Matthias Scheunemann

Biodistribution and catabolism of 18F-labeled neurotensin(8–13) analogs

4-([(18)F]fluoro)benzoyl-neurotensin(8-13) ((18)FB-Arg(8)-Arg(9)-Pro(10)-Tyr(11)- Ile(12)-Leu(13)-OH, 1) and two analogs stabilized in one and two positions ((18)FB-Arg(8)psi(CH(2)NH)Arg(9)-Pro(10)-Tyr(11)- Ile(12)-Leu(13)-OH, 2, (18)FB-Arg(8)psi(CH(2)NH)Arg(9)-Pro(10)-Tyr(11)-Tle(12)-Leu(13)-OH, 3) were synthesized in a radiochemical yield of 25-36% and a specific activity of 5-15 GBq/mmol. The peptides were evaluated in vitro and in vivo for their potential to image tumors overexpressing neurotensin receptor 1 (NTR1) by positron emission tomography (PET). All analogs exhibited in vitro binding affinity in the low nanomolar range to NTR1-expressing human tumors, measured by quantitative receptor autoradiography, HT-29 and WiDr cells, and to sections of tumors derived from these cell lines in mice. The radiotracers were internalized in the cells in vitro, and the fluorinated peptides were able to mobilize intracellular Ca(2+) of WiDr cells. In in vivo studies in rats and in mice bearing HT-29 cell tumors, only a moderate uptake of the radioligands into the studied tumors was observed, presumed to be due to degradation in vivo and fast elimination by the kidneys. In comparison with the other analogs, the specific tumor uptake expressed as tumor-to-muscle relation was highest for the radioligand 3. The blood clearance of 3 was reduced by co-injection of peptidase inhibitors. The catabolic pathways of the radiofluorinated peptides were elucidated. The results suggest that the high binding affinity to NTR1 and the stabilization against proteolytic degradation are not yet sufficient for tumor imaging by PET.

Technetium(v) and rhenium(v) complexes for 5-HT2A serotonin receptor binding: Structure-affinity considerations☆

Starting from the lead structure of ketanserin, a prototypic serotonin (5-HT) antagonist, new oxotechnetium(V) and oxorhenium(V) complexes were synthesized that are able to compete with [3H]ketan-serin in receptor-binding assays. To imitate organic 5-HT2 receptor ligands, fragments of ketanserin were combined with chelate moieties. Neutral compounds of the general formula [MOL1L2] (M = Tc, Re; L1 = HS-CH2CH2-S-CH2CH2-SH, N-(2-mercaptophenyl)salicylideneimine, N-(2-mercaptoethyl)-salicylideneimine, 3-(2-([N,N-bis(2-mercapto-S-ethyl)]-amino)ethyl)-2,4-(1H, 3H)-quinazolinedione and L2 = HS-R with R = subst. alkyl) were prepared by common action of a Tc(V) or Re(V) precursor with a mixture of equimolar amounts of a tridentate ligand L1 and a monodentate thiolate L2 bearing fragments of the lead structure. Lipophilic complexes consisting of a small S4 thiolate/thioether chelate unit, protonable nitrogen-containing spacer, and simple benzyl moiety significantly inhibited the specific binding of [3H]ketan-serin with IC50 values between 10 and 50 nM.

Structural modification of receptor-binding technetium-99m complexes in order to improve brain uptake

Low brain uptake is a generally accepted problem in developing technetium-99m brain receptor imaging agents. For a class of potential 5-HT2A receptorbinding agents we tried to improve the original low brain uptake of 0.4% injected dose (ID) in rats 5 min p.i. by modifying the lipophilic properties of the molecules. Because of the presence of a protonable nitrogen, which according to the pKa value leads to ionization of the molecule at blood pH, the pKa value was considered to be the parameter most suitable for adjustment of lipophilicity. Insertion of ether-oxygen in the molecule of five candidates lowers the apparent pKa value from 10.0 to 8.3 and dramatically increases the brain uptake to 1.3% ID at 5 min. The direct relationship between brain uptake and apparent pKa cannot be simply explained by the increase in the pKa-governed proportion of the neutral species.

