Radioligands for Tropomyosin Receptor Kinase (Trk) Positron Emission Tomography Imaging.

The tropomyosin receptor kinases family (TrkA, TrkB, and TrkC) supports neuronal growth, survival, and differentiation during development, adult life, and aging. TrkA/B/C downregulation is a prominent hallmark of various neurological disorders including Alzheimer's disease (AD). Abnormally expressed or overexpressed full-length or oncogenic fusion TrkA/B/C proteins were shown to drive tumorigenesis in a variety of neurogenic and non-neurogenic human cancers and are currently the focus of intensive clinical research. Neurologic and oncologic studies of the spatiotemporal alterations in TrkA/B/C expression and density and the determination of target engagement of emerging antineoplastic clinical inhibitors in normal and diseased tissue are crucially needed but have remained largely unexplored due to the lack of suitable non-invasive probes. Here, we review the recent development of carbon-11- and fluorine-18-labeled positron emission tomography (PET) radioligands based on specifically designed small molecule kinase catalytic domain-binding inhibitors of TrkA/B/C. Basic developments in medicinal chemistry, radiolabeling and translational PET imaging in multiple species including humans are highlighted.

Small Prosthetic Groups in 18F-Radiochemistry: Useful Auxiliaries for the Design of 18F-PET Tracers.

Prosthetic group (PG) applications in 18F-radiochemistry play a pivotal role among current 18F-labeling techniques for the development and availability of 18F-labeled imaging probes for PET (Wahl, 2002) (1). The introduction and popularization of PGs in the mid-80s by pioneers in 18F-radiochemistry has profoundly changed the landscape of available tracers for PET and has led to a multitude of new imaging agents based on simple and efficiently synthesized PGs. Because of the chemical nature of anionic 18F- (apart from electrophilic low specific activity 18F-fluorine), radiochemistry before the introduction of PGs was limited to simple nucleophilic substitutions of leaving group containing precursor molecules. These precursors were not always available, and some target compounds were either hard to synthesize or not obtainable at all. Even with the advent of recently introduced "late-stage fluorination" techniques for the 18F-fluorination of deactivated aromatic systems, PGs will continue to play a central role in 18F-radiochemistry because of their robust and almost universal usability. The importance of PGs in radiochemistry is shown by its current significance in tracer development and exemplified by an overview of selected methodologies for PG attachment to PET tracer molecules. Especially, click-chemistry approaches to PG conjugation, while furthering the historical evolution of PGs in PET tracer design, play a most influential role in modern PG utilization. All earlier and recent multifaceted approaches in PG development have significantly enriched the contingent of modern 18F-radiochemistry procedures and will continue to do so.

Imaging in vivo glutamate fluctuations with [(11)C]ABP688: a GLT-1 challenge with ceftriaxone.

Molecular imaging offers unprecedented opportunities for investigating dynamic changes underlying neuropsychiatric conditions. Here, we evaluated whether [(11)C]ABP688, a positron emission tomography (PET) ligand that binds to the allosteric site of the metabotropic glutamate receptor type 5 (mGluR5), is sensitive to glutamate fluctuations after a pharmacological challenge. For this, we used ceftriaxone (CEF) administration in rats, an activator of the GLT-1 transporter (EAAT2), which is known to decrease extracellular levels of glutamate. MicroPET [(11)C]ABP688 dynamic acquisitions were conducted in rats after a venous injection of either saline (baseline) or CEF 200 mg/kg (challenge). Binding potentials (BP(ND)) were obtained using the simplified reference tissue method. Between-condition statistical parametric maps indicating brain regions showing the highest CEF effects guided placement of microdialysis probes for subsequent assessment of extracellular levels of glutamate. The CEF administration increased [(11)C]ABP688 BP(ND) in the thalamic ventral anterior (VA) nucleus bilaterally. Subsequent microdialysis assessment revealed declines in extracellular glutamate concentrations in the VA. The present results support the concept that availability of mGluR5 allosteric binding sites is sensitive to extracellular concentrations of glutamate. This interesting property of mGluR5 allosteric binding sites has potential applications for assessing the role of glutamate in the pathogenesis of neuropsychiatric conditions.

