Development of molecular tools based on the dopamine D3 receptor ligand FAUC 329 showing inhibiting effects on drug and food maintained behavior.

Dopamine D3 receptor-mediated networks have been associated with a wide range of neuropsychiatric diseases, drug addiction and food maintained behavior, which makes D3 a highly promising biological target. The previously described dopamine D3 receptor ligand FAUC 329 (1) showed protective effects against dopamine depletion in a MPTP mouse model of Parkinson's disease. We used the radioligand [18F]2, a [18F]fluoroethoxy substituted analog of the lead compound 1 as a molecular tool for visualization of D3-rich brain regions including the islands of Calleja. Furthermore, structural modifications are reported leading to the pyrimidylpiperazine derivatives 3 and 9 displaying superior subtype selectivity and preference over serotonergic receptors. Evaluation of the lead compound 1 on cocaine-seeking behavior in non-human primates showed a substantial reduction in cocaine self-administration behavior and food intake.

In Vitro and In Vivo Characterization of Selected Fluorine-18 Labeled Radioligands for PET Imaging of the Dopamine D3 Receptor.

Cerebral dopamine D3 receptors seem to play a key role in the control of drug-seeking behavior. The imaging of their regional density with positron emission tomography (PET) could thus help in the exploration of the molecular basis of drug addiction. A fluorine-18 labeled D3 subtype selective radioligand would be beneficial for this purpose; however, as yet, there is no such tracer available. The three candidates [(18)F]1, [(18)F]2a and [(18)F]2b were chosen for in vitro and in vivo characterization as radioligands suitable for selective PET imaging of the D3 receptor. Their evaluation included the analysis of radiometabolites and the assessment of non-specific binding by in vitro rat brain autoradiography. While [(18)F]1 and [(18)F]2a revealed high non-specific uptake in in vitro rat brain autoradiography, the D3 receptor density was successfully determined on rat brain sections (n = 4) with the candidate [(18)F]2b offering a Bmax of 20.38 ± 2.67 pmol/g for the islands of Calleja, 19.54 ± 1.85 pmol/g for the nucleus accumbens and 16.58 ± 1.63 pmol/g for the caudate putamen. In PET imaging studies, the carboxamide 1 revealed low signal/background ratios in the rat brain and relatively low uptake in the pituitary gland, while the azocarboxamides [(18)F]2a and [(18)F]2b showed binding that was blockable by the D3 receptor ligand BP897 in the ventricular system and the pituitary gland in PET imaging studies in living rats.

Optimization and synthesis of an (18) F-labeled dopamine D3 receptor ligand using [(18) F]fluorophenylazocarboxylic tert-butylester.

There is still no efficient fluorine-18-labeled dopamine D3 subtype selective receptor ligand for studies with positron emission tomography. We aim at improving the D3 selectivity and hydrophilicity of a candidate ligand by changing the substitution pattern to a 2,3-dichlorophenylpiperazine and hydroxylation of the butyl chain. The compound [(18) F]3 exhibited D3 affinity of Ki = 3.6 nM, increased subtype selectivity (Ki (D2 /D3 ) = 60), and low affinity to 5-HT1A and α1 receptors (Ki (5-HT1A /D3 ) = 34; Ki (α1 /D3 ) = 100). The two-step radiosynthesis was optimized for analog [(18) F]4 by reducing the necessary concentration of the precursor amine (57 mM), which reacted with [(18) F]fluorophenylazocarboxylic tert-butylester under basic conditions. The optimization of the base (Cs 2 CO3 , 23 mM) and the adjustment of reaction temperature led to the radiochemical yield of 63% after 5 min at 35°C. The optimized reaction conditions were transferred on to the synthesis of [(18) F]3 with an overall non-decay corrected yield of 8-12% in a specific activity of 32-102 GBq/µmol after a total synthesis time of 30-35 min. This provides a D 3 radioligand candidate with improved attributes concerning selectivity and radiosynthesis for further preclinical studies.

Synthesis and evaluation of fluoro substituted pyridinylcarboxamides and their phenylazo analogues for potential dopamine D3 receptor PET imaging.

A series of fluoro substituted pyridinylcarboxamides and their phenylazo analogues with high affinity and selectivity for the dopamine D3 receptor was synthesized by the use of 6-fluoropyridine-3-carbonyl chloride (1) and fluorophenylazocarboxylic ester (2). Several of these compounds (9a-e and 10a-h) have been evaluated in vitro, among which 9b, 10a, 10c and 10d proved to have at least single-digit nanomolar affinity for D3. They also exhibit considerable selectivity over the other dopamine receptor subtypes and noteworthy selectivity over the structurally related serotonin receptor subtypes 5-HT(1A) and 5-HT2, offering potential radiotracers for positron emission tomography.