No carrier added preparations of ‘3 + 1’ mixed-ligand 99mTc complexes

Abstract The no carrier added (n.c.a.) preparation of potentially receptor-binding ‘3 + 1’ mixed-ligand technetium complexes has not so far been successfully accomplished. This article deals with our results in the preparation of n.c.a. Tc complexes with tridentate S-S-S or S-N-S ligands and a series of bulky monothiolato ligands. It was found that Tc(V) gluconate or Tc(V) ethylene glycolate are suitable precursors for the complex formation. In a two-step procedure consisting of a reaction of the monothiolato ligand with the precursor and subsequent addition of the tridentate ligand, the desired ‘3 + 1’ mixed-ligand complexes are formed with yields of up to 90%. Low ligand concentrations and pH 9–10 promote the formation of the technetium compounds. A comparison of their analytical properties (TLC, HPLC) and biodistribution data of carrier added and no carrier added technetium complexes show the identity of the investigated compounds.

Radiosynthesis and first evaluation in mice of [18F]NS14490 for molecular imaging of α7 nicotinic acetylcholine receptors

[(18)F]NS14490, a new potential radiotracer for neuroimaging of α7 nicotinic acetylcholine receptors (α7 nAChRs), was synthesized and evaluated in vitro and in vivo. Radioligand binding studies using [(3)H]methyllycaconitine and NS14490 as competitor showed a good target affinity (K(i,α7) = 2.5 nM) and a high selectivity towards other nAChRs. Radiosynthesis of [(18)F]NS14490 was performed by two different labelling procedures: a two-step synthesis using a prosthetic group, which led to 7% labelling yield, and the convenient direct nucleophilic substitution of the corresponding tosylate precursor, which resulted in 70% labelling yield. After optimisation of the isolation, purification and formulation process, biodistribution studies were performed in CD-1 mice. The brain uptake of [(18)F]NS14490 was comparably low (0.16% ID g(-1) wet weight at 5 min p.i.). The radiotracer showed a high metabolic stability in plasma and brain. Also, the target specificity was proven by pre-administration of a highly affine α7 ligand providing a rationale basis for further in vivo evaluation.

Neuroimaging of the vesicular acetylcholine transporter by a novel 4-[18F]fluoro-benzoyl derivative of 7-hydroxy-6-(4-phenyl-piperidin-1-yl)-octahydro-benzo[1,4]oxazines

Phenylpiperidinyl-octahydro-benzo[1,4]oxazines represent a new class of conformationally restrained vesamicol analogues. Derived from this morpholine-fused vesamicol structure, a new fluorine-18-labeled 4-fluorobenzoyl derivative ([(18)F]FBMV) was synthesized with an average specific activity of 75 GBq/micromol and a radiochemical purity of 99%. The radiolabeling method included an exchange reaction of a 4-nitro group of the precursor by fluorine-18, a reduction procedure to eliminate excess of the nitro compound, followed by a high-performance liquid chromatography purification. [(18)F]FBMV demonstrates (i) a moderate lipophilic character with a logD(pH7.0) 1.8+/-0.10; (ii) a considerable binding affinity to the vesicular acetylcholine transporter (VAChT) (K(i)=27.5 nM), as determined using PC12 cells transfected with a VAChT cDNA, and a low affinity to sigma(1,2) receptors (K(i) >3000 nM); (iii) a good uptake into the rat and pig brains; (iv) a typical accumulation in the VAChT-containing brain regions; and (v) an approximately 20% reduction in cortical tracer binding after a specific cholinergic lesion using 192IgG-saporin. [(18)F]FBMV exhibits another PET marker within the group of vesamicol derivatives that demonstrates potentials in imaging brain cholinergic deficits, while its usefulness in clinical practice must await further investigation.