¹8F-labeled silicon-based fluoride acceptors: potential opportunities for novel positron emitting radiopharmaceuticals.

BACKGROUND: Over the recent years, radiopharmaceutical chemistry has experienced a wide variety of innovative pushes towards finding both novel and unconventional radiochemical methods to introduce fluorine-18 into radiotracers for positron emission tomography (PET). These "nonclassical" labeling methodologies based on silicon-, boron-, and aluminium-(18)F chemistry deviate from commonplace bonding of an [(18)F]fluorine atom ((18)F) to either an aliphatic or aromatic carbon atom. One method in particular, the silicon-fluoride-acceptor isotopic exchange (SiFA-IE) approach, invalidates a dogma in radiochemistry that has been widely accepted for many years: the inability to obtain radiopharmaceuticals of high specific activity (SA) via simple IE. METHODOLOGY: The most advantageous feature of IE labeling in general is that labeling precursor and labeled radiotracer are chemically identical, eliminating the need to separate the radiotracer from its precursor. SiFA-IE chemistry proceeds in dipolar aprotic solvents at room temperature and below, entirely avoiding the formation of radioactive side products during the IE. SCOPE OF REVIEW: A great plethora of different SiFA species have been reported in the literature ranging from small prosthetic groups and other compounds of low molecular weight to labeled peptides and most recently affibody molecules. CONCLUSIONS: The literature over the last years (from 2006 to 2014) shows unambiguously that SiFA-IE and other silicon-based fluoride acceptor strategies relying on (18)F(-) leaving group substitutions have the potential to become a valuable addition to radiochemistry.

Cholinergic Depletion in Alzheimer's Disease Shown by [ (18) F]FEOBV Autoradiography.

Rationale. Alzheimer's Disease (AD) is a neurodegenerative condition characterized in part by deficits in cholinergic basalocortical and septohippocampal pathways. [(18)F]Fluoroethoxybenzovesamicol ([(18)F]FEOBV), a Positron Emission Tomography ligand for the vesicular acetylcholine transporter (VAChT), is a potential molecular agent to investigate brain diseases associated with presynaptic cholinergic losses. Purpose. To demonstrate this potential, we carried out an [(18)F]FEOBV autoradiography study to compare postmortem brain tissues from AD patients to those of age-matched controls. Methods. [(18)F]FEOBV autoradiography binding, defined as the ratio between regional grey and white matter, was estimated in the hippocampus (13 controls, 8 AD) and prefrontal cortex (13 controls, 11 AD). Results. [(18)F]FEOBV binding was decreased by 33% in prefrontal cortex, 25% in CA3, and 20% in CA1. No changes were detected in the dentate gyrus of the hippocampus, possibly because of sprouting or upregulation toward the resilient glutamatergic neurons of the dentate gyrus. Conclusion. This is the first demonstration of [(18)F]FEOBV focal binding changes in cholinergic projections to the cortex and hippocampus in AD. Such cholinergic synaptic (and more specifically VAChT) alterations, in line with the selective basalocortical and septohippocampal cholinergic losses documented in AD, indicate that [(18)F]FEOBV is indeed a promising ligand to explore cholinergic abnormalities in vivo.

Concordance between in vivo and postmortem measurements of cholinergic denervation in rats using PET with [18F]FEOBV and choline acetyltransferase immunochemistry.

BACKGROUND: Fluorine-18 fluoroethoxybenzovesamicol ([18F]FEOBV) is a radioligand for the selective imaging of the vesicular acetylcholine transporter with positron emission tomography (PET). The current study demonstrates that pathological cortical cholinergic deafferentation can be quantified in vivo with [18F]FEOBV PET, yielding analogous results to postmortem histological techniques. METHODS: Fifteen male rats (3 months old) underwent a cerebral infusion of 192 IgG-saporin at the level of the nucleus basalis magnocellularis. They were scanned using [18F]FEOBV PET, then sacrificed, and their brain tissues collected for immunostaining and quantification of cholinergic denervation using optical density (OD). RESULTS: For both PET binding and postmortem OD, the highest losses were found in the cortical areas, with the highest reductions in the orbitofrontal, sensorimotor, and cingulate cortices. In addition, OD quantification in the affected areas accurately predicts [18F]FEOBV uptake in the same regions when regressed linearly. CONCLUSIONS: These findings support [18F]FEOBV as a reliable imaging agent for eventual use in human neurodegenerative conditions in which cholinergic losses are an important aspect.

