Carsten Hocke

Labeling and Glycosylation of Peptides Using Click Chemistry: A General Approach to 18F-Glycopeptides as Effective Imaging Probes for Positron Emission Tomography

In the field of molecular imaging, positron emission tomography (PET) has emerged as an imaging modality with excellent sensitivity for in vivo studies. PET labeling is challenging since short-lived positron-emitting isotopes such as F and C are used as labeling agents. The optimization and efficient application of rapid and reliable labeling strategies are prerequisites for obtaining access to new radiopharmaceuticals for both research and clinical trials. Bioactive peptides that specifically address molecular targets in vivo represent an important class of PET tracers to facilitate predictive imaging and PET-guided therapy. Diverse strategies for the synthesis of peptide-based radiopharmaceuticals using F-labeled prosthetic groups have been elaborated, including chemoselective oxime conjugation and the use of F-labeled maleimide derivatives as cysteinereactive reagents. Following the concept of click chemistry introduced by Sharpless et al., the Huisgen [3+2] azide– alkyne cycloaddition has been adapted to F-radiosynthetic methods in order to take advantage of its selectivity, reliability, and speed under aqueous mild Cupromoted reaction conditions. The versatility of peptide imaging agents is frequently hampered by their instability in vivo because of rapid degradation by endogenous peptidases. As an example, the synthesis of radiolabeled peptide-based imaging agents for the neurotensin receptor-1 (NTR-1), which is overexpressed in a number of human cancers, requires modifications to improve the metabolic stability. Synthetic approaches to RGD tracers targeting avb3 integrin, which plays a key role in angiogenesis, capitalize on the pioneering studies by Kessler et al. , who successfully developed cyclic pentameric RGD peptides that selectively recognize integrin avb3. [9] Various radiolabeled cyclic RGD peptides have been described. Among these, [F]galactoRGD has been extensively evaluated in clinical studies. Since glycosylation of peptides is known to frequently improve the biokinetic and in vivo clearance properties, [F]galacto-RGD and further radiopeptides have been approached. 13] However, the multistep radiosynthesis of [F]galacto-RGD is time-consuming and laborious. In proposals to overcome this drawback, F-labeling by 2-deoxy-2[F]fluoroglucose (FDG) has been discussed. 15] The major disadvantages of the F-peptide-labeling strategies currently used are 1) harsh reaction conditions, 2) laborious multiple-step syntheses with a limited decayuncorrected radiochemical yield (RCY), which would complicate the automation for large-scale production, and 3) lipophilic derivatization, which impair the biokinetic properties of the tracer. Based on our previous work on click chemistry in drug discovery and the synthesis of b-mannosyl azides, we herein present an efficient strategy toward F-labeling with concomitant glycosylation for the synthesis of F-glycopeptides as imaging agents for PET. We combined this strategy with the development of a metabolically stable glycopeptoid analogue of NT(8–13), which is the highly potent C-terminal hexapeptide of the natural agonist neurotensin (NT). As a proof of concept, two F-glycopeptides derived from NT(8– 13) and c(RGDfPra), respectively, were applied to biodistribution studies and mPET for imaging NTR and avb3-integrin expression in vivo using xenograft nude mice models. In detail, 2-deoxy-2-fluoroglucosyl azide (3) could be obtained starting from tetraacetylated 2-deoxy-2-fluoroglucose. The glucosyl azide 3 was applied for the Cu-catalyzed azide–alkyne coupling with a series of alkyne-functionalized peptides to evaluate the influence of the appended glycosyl residue on receptor recognition. Commercially available propargylglycine (Pra) was introduced by solid-phase-supported synthesis at position X into the sequence of the bioactive peptide c(RGDfX) and at the N terminus of NT(8– 13) and metabolically stabilized derivatives thereof. Considering our studies on the influence of peptide backbone modifications and ligand conformation on affinity changes for a series of NT(8–13) analogues, metabolic stabilization was envisioned by alteration of three amino acids in the sequence [*] Dr. S. Maschauer, Dr. C. Hocke, Prof. T. Kuwert, Dr. O. Prante Nuklearmedizinische Klinik, Labor f r Molekulare Bildgebung Friedrich-Alexander-Universit t Erlangen-N rnberg Schwabachanlage 6, 91054 Erlangen (Germany) Fax: (+ 49)9131-853-4325 E-mail: olaf.prante@uk-erlangen.de