Biodistribution studies of two 18F-labeled pyridinylphenyl amides as subtype selective radioligands for the dopamine D3 receptor.

INTRODUCTION: Dopamine D3 receptors are implicated in various neuropsychiatric diseases, drug abuse and alcoholism, but specific agents for D3 molecular imaging are lacking. We evaluated two in vitro selective fluorine-18-labeled radioligand candidates ([(18)F]5 and [(18)F]6) for positron emission tomography (PET) imaging of D3 receptor availability in the brain. METHODS: Biodistribution was evaluated in Sprague-Dawley rats using ex vivo autoradiography and small-animal PET. Protein binding studies were conducted in human plasma and cerebrospinal fluid. RESULTS: [(18)F]5 showed rapid blood-brain barrier penetration and fast washout after intravenous injection, whereas the rat brain penetration of [(18)F]6 was lower. The total distribution volume (VT) of [(18)F]5 was 20-26 mL g(-1) throughout brain. Co-injection with the D3 antagonist BP897 resulted in globally increased cerebral washout of [(18)F]5 and [(18)F]6, but SUV analysis and parametric mapping of binding potential (BPND) relative to the cerebellum did not reveal specific binding of either ligand in D3-rich brain regions, i.e. the ventral striatum. However, there was substantial displaceable binding of [(18)F]5, and to a lesser extent [(18)F]6, in the pituitary. CONCLUSION: These radioligands reveal dopamine D3 receptors in the pituitary, but are not suitable for PET imaging of in brain, possibly due to low specific signal relative to the globally high VT.

Systematic evaluation of phantom fluids for simultaneous PET/MR hybrid imaging.

UNLABELLED: With the recent advent of integrated PET/MR hybrid systems, the need for simultaneous PET and MR phantom measurements arises. Phantom fluids that are used in stand-alone MR systems, especially in larger phantoms and at a high magnetic field strength, are not necessarily applicable in PET imaging and vice versa. In this study, different approaches to fluid selection were considered and systematically evaluated with respect to their usability for simultaneous PET/MR phantom imaging. METHODS: Demineralized water, water with increased electrical conductivity, a water-oil emulsion, and monoethylene and triethylene glycol were investigated in MR and PET measurements using the most common PET tracer (18)F-FDG. As an alternative to (18)F-FDG, a modified PET tracer ((18)F-fluoride Kryptofix 222 complex) was investigated toward its ability to dissolve in pure oil, which provides good signal homogeneity in MR imaging. Measurements were performed on a 3.0 T integrated PET/MR whole-body system using a National Electrical Manufacturers Association quality-standard phantom. RESULTS: All tested fluids dissolved the radiotracer (18)F-FDG homogeneously. Regarding their suitability for MR at 3.0 T, all fluids significantly improved the homogeneity compared to pure water (increase of excitation flip angle within the tested phantom by a factor of 2.0). When the use of (18)F-FDG was preferred, triethylene glycol provided the best compromise (flip angle increase by a factor of 1.13). The potential alternative tracer, (18)F-fluoride Kryptofix 222 complex, dissolved in pure oil; however, it is not optimal in its tested composition because it accumulates at the bottom of the phantom during the time of measurement. CONCLUSION: This study provides a systematic approach toward phantom fluid selection for imaging a given quality-standard body phantom--and phantoms of comparable size--at 3.0 T. For simultaneous PET/MR scans using the standard tracer (18)F-FDG, an alternative fluid to water and oil is proposed that serves as a viable option for both imaging modalities. Nevertheless, when water is preferred, ways to improve MR image homogeneity are presented. The tested alternative PET tracer enables the use of pure oil in combined scans, but the tracer composition needs to be optimized for phantom measurement applications.

A series of 18F-labelled pyridinylphenyl amides as subtype-selective radioligands for the dopamine D3 receptor.

Synthesis, biological activity, and structure-selectivity relationship (SSR) studies of a novel series of potential dopamine D3 receptor radioligands as imaging agents for positron emission tomography (PET) are reported. Considering a structurally diverse library of D3 ligands, SSR studies were performed for a new series of fluorinated pyridinylphenyl amides using CoMFA and CoMSIA methods. The in vitro D3 affinities of the predicted series of biphenyl amide ligands 9 a-d revealed single-digit to sub-nanomolar potencies (K(i)=0.52-1.6 nM), displaying excellent D3 selectivity over the D2 subtype of 110- to 210-fold for the test compounds 9 a-c. Radiofluorination by nucleophilic substitution of Br or NO(2) by (18)F led to radiochemical yields of 66-92 % for [(18)F]9 a-d. However, the specific activities of [(18)F]9 b and [(18)F]9 d were insufficient, rendering their use for in vivo studies impossible. Biodistribution studies of [(18)F]9 a and [(18)F]9 c using rat brain autoradiography revealed accumulation in the ventricles, thus indicating insufficient biokinetic properties of [(18)F]9 a and [(18)F]9 c for D3 receptor imaging in vivo.