Design of brain imaging agents for positron emission tomography: do large bioconjugates provide an opportunity for in vivo brain imaging?

The development of brain imaging agents for positron emission tomography and other in vivo imaging modalities mostly relies on small compounds of low MW as a result of the restricted transport of larger molecules, such as peptides and proteins, across the blood-brain barrier. Besides passive transport, only a few active carrier mechanisms, such as glucose transporters and amino acid transporters, have so far been exploited to mediate the accumulation of imaging probes in the brain. An important question for the future is whether some of the abundant active carrier systems located at the blood-brain barrier can be used to shuttle potential, but non-crossing, imaging agents into the brain. What are the biological and chemical constrictions toward such bioconjugates and is it worthwhile to persue such a delivery strategy?

One-step (18)F-labeling of peptides for positron emission tomography imaging using the SiFA methodology.

Here we present a procedure to label peptides with the positron-emitting radioisotope fluorine-18 ((18)F) using the silicon-fluoride acceptor (SiFA) labeling methodology. Positron emission tomography (PET) has gained high importance in noninvasive imaging of various diseases over the past decades, and thus new specific imaging probes for PET imaging, especially those labeled with (18)F, because of the advantageous properties of this nuclide, are highly sought after. N-terminally SiFA-modified peptides can be labeled with (18)F(-) in one step at room temperature (20-25 °C) or below without forming side products, thereby producing satisfactory radiochemical yields of 46 ± 1.5% (n = 10). The degree of chemoselectivity of the (18)F-introduction, which is based on simple isotopic exchange, allows for a facile cartridge-based purification fully devoid of HPLC implementation, thereby yielding peptides with specific activities between 44.4 and 62.9 GBq µmol(-1) (1,200-1,700 Ci mmol(-1)) within 25 min.

Synthesis of [(18)F]SiFB: a prosthetic group for direct protein radiolabeling for application in positron emission tomography.

N-Succinimidyl 3-(di-tert-butyl[(18)F]fluorosilyl)benzoate ([(18)F]SiFB) is a highly reactive prosthetic group for radiolabeling of proteins for use in positron emission tomography (PET). It is similar to N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB), the 'gold-standard' prosthetic group for protein (18)F-labeling, but can be synthesized using a much shorter and technically easier procedure. A recently reported simple procedure to obtain anhydrous (18)F- by avoiding time-consuming azeotropic drying is applied with a slight modification to prevent basic hydrolysis of the active N-hydroxysuccinimide (NHS) ester moiety of [(18)F]SiFB. The labeling of [(18)F]SiFB is performed by a fast (18)F-(19)F isotopic exchange (IE) reaction at room temperature (20-25 °C) within 30 min. [(18)F]SiFB is purified using a C18 cartridge instead of HPLC, further decreasing the overall time required for protein labeling. High specific activities > 18.5 GBq µmol(-1) (> 500 Ci mmol(-1)) can be obtained. Finally, incubation of [(18)F]SiFB with the desired protein in an aqueous solution at pH 9, followed by HPLC purification, provides the final solution of the labeled protein ready for in vivo applications.

Protein labeling with the labeling precursor [(18)F]SiFA-SH for positron emission tomography.

Proteins previously derivatized with the cross-coupling reagent sulfo-SMCC (4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxy-succinimide ester sodium salt) can be easily labeled in high radiochemical yields with the silicon-fluoride acceptor (SiFA) reagent [(18)F]SiFA-SH, obtained via isotopic exchange, by thiol-maleimide coupling chemistry (n = 10). The specific activity of SiFA-SH obtained in a one-step labeling reaction was > 18.5 GBq µmol(-1) (> 500 Ci mmol(-1)). The number of SiFA building blocks per protein molecule is defined by the previously introduced number of maleimide groups, which can be determined by a simple and convenient Ellman's assay. Not more than two maleimide groups are introduced using sulfo-SMCC, thereby keeping the modification of the protein low and preserving its biological activity.

