P. A. Schubiger

Stabilization of neurotensin analogues: effect on peptide catabolism, biodistribution and tumor binding

Neurotensin (NT) receptors in pancreatic and other neuroendocrine tumors are promising targets for imaging and therapeutic purposes. Here, we report on the effect of distinct changes in the peptide chain on catabolism in vitro for five radiolabeled [99mTc] neurotensin analogues having high affinity for neurotensin receptors. Substitution of NT(1-7) by (NalphaHis)Ac--the Tc-binding moiety--combined with a reduced bond 8-9 (CH2NH), N-methylation of peptide bonds or replacement of Ile(12) by tertiary leucin (Tle) led to peptide stabilization of various degrees. Biodistribution studies in nude mice bearing HT29 xenografts showed higher tumor uptake with more stable peptides, yielding high tumor to blood ratios of up to 70.

NanoPET imaging of [18F]fluoromisonidazole uptake in experimental mouse tumours

PurposeThe purpose of this study was to assess the potential and utility of ultra-high-resolution hypoxia imaging in various murine tumour models using the established hypoxia PET tracer [18F]fluoromisonidazole ([18F]FMISO).Methods[18F]FMISO PET imaging was performed with the dedicated small-animal PET scanner NanoPET (Oxford Positron Systems) and ten different human tumour xenografts in nude mice as well as B16 melanoma tumours in syngeneic Balb/c mice. For comparison, [18F]fluorodeoxyglucose ([18F]FDG) PET scans were also performed in the mice bearing human tumour xenografts.ResultsIn 10 out of 11 experimental tumour models, [18F]FMISO PET imaging allowed clear-cut visualisation of the tumours. Inter- and intratumoural heterogeneity of tracer uptake was evident. In addition to average TMRR (tumour-to-muscle retention ratio including all voxels in a volume of interest (VOI)), the parameters TMRR75% and TMRR5 (tumour-to-muscle retention ratio including voxels of 75% or more of the maximum radioactivity in a VOI and the five hottest pixels, respectively) also served as measures for quantifying the heterogeneous [18F]FMISO uptake in the tumours. The variability observed in [18F]FMISO uptake was related neither to tumour size nor to the injected mass of the radiotracer. The pattern of normoxic and hypoxic regions within the human tumour xenografts, however, correlated with glucose metabolism as revealed by comparison of [18F]FDG and [18F]FMISO images.ConclusionThis study demonstrates the feasibility and utility of [18F]FMISO for imaging murine tumour models using NanoPET.

Electrophysiological study, biodistribution in mice, and preliminary PET evaluation in a rhesus monkey of 1-amino-3-[18F]fluoromethyl-5-methyl-adamantane (18F-MEM): a potential radioligand for mapping the NMDA-receptor complex.

The effect of the fluorinated memantine derivative and NMDA receptor antagonist, 1-amino-3-fluoromethyl-5-methyl-adamantane (19F-MEM), at the NMDA receptor ion channel was studied by patch clamp recording. The results showed that 19F-MEM is a moderate NMDA receptor channel blocker. A procedure for the routine preparation of the 18F-labelled analog 18F-MEM has been developed using a two-step reaction sequence. This involves the no-carrier-added nucleophilic radiofluorination of 1-[N-(tert-butyloxy)carbamoyl]-3-(toluenesulfonyloxy)methyl- 5-methyl-adamantane and the subsequent cleavage of the BOC-protecting group using aqueous HCI. The 18F-MEM was obtained in 22 +/- 7% radiochemical yield (decay-corrected to EOB) in a total synthesis time including HPLC purification of 90 min. A biodistribution study after i.v. injection of 18F-MEM in mice showed a fast clearance of radioactivity from blood and relatively high initial uptake in the kidney and in the lung, which gradually decreased with time. The brain uptake was high (up to 3.6% ID/g, 60 min postinjection) with increasing brain-blood ratios: 2.40, 5.10, 6.33, and 9.27 at 5, 30, 60, and 120 min, respectively. The regional accumulation of the radioactivity in the mouse brain was consistent with the known distribution of the PCP recognition site. Preliminary PET evaluation of the radiotracer in a rhesus monkey demonstrated good uptake and prolonged retention in the brain, with a plateau from 35 min onwards p.i. in the NMDA receptor-rich regions (frontal cortex, striata, and temporal cortex). Delineation of the hippocampus, a region known to contain a high density of NMDA receptors, was not possible owing to the resolution of the PET tomograph. The regional brain uptake of 18F-MEM was changed by memantine and by a pharmacological dose of (+)-MK-801, indicating competition for the same binding sites. In a preliminary experiment, haloperidol, a dopamine D2 and sigma receptor antagonist, decreased the binding of 18F-MEM from the brain regions examined, suggesting that binding was also occurring to the sigma recognition sites.