Synthesis of a (68)ga-labeled peptoid-Peptide hybrid for imaging of neurotensin receptor expression in vivo.

The neurotensin receptor subtype 1 (NTS1) represents an attractive molecular target for imaging various tumors. Positron emission tomography (PET) gained widespread importance due to its sensitivity. We combined the design of a metabolically stable neurotensin analogue with a (68)Ga-radiolabeling approach. The (68)Ga-labeled peptoid-peptide hybrid [(68)Ga]3 revealed high stability, specific tumor uptake (0.7%ID/g, 65 min p.i.), and advantageous biokinetics in vivo using HT29 tumor-bearing nude mice. Because of the ability to internalize into NTS1-expressing tumor cells, [(68)Ga]3 proved to be highly suitable for a reliable and practical visualization of NTS1-expressing tumors in vivo by small animal PET.

Synthesis, radiofluorination, and in vitro evaluation of pyrazolo[1,5-a]pyridine-based dopamine D4 receptor ligands: discovery of an inverse agonist radioligand for PET.

A series of fluoro-substituted analogs structurally derived from the aminomethyl-substituted pyrazolo[1,5- a]pyridine lead compounds 9 (FAUC 113) and 10 (FAUC 213) were synthesized and evaluated as high-affinity D 4 receptor (D 4R) ligands ( 3a- 3h, K i = 1.3-28 nM). The para-fluoroethoxy-substituted derivatives 3f and 3h revealed an outstanding D 4 subtype selectivity of more than 3 orders of magnitude over both congeners D 2 and D 3 combined with inverse agonism at D 4R. The corresponding (18)F-labeled radioligands revealed high serum stability in vitro and log P values of 2-3. In vitro rat brain autoradiography showed specific binding of [ (18)F]3h in distinct brain regions, including the gyrus dentate of the hippocampus, that were inhibited by both eticlopride (65-80%) and the selective D 4R antagonist 10 (78-93%). The observed binding pattern was mainly consistent with the known D 4R distribution in the rat brain. Thus, [(18)F]3h (FAUC F41) represents a potential radioligand for studying the D 4R in vivo by positron emission tomography (PET).

18F-Labeled FAUC 346 and BP 897 derivatives as subtype-selective potential PET radioligands for the dopamine D3 receptor.

Disturbances of neutrotransmission at the dopamine D3 receptor are related to several neuropsychiatric diseases and in particular to drug addiction. Herein, we report the computer-assisted prediction of D3 selectivities of new fluoroalkoxy-substituted receptor ligands by means of 3D-QSAR analysis. As close analogues of the D3-selective lead compound FAUC 346 and BP 879, the (19)F-substituted test compounds 4 a-d were synthesized and evaluated. In vitro investigation of their binding characteristics in transfected Chinese Hamster Ovary (CHO) cells led to excellent K(i) values between 0.12 and 0.69 nM at the dopamine D3 subtype. The benzothiophene-substituted carboxamide 4 a (K(i)=0.12 nM) displayed 133 and 283-fold selectivity over the structurally related D2(Long) and D4 subtypes, respectively. Mitogenesis assays showed the behavior of partial agonists. Based on these data, we synthesized the [(18)F]fluoroethoxy-substituted radioligands [(18)F]4 a-d. The N-[4-[4-(2-hydroxyphenyl)piperazin-1-yl]butyl]-2-carboxamides 3 a-d were prepared and labeled with 2-[(18)F]fluoroethyltosylate in a two-step procedure. Optimization of the (18)F-labeling conditions led to radiochemical yields between 24 and 65 %.

Nucleophilic 18F-fluorination of heteroaromatic iodonium salts with no-carrier-added [18F]fluoride.

Diaryliodonium salts containing the 2-thienyl group as an example of an electron-rich heteroaromatic moiety proved to be very potent precursors for the nucleophilic, regioselective no-carrier-added (nca) radiofluorination of various arenes. It even allowed the nucleophilic introduction of nca [18F]fluoride into electron-rich arene compounds in one step. The influences of the substitution pattern, of counteranions, and of different reaction conditions were studied. Effects of counterions could be explained by the influence of solvent on ion pair separation of precursor salts. Different aryl(2-thienyl)iodonium salts were used as precursors, where the homoaromatic group systematically varied from bearing electron-deficient to electron-rich substituents. Relative rates of exchange kinetics correlated linearly with Hammett constants of the appropriate substituents confirming a nucleophilic aromatic substitution reaction of high reactivity and low selectivity.