PET imaging of cholinergic deficits in rats using [18F]fluoroethoxybenzovesamicol ([18F]FEOBV).

[(18)F]fluoroethoxybenzovesamicol ([(18)F]FEOBV) is one of the most promising radioligands for imaging the vesicular ACh transporter (VAChT) with positron emission tomography (PET). We report here that this method can detect subtle cholinergic terminals losses such as those associated with aging, or those following a partial lesion of the nucleus basalis magnocellularis (NBM). Twenty-one adult rats were evenly distributed in three groups including 1) aged rats (18 months); 2) young rats (3 months); and 3) rats with unilateral lesion of the NBM, following a local stereotaxic infusion of 192 IgG-saporin. In both normal and lesioned rats, our results revealed the highest [(18)F]FEOBV binding to be in the striatum, followed by similar values in both frontal cortex and thalamus, while lower values were observed in both hippocampus and temporo-parietal cortex. This binding distribution is consistent with the known anatomy of brain cholinergic systems. In the lesioned rats, [(18)F]FEOBV binding was found to be reduced mostly in the ventral frontal cortex on the side of the lesion, but some reductions were also observed in the homologous region of the contralateral hemisphere. Aging was found to be associated with a [(18)F]FEOBV binding reduction limited to the hippocampus of both hemispheres. [(18)F]FEOBV appears to be a very promising marker for the in vivo quantification of the brain VAChT; PET imaging of this agent allows in vivo detection of both physiological and pathological reductions of cholinergic terminals density.

Fully automated SPE-based synthesis and purification of 2-[18F]fluoroethyl-choline for human use.

INTRODUCTION: 2-[(18)F]Fluoroethyl-choline ([(18)F]FECH) is a promising tracer for the detection of prostate cancer as well as brain tumors with positron emission tomography (PET). [(18)F]FECH is actively transported into mammalian cells, becomes phosphorylated by choline kinase and gets incorporated into the cell membrane after being metabolized to phosphatidylcholine. So far, its synthesis is a two-step procedure involving at least one HPLC purification step. To allow a wider dissemination of this tracer, finding a purification method avoiding HPLC is highly desirable and would result in easier accessibility and more reliable production of [(18)F]FECH. METHODS: [(18)F]FECH was synthesized by reaction of 2-bromo-1-[(18)F]fluoroethane ([(18)F]BFE) with dimethylaminoethanol (DMAE) in DMSO. We applied a novel and very reliable work-up procedure for the synthesis of [(18)F]BFE. Based on a combination of three different solid-phase cartridges, the purification of [(18)F]BFE from its precursor 2-bromoethyl-4-nitrobenzenesulfonate (BENos) could be achieved without using HPLC. Following the subsequent reaction of the purified [(18)F]BFE with DMAE, the final product [(18)F]FECH was obtained as a sterile solution by passing the crude reaction mixture through a combination of two CM plus cartridges and a sterile filter. The fully automated synthesis was performed using as well a Raytest SynChrom module (Raytest, Germany) or a Scintomics HotboxIII module (Scintomics, Germany). RESULTS: The radiotracer [(18)F]FECH can be synthesized in reliable radiochemical yields (RCY) of 37±5% (Synchrom module) and 33±5% (Hotbox III unit) in less than 1 h using these two fully automated commercially available synthesis units without HPLC involvement for purification. Detailed quality control of the final injectable [(18)F]FECH solution proved the high radiochemical purity and the absence of Kryptofix2.2.2, DMAE and DMSO used in the course of synthesis. Sterility and bacterial endotoxin testing following standard procedures verified that the described production method for [(18)F]FECH is suitable for human applications. CONCLUSIONS: The routine production of [(18)F]FECH with sufficient RCYs was established by reliable and fast solid-phase extraction purifications of both the secondary labeling precursor [(18)F]BFE and the final product [(18)F]FECH, avoiding complex and sensitive HPLC equipment. The purity of the product was >95%, rendering the tracer suitable for human application. The newly developed purification procedure for [(18)F]BFE significantly reduces the complexity of the automated synthesis unit, hence reducing the cost for routine production in a clinical setup and allowing easy transfer to different synthesis modules.

