced by chemotherapy in hypopharynx cancer. (11)C-Methionine PET measurements correlate with end-of-treatment response evaluated with MRI and may thus be helpful to physicians in treatment planning by avoiding unnecessary chemotherapy courses for nonresponding patients.