A new 18F-labeled fluoroacetylmorpholino derivative of vesamicol for neuroimaging of the vesicular acetylcholine transporter

With the aim of producing selective radiotracers for in vivo imaging of the vesicular acetylcholine transporter (VAChT) using positron mission tomography (PET), here, we report synthesis and analysis of a new class of conformationally constrained vesamicol analogues with moderate lipophilicity. The sequential ring opening on trans-1,4-cyclohexadiene dioxide enabled an approach to synthesize 6-arylpiperidino-octahydrobenzo[1,4]oxazine-7-ols [morpholino vesamicols]. The radiosynthesis of the [18F]fluoroacetyl-substituted derivative ([18F]FAMV) was achieved starting from a corresponding bromo precursor [2-Bromo-1-[7-hydroxy-6-(4-phenyl-piperidin-1-yl)-octahydro-benzo[1,4]oxazin-4-yl]-ethanone] and using a modified commercial computer-controlled module system with a radiochemical yield of 27+/-4%, a high radiochemical purity (99%) and a specific activity of 35 GBq/micromol. In competitive binding assays using a PC12 cell line overexpressing VAChT and [3H]-(-) vesamicol, 2-fluoro-1-[7-hydroxy-6-(4-phenyl-piperidin-1-yl)-octahydro-benzo[1,4]oxazin-4-yl]-ethanone (FAMV) demonstrated a high selectivity for binding to VAChT (K(i): 39.9+/-5.9 nM) when compared to its binding to sigma 1/2 receptors (Ki>1500 nM). The compound showed a moderate lipophilicity (logD (pH 7)=1.9) and a plasma protein binding of 49%. The brain uptake of [18F]FAMV was about 0.1% injected dose per gram at 5 min after injection and decreased continuously with time. Notably, an increasing accumulation of radioactivity in the lateral brain ventricles was observed. After 1 h, the accumulation of [18F]FAMV, expressed as ratio to the cerebellum, was 4.5 for the striatum, 2.0 for the cortical and 1.5 for the hippocampal regions, measured on brain slices using ex vivo autoradiography. At the present time, 75% of [18F]FAMV in the plasma was shown to be metabolized to various hydrophilic compounds, as detected by high-performance liquid chromatography. The degradation of [18F]FAMV was also detected in brain extracts as early as 15 min post injection (p.i.) and increased to 50% at 1 h postinjection. In conclusion, although the chemical properties of [18F]FAMV and the selectivity of binding to VAChT appear to be promising indicators of a useful PET tracer for imaging VAChT, a low brain extraction, in combination with only moderate specific accumulation in cholinergic brain regions and an insufficient in vivo stability prevents the application of this compound for neuroimaging in humans.

A 18F‐Labeled Fluorobutyl‐Substituted Spirocyclic Piperidine Derivative as a Selective Radioligand for PET Imaging of Sigma1 Receptors

In this study, we synthesized and evaluated a new spirocyclic piperidine derivative 3, containing a 4‐fluorobutyl side chain, as a PET radioligand for neuroimaging of σ1 receptors. In vitro, compound 3 displayed high affinity for σ1 receptors (Ki=1.2 nM) as well as high selectivity. [18F]3 radiosynthesis was performed from the corresponding tosylate precursor, with high radiochemical yield (45–51 %), purity (>98 %), and specific activity (>201 GBq μmol−1). Metabolic stability of [18F]3 in the brain of CD‐1 mice was verified, and no penetration of peripheral radiometabolites into the cerebral tissue was observed. Results of ex vivo autoradiography revealed that the distribution of [18F]3 in the brain corresponded to regions with high σ1 receptor density. The highest region‐specific total‐to‐nonspecific ratio was determined in the facial nucleus (4.00). Biodistribution studies indicated rapid and high levels in brain uptake of [18F]3 (2.2 % ID per gram at 5 min p.i.). Pre‐administration of haloperidol significantly inhibited [18F]3 uptake into the brain and σ1 receptor‐expressing organs, further confirming in vivo target specificity.