In vitro and initial in vivo evaluation of (68)Ga-labeled transferrin receptor (TfR) binding peptides as potential carriers for enhanced drug transport into TfR expressing cells.

PURPOSE: The transferrin receptor (TfR) is one of the most attractive targets to overcome the blood-brain barrier (BBB). It has recently been shown that THRPPMWSPVWP binds to the TfR and is subsequently internalized into TfR-expressing cells. Here, we evaluated the ability of THRPPMWSPVWP to become internalized into human TfR-expressing cells via endocytosis to determine its potential to act as a carrier system for the transport of small molecules across the BBB. PROCEDURES: To validate the underlying concept of a conjugate consisting of a small brain imaging tracer and a large peptidic carrier molecule, a conjugate of the high affinity D2 receptor ligand fallypride and the TfR targeting peptide THRPPMWSPVWP has been synthesized. Furthermore, two derivatives of THRPPMWSPVWP were labeled with (68)Ga in high radiochemical yields (>96%) and a radiochemical purity of 96-98% and evaluated in vitro and in vivo. RESULTS: The fallypride-THRPPMWSPVWP conjugate still displayed a K(i) of 27 nM. The uptake of the (68)Ga-labeled peptides into TfR-bearing cells was investigated using U87MG and HT-29 cells to assess the capability of the peptide to act as a carrier molecule targeting the TfR. The in vitro binding studies revealed negligible uptake of the tested (68)Ga-labeled conjugates ranging from 0.08% to 0.66% after 60 min incubation at 37°C. Initial in vivo experiments with (68)Ga-DOTA-S-maleimido-THRPPMWSPVWP in two healthy rats showed a mean brain uptake of 0.037% injected dose per gram, confirming the results obtained in vitro. CONCLUSION: These results suggest that the accumulation of the (68)Ga-radiolabeled conjugates of the TfR-binding peptide THRPPMWSPVWP into TFR expressing human cell lines is nonspecific and too low to render this peptide suitable as a possible carrier molecule for a receptor-mediated transport of compounds across the BBB.

Basal opioid receptor binding is associated with differences in sensory perception in healthy human subjects: a [18F]diprenorphine PET study.

The endogenous opioid system is involved in many body functions including pain processing and analgesia. To determine the role of basal opioid receptor availability in the brain in pain perception, twenty-three healthy subjects underwent positron emission tomography (PET) utilizing the subtype-nonselective opioid antagonist [(18)F]diprenorphine, quantitative sensory testing (QST) and the cold pressor test. Binding potentials (BPs) were calculated using a non-invasive reference tissue model and statistical parametric mapping was applied for t-statistical analysis on a voxelwise basis. We found that cold pain-sensitive subjects present a significantly lower BP in regions including the bilateral insular cortex and the left orbitofrontal cortex. In addition, correlation analysis revealed an inverse correlation between opioid BP in the bilateral motor and premotor region and perceptual wind-up. These findings indicate that interindividual differences in pain perception are partially accounted for by basal opioid receptor availability. A secondary aim of this study was to investigate the contribution of basal opioid receptor availability to the perception of non-nociceptive stimuli. The following negative correlations between regional opioid BP and scores of QST parameters were found: BP in the right premotor cortex and scores of alternating cold and warm stimuli, BP in the left midcingular cortex and scores of cold detection threshold, BP in the left insula and scores of mechanical detection threshold. These results suggest that the opioid receptor system is involved in the perception not only of pain but also of non-painful somatosensory stimuli.

Improved work-up procedure for the production of [(18)F]flumazenil and first results of its use with a high-resolution research tomograph in human stroke.