Structure-selectivity investigations of D2-like receptor ligands by CoMFA and CoMSIA guiding the discovery of D3 selective PET radioligands.

Elucidation of the physiological role of the D3 receptor and its distribution in the brain using positron emission tomography (PET) is hampered by the lack of bioavailable subtype selective tracer ligands. To develop appropriate D3 radioligands, we designed an integrative procedure involving the elucidation of structural features determining D3 selectivity over both congeners D2 and D4 by comparative molecular analysis. Thus, we have successfully generated CoMFA and CoMSIA models based on the affinitiy differences of a series of 79 ligands representing a broad range of selectivities. These models yielded highly significant cross-validations (q2cv(D3/D2) = 0.86; q2cv(D3/D4) = 0.92) and excellent predictions of a 16-ligand test set (r2pred = 0.79-0.93). Exploiting this information, synthesis and receptor binding studies directed us to the fluorinated lead compounds 78 and 79, featuring subnanomolar D3 affinities and considerable selectivities over D2 and D4 and, subsequently, to the subtype selective PET tracers [18F]78 and [18F]79.

Synthesis and evaluation of 18F-labeled dopamine D3 receptor ligands as potential PET imaging agents.

A series of fluoro substituted aryl carboxamides was synthesized revealing high affinity for the dopamine D3 receptor. In contrast to 2-methoxy substitution, a 2,3-dichloro substitution pattern at the phenylpiperazine moiety induces a 10-fold increase of D3 affinity which is expressed by Ki values of 0.53, 1.1, and 9.0 nM for 8b, 8d, and 8f. Applying aromatic 18F-for-Br(Cl) substitution, high radiochemical yields between 76-82% were obtained for [18F]8c-f. The most promising ligand, [18F]8d, was used as imaging agent of the D3 receptor in vitro. However, due to the lack of specific binding, further studies should aim at the development of radioligands with improved D3 receptor selectivity.

Synthesis and radioiodination of selective ligands for the dopamine D3 receptor subtype.

Starting from FAUC 365, a series of iodine substituted heteroaryl carboxamides has been synthesized revealing high affinity and selectivity for the dopamine D3 receptor. Binding data showed a 15-560-fold selectivity for the dopamine D3 over D2. A 2,3-dichloro substitution pattern on the phenylpiperazine moiety led to the highest subtype selectivity, whereas the 2-methoxy substituted compounds showed superior D3 affinity. Suitable precursors were radioiodinated with high radiochemical yields (53-85%) leading to potential imaging agents for the D3 receptor by SPET.

Characterization of uptake of 3-[(131)I]iodo-alpha-methyl-L-tyrosine in human monocyte-macrophages.

The radiopharmaceutical 3-[(123)I]iodo-alpha-methyl-L-tyrosine ([(123)I]IMT) can be used to study amino acid transport by single-photon emission tomography (SPET). In order to evaluate the potential contribution of [(123)I]IMT accumulation in macrophages to overall uptake values measured in neoplastic lesions in vivo, we studied the mechanisms governing the uptake of this tracer by human monocyte-macrophages (HMMs). HMMs were isolated from healthy human donors by density gradient centrifugation using Ficoll methods. The human glioblastoma cell line U-138 MG (GLIOs) was obtained from American Type Culture Collection. Using multiwell dishes, cells were incubated in phosphate buffered saline or an equivalent sodium-free buffer with 50 kBq [(131)I]IMT per well. [(131)I]IMT uptake was quantified as % injected dose per mass of protein within each culture well. Several natural and artificial amino acids were used as potential transport inhibitors both in sodium-containing and sodium-free medium. [(131)I]IMT uptake was significantly lower in HMMs than in GLIOs (34 +/- 2 %/mg (40 min) vs. 507 +/- 50 %/mg at 30 minutes of incubation, respectively; p < 0.01). Endotoxin (LPS) significantly increased [(131)I]IMT uptake in HMMs by a factor of approximately 2. Transport into non-stimulated HMMs was exclusively sodium-independent and inhibitable by BCH, but not by MeAIB. Under LPS stimulation exclusively, there was in addition also a sodium-dependent inhibition of [(131)I]IMT uptake by L-arginine and MeAIB, albeit to a minor extent. [(131)I]IMT accumulation in HMMs is mainly mediated via an L-like amino acid transport system and increases on HMM activation by LPS. LPS may induce an additional Na(+)-dependent transport system in HMMs. The considerably lower [(131)I]IMT uptake in HMMs than in GLIOs suggests that overall uptake values of this tracer measured by SPET in tumors are not significantly affected by [(123)I]IMT accumulation in macrophages within the neoplastic lesion.