Assessment of the binding properties of Granuloszint

Abstract123I-Granuloszint (a murine monoclonal antibody — called AK-47 — against NCA-95 glycoprotein of granulocytes) has been proved to be a very convenient and successful radiopharmaceutical for visualizing infectious diseases. For a broad introduction in routine nuclear medicine it was necessary to optimize the labelling method and to determine in vitro exactly those biological and binding parameters which are relevant for an effective application in vivo. Binding to granulocytes has been shown to be specific and saturable (non specific binding about 10%) and is not via the Fc part of the antibody. The investigation of the binding properties of 125I-labelled AK-47 gave the following results: affinity constant 5×108 M-1, 20000–200000 epitopes per granulocyte and an immunoreactivity of more than 90%. Labelling with 123I reduced the immunoreactivity to 40%. The Lindmo method and immunoblotting are used as quality control to check the likely in vivo behaviour of the labelled antibody. There is a good correspondence between the results from the two methods. With our special labelling method and the different in vitro tests we have found a reliable way to control the production and to assure an optimal binding behaviour of 123I-Granuloszint.

Positron emission tomography in drug evaluation: Influence of three different catechol-O-methyltransferase inhibitors on metabolism of [NCA] 6-[18F]fluoro-l-dopa in rhesus monkey

We compared the influence of three different catechol-O-methyltransferase (COMT) inhibitors (CGP 28014, OR-611 and Ro 40-7592) on the metabolism of no-carrier-added (NCA) 6-[18F]fluoro-L-dopa (6-FDOPA) in one Rhesus monkey. All three COMT inhibitors improved 6-FDOPA availability in plasma, increased the specific uptake in the brain and thus improved 6-FDOPA uptake measurements using positron emission tomography (PET). Best results were obtained with Ro 40-7592.

Synthesis and preliminary in vitro evaluation of a new memantine derivative 1-amino-3-[18F]fluoromethyl-5-methyl-adamantane: A potential ligand for mapping the N-methyl-D-aspartate receptor complex

The new memantine derivative 1-amino-3-[18F]fluoromethyl-5-methyl-adamantane (18F-MEM) was prepared in a two-step reaction sequence for evaluation as a PET tracer. This involves the no-carrier-added nucleophilic radiofluorination of 1-[N-(tert-butyloxy)-carbamoyl]-3-(toluenesulfonyloxy)methyl-5-methyl-adamantane (3) with K18F/Kryptofix 2.2.2 in DMSO and the subsequent deprotection of the resulting 18F-BOC-MEM by addition of aqueous HCl. 18F-MEM was obtained after purification by reversed phase HPLC in 22±7 % radiochemical yield (decay corrected to EOB) with a radiochemical purity > 99% and a total synthesis time of 100 min. 18F-MEM is stable up to 6 h in aqueous solution at room temperature and revealed appropriate lipophilicity for good diffusion through the blood-brain-barrier. In vitro studies with the non-radioactive analog, 1-amino-3-fluoromethyl-5-methyl-adamantane (19F-MEM) indicated that this compound binds selectively to the phencyclidine (PCP) binding site within the NMDA receptor complex.