INTRODUCTION: The central benzodiazepine receptor (cBZR)-gamma-aminobutyric acid (GABA(A)) receptor complex in the human brain plays an important role in many neurological and psychiatric disorders. (18)F-Labeled flumazenil ([(18)F]FZ) provides a potentially useful tracer to investigate those disorders by means of positron emission tomography (PET). METHODS: [(18)F]Flumazenil was synthesized from its nitro-precursor Ro 15-2344 in DMF at high temperatures between 150 degrees C and 160 degrees C. Other solvents like acetonitrile and dimethylsulfoxide were also investigated as reaction media. A new HPLC method for the final purification of [(18)F]FZ was developed to circumvent some difficulties associated with a previously published procedure sometimes led to a contamination of [(18)F]FZ with Ro 15-2344. The final purification of the radiotracer was achieved using a Waters Symmetry Prep C18 HPLC column with elution with 0.05 M sodium acetate (NaOAc) buffer (pH 5)/THF/MeOH (80:10:10). RESULTS: [(18)F]FZ could be synthesized in reproducible radiochemical yields (RCYs) of 15-20% (decay corrected to EOB) after 80 min overall synthesis time. The synthesized [(18)F]FZ was applied for the first time in a human PET study in a patient with ischemic right middle cerebral artery stroke using the HRRT high-resolution research scanner (Siemens Medical Solution, Knoxville, TN, USA). CONCLUSIONS: [(18)F]FZ is a potentially useful GABA receptor-binding PET ligand. A modified procedure for its preparation in reproducibly high radiochemical yields has been described and the [(18)F]FZ thus produced has been used successfully in a pilot clinical study.

[18F]SiFA-isothiocyanate: a new highly effective radioactive labeling agent for lysine-containing proteins.

A highly efficient (18)F-labeling synthon for universal protein labeling is reported. Diverse (18)F-labeled proteins of 66-144 kDa were prepared with [(18)F]SiFA-isothiocyanate synthesized by an isotopic (19)F for (18)F exchange at the silicon atom. Overall preparative radiochemical yields were 20-40 % after 40-50 min. No bone uptake of (18)F radioactivity was detected until 90 min post-injection of (18)F-SiFA-RSA; this demonstrates the metabolic stability of the [(18)F]SiFA moiety.

Kit-like 18F-labeling of proteins: synthesis of 4-(di-tert-butyl[18F]fluorosilyl)benzenethiol (Si[18F]FA-SH) labeled rat serum albumin for blood pool imaging with PET.

Radiosyntheses of 18F-radiopharmaceuticals for positron emission tomography (PET) normally require an extraordinarily high effort of technical equipment and specially trained personnel. We recently reported a novel method for the introduction of fluorine-18 into peptides for PET-imaging based on silicon-18F-chemistry (SiFA technique). We herewith introduce the first SiFA-based Kit-like radio-fluorination of a protein (rat serum albumin,RSA) and demonstrate its usefulness for in vivo imaging with microPET in normal rats as well as in a rat heterotropic transplanted heart model. As a labeling agent, we prepared 4-(di-tert-butyl[18F]fluorosilyl)benzenethiol (Si[18F]FASH)by simple isotopic exchange in 40-60% radiochemical yield (RCY) and coupled it directly to a Sulfo-SMCC derivatized RSA in an overall RCY of 12% within 20-30 min. The technically simple labeling procedure does not require any elaborated purification procedures and is a straightforward example of a successful application of Si-18F chemistry for in vivo imaging with PET.

para-Functionalized aryl-di-tert-butylfluorosilanes as potential labeling synthons for (18)F radiopharmaceuticals.

Broad spectrum: Novel para-functionalized aryl-di-tert-butylfluorosilanes, p-(tBu(2)FSi)C(6)H(4)X (X=functional group), have been made available and broaden the spectrum of silicon-based (18)F acceptors (SiFAs) for potential PET applications. For example, the [(18)F]maleimido derivative 1 has been employed for the synthesis of [(18)F]1- labeled rat serum albumin (RSA), the applicability of which for PET has been verified by in vivo experiments.The syntheses of the functionalized triorganofluorosilanes tBu(2)(p-XC(6)H(4))SiF (3 a, X=SH; 4 a, X=NCS; 4 b, X=NCO; 5, X=NC(4)H(2)O(2); 7, X=COOH; 8 a, X=COONC(4)H(4)O(2); 8 b, X=COOC(6)F(5)) are reported. These compounds display potential as silicon-based fluoride acceptors (SiFAs). The molecular structures of compounds 5, 7, and 8 a have been determined by single-crystal X-ray diffraction studies. With the exception of compounds 8 a and 8 b, all of the compounds could be (18)F-labeled by isotopic exchange in good to high radiochemical yields (RCY) with good to excellent specific activities. As proof of applicability, the maleimido-functionalized SiFA derivative 5, which is specific for thiol groups, has been used for the labeling of rat serum albumin (RSA) that had been derivatized with 2-iminothiolane. The incorporation of [(18)F]5 into the derivatized RSA reached a maximum yield after 30 min at ambient temperature. After purification, the [(18)F]RSA was evaluated in a healthy rat by means of muPET and displayed an expedient in vivo stability over 180 min.