NEMA NU2-2001 guided performance evaluation of four Siemens ECAT PET scanners

The NEMA NU 2-2001 protocol has been especially designed for the performance evaluation of whole-body positron emission tomography (PET) measurements with scanners acquiring data in three-dimensional (3-D) mode without septa or with septa retracted. We report on the comparative NEMA NU 2-2001 performance tests on four different Siemens/CTI ECAT PET scanners, the two bismuth germanate oxide scanners HR+ and EXACT and the two lutetium oxyorthosilicate scanners ACCEL and EMERGE. All scanners were operated in 3-D mode only. Until now, no complete NEMA NU 2-2001 evaluations have been reported for the HR+ and EXACT. For the ACCEL and the EMERGE, these are the first published performance characteristics as far as we know. The HR+ showed a superior spatial resolution, whereas the ACCEL had the best countrate [noise equivalent count (NEC)] performance above a radioactivity concentration of 2 kBq/cc. The sensitivity of the full-ring scanners did not differ much with numbers between 5982 and 7180 cps/MBq. The sensitivity of the partial-ring scanner EMERGE was 2279 cps/MBq. Although the EMERGE yields lower NEC rates than the EXACT for radioactivity concentrations less than 10 kBq/cc, it is favorable beyond this cross over point. The scatter fraction of all four scanners was about 48%. The lesions to be examined by the image quality test could be seen best with the ACCEL. The NEMA NU 2-2001 performance tests proved to be a valuable guideline for this instrumental comparison.

Systematic Evaluation of Phantom Fluids for Simultaneous PET/MR Hybrid Imaging

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 18F-FDG. As an alternative to 18F-FDG, a modified PET tracer (18F-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 18F-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 18F-FDG was preferred, triethylene glycol provided the best compromise (flip angle increase by a factor of 1.13). The potential alternative tracer, 18F-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 18F-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.

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 19F‐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 Ki values between 0.12 and 0.69 nM at the dopamine D3 subtype. The benzothiophene‐substituted carboxamide 4 a (Ki=0.12 nM) displayed 133 and 283‐fold selectivity over the structurally related D2Long and D4 subtypes, respectively. Mitogenesis assays showed the behavior of partial agonists. Based on these data, we synthesized the [18F]fluoroethoxy‐substituted radioligands [18F]4 a–d. The N‐[4‐[4‐(2‐hydroxyphenyl)piperazin‐1‐yl]butyl]‐2‐carboxamides 3 a–d were prepared and labeled with 2‐[18F]fluoroethyltosylate in a two‐step procedure. Optimization of the 18F‐labeling conditions led to radiochemical yields between 24 and 65 %.

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.

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 (Ki=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 NO2 by 18F led to radiochemical yields of 66–92 % for [18F]9 a–d. However, the specific activities of [18F]9 b and [18F]9 d were insufficient, rendering their use for in vivo studies impossible. Biodistribution studies of [18F]9 a and [18F]9 c using rat brain autoradiography revealed accumulation in the ventricles, thus indicating insufficient biokinetic properties of [18F]9 a and [18F]9 c for D3 receptor imaging in vivo.

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 [18F]1, [18F]2a and [18F]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 [18F]1 and [18F]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 [18F]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 [18F]2a and [18F]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 18F-labeled dopamine D3 receptor ligand using [18F]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.

Tissue distribution of radioiodinated FAUC113 Assessment of a pyrazolo(1,5- a ) pyridine based dopamine D4 receptor radioligand candidate

AIM Disturbances of the D4 receptor subtype have been implicated in the genesis of a broad range of psychiatric disorders. In order to assess the suitability of a radioiodinated analogue of the D4-selective ligand FAUC 113 for tracer studies in vivo, we investigated the in-vivo stability, biodistribution and brain-uptake of 7-(131)I-FAUC 113 in Sprague-Dawley rats. METHODS Radiolabelling was carried out with high radiochemical yield and specific activity. After intravenous injection, blood and tissue samples, taken at designated time intervals, were collected for analysis. Analyses of metabolites were performed by radio-hplc and radio-tlc. For in-vivo evaluation, sagittal cryo-sections of the rat brain were investigated by in-vitro and ex-vivo autoradiography on a mu-Imager system. RESULTS 7-(131)I-FAUC 113 was rapidly cleared from blood. Highest uptake was observed in kidney (0.603 +/- 0.047% ID/g, n = 4) and liver (0.357 +/- 0.070% ID/g, n = 4) at 10 min p.i.; 7-(131)I-FAUC 113 displayed rapid uptake (0.21-0.26% ID/g) and fast clearance in various brain regions consistent with the determined logP-value of 2.36 +/- 0.15 (n = 4). In-vivo stability of 7-(131)I-FAUC 113 was confirmed in the frontal cortex (>95%). Ex-vivo autoradiography revealed a frontal cortex-to-cerebellum ratio of 1.57 +/- 0.13 at 10 min p.i. (n = 6). Coinjection with L-750667 could not suppress any putative specific binding of 7-(131)I-FAUC 113. In-vitro autoradiography using authentic 7-iodo-FAUC 113 or L-750667 failed to cause significant displacement of the radioligand. CONCLUSIONS Radioiodinated FAUC 113 does not allow imaging of D4 receptors in the rat brain in vivo nor in vitro. Further work should aim at the development of selective dopamine D4 radioligands with improved tracer characteristics, such as receptor affinity and subtype selectivity, specific activity or blood-brain barrier permeability.