Radiosynthesis of [11C]brofaromine, a potential tracer for imaging monoamine oxidase A

Brofaromine (4-5(-methoxy-7-bromobenzofuranyl)-2-piperidine-HCl) is a potent and selective inhibitor of monoamine oxidase (MAO) A. Two methods for its synthesis and a preliminary positron emission tomography (PET) evaluation in monkey brain are described. The first method, at low carrier concentration of CO2, consisted of direct O-methylation of (4-(5-hydroxy-7-bromobenzofuranyl)-2-piperidine). The total radiochemical yield achieved ranged from 30 to 50% (from end of bombardment [EOB] and decay corrected) with an overall synthesis time of 45 min. The second approach, with high carrier amounts of CO2 arising from inherent target problems, was accomplished in a three-step route involving protection of secondary amino functionality, O-methylation and deprotection. The total radiochemical yield was 10% (from EOB and decay corrected) with a total synthesis time of 70 min. For both methods methylation was achieved using the classical methylating agent [11C]CH3I, and radiochemical purity was higher than 98%. PET evaluation of the radioligand in a Rhesus monkey showed a high uptake of radioactivity in the brain. Using the irreversible MAO-A inhibitor clorgyline and reversible MAO-A inhibitors moclobemide and brofaromine, three blockade experiments were designed to determine the extent of specific binding of [11C]brofaromine to MAO-A. No apparent decrease in accumulation of radioactivity in the monkey brain was observed when compared to a baseline scan.

[123I18F] N-(2-aminoethyl)-5-halogeno-2-pyridinecarbox-amides, site specific tracers for MAO-B mapping with SPET and PET☆

Abstract After the successful 123 I-labelling of Ro 43-0463 the new radiopharmaceutical was tested in animals and humans. Both investigations proved the binding of the labelled compound to cerebral MAO-B. SPET investigations resulted in an imaging of the MAO-B distribution in the human brain. This prompted us to develop the labelling of this type of MAO-B inhibitor with 18 F enabling us to transfer our results to PET.

123I-labeling and evaluation of Ro 43–0463, a spet tracer for MAO-B imaging

Using the copper assisted halogen exchange the MAO-B inhibitor Ro 43-0463, N-(2-aminoethyl)-5-iodo-2-pyridinecarboxamide, was labelled with 123I as well as with 125I to allow in vitro and in vivo investigations including SPET with healthy volunteers. Ro 43-0463 is known to inhibit reversibly and specifically MAO-B, having an IC50 of 3 x 10(-8) Mol/L. The labeling in the presence of CuSO4 and ascorbic acid was optimised, varying time (30 to 105 min), precursor concentration (1-3.5 mg) and temperature (130-200 degrees C). The labeling yield ranged between 60 and 70%. Purification was achieved with Lichrosorb RP-18 (5 micron, 250 x 8 mm) and 1.5 mL/min 0.36 M H3PO4/EtOH 97/3 [0.01 M (NH4)2HPO4]. After neutralisation and sterile filtration the final activity concentration ranged between 18.5 and 37 MBq/mL. Biodistribution studies showed a brain to blood ratio greater than 1 within 1 h p.i. The main radiation burden calculated from these animal data is to alimentary and excretory organs and the ovaries. Autoradiography was performed using rat brain slices and 5 nM [125I]Ro 43-0463 in TRIS-buffer pH 7.4 for 90 min at 20 degrees C. Its radioactivity pattern corresponds to the known distribution of MAO-B in the rat brain. By displacement with L-deprneyl the highly specific binding of R0 43-0463 was proven in vitro. SPECT studies with normal volunteers corresponded with the pattern found in autoradiography.

11C-radiolabeling of hallucinogenic psilocin, a potential radioligand for studying the role of serotonin receptors in psychotic symptom formation

The desmethyl compound, 4-hydroxy-N-methyltryptamine (4), was synthesized via a four-step reaction sequence starting from 4-benzyloxyindole (1). Psilocin (4-hydroxy-N,N-dimethyltryptamine), an indole hallucinogen, was labeled by N-monomethylation of the side chain using the classical methylating agent [ 11 C]CH 3 I in 20±5% (decay corrected from [ 11 C]CH 3 I) radiochemical yield. Specific activities obtained ranged from 900-2300Ci/mmol at EOS (end of synthesis). The radiosynthesis, semi-preparative HPLC and formulation were completed in an average time of 45 minutes.