Human concentrative nucleoside transporter 1-mediated uptake of 5-azacytidine enhances DNA demethylation.

The DNA methyltransferase inhibitors 5-azacytidine (5-azaCyd) and 5-aza-2'-deoxycytidine have found increasing use for the treatment of myeloid leukemias and solid tumors. Both nucleoside analogues must be transported into cells and phosphorylated before they can be incorporated into DNA and inactivate DNA methyltransferases. The members of the human equilibrative and concentrative nucleoside transporter families mediate transport of natural nucleosides and some nucleoside analogues into cells. However, the molecular identity of the transport proteins responsible for mediating the uptake of 5-azanucleosides has remained unknown. To this end, we have generated a stably transfected Madin-Darby canine kidney strain II cell line expressing recombinant hCNT1. An antiserum directed against hCNT1 specifically detected the protein in the apical membrane of hCNT1-expressing Madin-Darby canine kidney cells. Using [14C]5-azaCyd, we show here that hCNT1 mediated the Na+-dependent uptake of this drug with a Km value of 63 micromol/L. Na+-dependent transport of radiolabeled cytidine, uridine, and 5-fluoro-5'-deoxyuridine further showed the functionality of the transporter. hCNT1-expressing cells were significantly more sensitive to 5-azaCyd, and drug-dependent covalent trapping of DNA methyltransferase 1 was substantially more pronounced. Importantly, these results correlated with a significant sensitization of hCNT1-expressing cells toward the demethylating effects of 5-azaCyd and 5-aza-2'-deoxycytidine. In conclusion, our study identifies 5-azaCyd as a novel substrate for hCNT1 and provides direct evidence that hCNT1 is involved in the DNA-demethylating effects of this drug.

Opioid receptor PET reveals the psychobiologic correlates of reward processing.

UNLABELLED: Little is known about the neurobiologic correlates of human personality. On the basis of the key role of the central opioidergic system in addiction and substance abuse, we investigated the relationship between certain personality traits that are supposed to be relevant in addiction and the opioid receptor status in healthy subjects. METHODS: We investigated 23 healthy male volunteers who were extensively clinically tested to exclude substance abuse. All of the subjects underwent 1 PET scan with the subtype-nonselective opioidergic radioligand 18F-fluoroethyl-diprenorphine under resting conditions without sensory or cognitive stimulation. Subsequently, the subjects were psychologically tested for the personality traits novelty seeking, harm avoidance, reward dependence, and persistence, according to Cloninger's biosocial model of personality. The binding potential (BP) as a parameter of regional cerebral opioid receptor availability was computed by means of the modified Logan plot using the occipital cortex as a reference region. Further imaging data analysis was performed using statistical parametric mapping; after stereotactic normalization, the correlations were calculated between the regional BP and the psychologic scores on a voxel-by-voxel basis. RESULTS: The correlation analysis between personality dimensions and opioid receptor availability showed a significant (P < 0.001) positive correlation between the scores of reward dependence and the BP of the bilateral ventral striatum with nucleus accumbens (z scores, 4.52 and 4.33, respectively). The additionally performed region-of-interest-based correlation analysis yielded correlation coefficients of r = 0.84 and r = 0.81 for the left and right ventral striata, respectively. No further significant correlations were detectable between the other personality dimensions and cerebral opioid receptor binding. CONCLUSION: In healthy subjects, personality traits, which might be predisposing for addictive behavior, are correlated to the opioidergic neurotransmission in core structures of the human reward system.