Radiosynthesis and Radiotracer Properties of a 7-(2-[18F]Fluoroethoxy)-6-methoxypyrrolidinylquinazoline for Imaging of Phosphodiesterase 10A with PET

Phosphodiesterase 10A (PDE10A) is a key enzyme of intracellular signal transduction which is involved in the regulation of neurotransmission. The molecular imaging of PDE10A by PET is expected to allow a better understanding of physiological and pathological processes related to PDE10A expression and function in the brain. The aim of this study was to develop a new 18F-labeled PDE10A ligand based on a 6,7-dimethoxy-4-pyrrolidinylquinazoline and to evaluate its properties in biodistribution studies. Nucleophilic substitution of the 7-tosyloxy-analogue led to the 7-[18F]fluoroethoxy-derivative [18F]IV with radiochemical yields of 25% ± 9% (n = 9), high radiochemical purity of ≥99% and specific activities of 110–1,100 GBq/μmol. [18F]IV showed moderate PDE10A affinity (KD,PDE10A = 14 nM) and high metabolic stability in the brain of female CD-1 mice, wherein the radioligand entered rapidly with a peak uptake of 2.3% ID/g in striatum at 5 min p.i. However, ex vivo autoradiographic and in vivo blocking studies revealed no target specific accumulation and demonstrated [18F]IV to be inapplicable for imaging PDE10A with PET.

Assessment of the in vitro and in vivo properties of a 99mTc-labeled inhibitor of the multidrug resistant gene product P-glycoprotein

Overexpression of P-glycoprotein (Pgp), which is present in the plasma membrane of various tumor cells and in several normal cell types, contributes to the multidrug resistance (MDR) phenotype of many human cancers. As a prerequisite for therapy, the expression of Pgp must be studied. The available clinical radiopharmaceuticals for studying the expression of Pgp include the lipophilic (99m)Tc cations (sestamibi, tetrofosmin) as well as [(99m)Tc]Q57, [(99m)Tc]Q58, and [(99m)Tc]Q63. Here we describe the in vitro and in vivo properties of the structurally different complex (3-thiapentane-1, 5-dithiolato)[[N-(3-phenylpropyl)-N-2(3-quinazoline-2, 4-dionyl)-ethyl]amino-ethylthiolato¿ oxotechnetium(V) ((99/99m)Tc1) as a potential inhibitor of Pgp. (99)Tc1 enhances the net cell accumulation of Pgp substrates [(3)H]vinblastine, [(3)H]vincristine, [(3)H]colchicine, [(99m)Tc]sestamibi, and [(99m)Tc]tetrofosmin in rat brain endothelial cells (RBE4), an immortalized endothelial cell line that expresses Pgp. In addition, the cell accumulation of (99m)Tc1 could be increased by verapamil and reserpine, which are known Pgp inhibitors. A multitracer approach was used to study the side effects of (99)Tc1 on cell metabolism. The cells were simultaneously incubated with [(99m)Tc]sestamibi, 2-[(18)F]fluoro-2-deoxyglucose ([(18)F]FDG), and various (3)H-labeled tracers. Two-dimensional scatter plots of [(99m)Tc]sestamibi uptake/[(18)F]FDG uptake show typical changes of known Pgp inhibitors including (99)Tc1. The effects of (99)Tc1 on the in vivo distribution of [(99m)Tc]sestamibi and [(18)F]FDG in rats also are comparable with the effects of verapamil, an established Pgp inhibitor and calcium channel blocker. We conclude that (99/99m)Tc1 is a transport substrate and a potential inhibitor of Pgp. Our approach may be useful in the design of further radiotracers with specificity to Pgp.