Synthesis, Biodistribution and In vitro Evaluation of Brain Permeable High Affinity Type 2 Cannabinoid Receptor Agonists [11C]MA2 and [18F]MA3.

The type 2 cannabinoid receptor (CB2) is a member of the endocannabinoid system and is known for its important role in (neuro)inflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([11C]MA2) and a fluorine-18 ([18F]MA3) labeled analog of a highly potent N-arylamide oxadiazole CB2 agonist (EC50 = 0.015 nM). MA2 and MA3 behaved as potent CB2 agonist (EC50: 3 nM and 0.1 nM, respectively) and their in vitro binding affinity for hCB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group) was found to have higher binding affinity and EC50 values when compared to the originally reported trifluoromethyl analog 12. [11C]MA2 and [18F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [11C]MA2 and [18F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However, in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted.

New Chelators for Low Temperature Al(18)F-Labeling of Biomolecules.

The Al(18)F labeling method is a relatively new approach that allows radiofluorination of biomolecules such as peptides and proteins in a one-step procedure and in aqueous solution. However, the chelation of the {Al(18)F}(2+) core with the macrocyclic chelators NOTA or NODA requires heating to 100-120 °C. Therefore, we have developed new polydentate ligands for the complexation of {Al(18)F}(2+) with good radiochemical yields at a temperature of 40 °C. The stability of the new Al(18)F-complexes was tested in phosphate buffered saline (PBS) at pH 7.4 and in rat serum. The stability of the Al(18)F-L3 complex was found to be comparable to that of the previously reported Al(18)F-NODA complex up to 60 min in rat serum. Moreover, the biodistribution of Al(18)F-L3 in healthy mice showed the absence of in vivo defluorination since no significant bone uptake was observed, whereas the major fraction of activity at 60 min p.i. was observed in liver and intestines, indicating hepatobiliary clearance of the radiolabeled ligand. The acyclic chelator H3L3 proved to be a good lead candidate for labeling of heat-sensitive biomolecules with fluorine-18. In order to obtain a better understanding of the different factors influencing the formation and stability of the complex, we carried out more in-depth experiments with ligand H3L3. As a proof of concept, we successfully conjugated the new AlF-chelator with the urea-based PSMA inhibitor Glu-NH-CO-NH-Lys to form Glu-NH-CO-NH-Lys(Ahx)L3, and a biodistribution study in healthy mice was performed with the Al(18)F-labeled construct. This new class of AlF-chelators may have a great impact on PET radiochemical space as it will stimulate the rapid development of new fluorine-18 labeled peptides and other heat-sensitive biomolecules.

Development and biological evaluation of 99mTc-sulfonamide derivatives for in vivo visualization of CA IX as surrogate tumor hypoxia markers.

In vivo visualization of tumor hypoxia related markers, such as the endogenous transmembrane protein CA IX may lead to novel therapeutic and diagnostic applications in the management of solid tumors. In this study 4-(2-aminoethyl)benzene sulfonamide (AEBS, K(i) = 33 nM for CA IX) has been conjugated with bis(aminoethanethiol) (BAT) and mercaptoacetyldiglycine (MAG2) tetradendate ligands and the conjugates radiolabelled with (99m)Tc, to obtain anionic and neutral (99m)Tc-labeled sulfonamide derivatives, respectively. The corresponding rhenium analogues were also prepared and showed good inhibitory activities against hCA IX (K(i) = 59-66 nM). In addition, a second generation bis AEBS was conjugated with MAG2 and labeled with (99m)Tc, and the obtained diastereomers were also evaluated in targeting CA IX. Biodistribution studies in mice bearing HT-29 colorectal xenografts revealed a maximum tumor uptake of <0.5% ID/g at 0.5 h p.i for all the tracers. In vivo radiometabolite analysis indicated that at 1 h p.i. MAG2 tetradendate ligands were more stable in plasma (>50% intact) compared to the neutral complex (28% intact). This preliminary data suggest that negatively charged (99m)Tc-labeled sulfonamide derivatives with modest lipophilicity and longer circulation time could be promising markers to target CA IX.

18F-MK-9470 PET imaging of the type 1 cannabinoid receptor in prostate carcinoma: a pilot study.

BACKGROUND: Preclinical and histological data show overexpression of the type 1 cannabinoid receptor (CB1R) in prostate carcinoma (PCa). In a prospective study, the feasibility of 18F-MK-9470 positron emission tomography (PET) imaging in patients with primary and metastatic PCa was evaluated. METHODS: Eight patients were included and underwent 18F-MK-9470 PET/CT imaging. For five patients with primary PCa, dynamic PET/CT imaging was performed over three acquisition intervals (0 to 30, 60 to 90 and 120 to 150 min post-injection). In malignant and benign prostate tissue regions, time activity curves of the mean standardized uptake value (SUVmean) were determined as well as the corresponding area under the curve to compare 18F-MK-9470 uptake over time. Muscle uptake of 18F-MK-9470 was used as reference for non-specific binding. Magnetic resonance imaging (MRI) was used as anatomical reference and for delineating intraprostatic tumours. Histological and immunohistochemical (IHC) examination was performed on the whole-mount histopathology sections of four patients who underwent radical prostatectomy to assess the MRI-based tumour versus benign tissue classification. For three patients with proven advanced metastatic disease, two static PET/CTs were performed 1 and 3 h post-injection. 18F-MK-9470 uptake was evaluated in bone lesions of metastatic PCa by comparing SUVmean values of metastases with these of the contralateral bone tissue. RESULTS: 18F-MK-9470 uptake was significantly higher in benign and malignant prostate tissue compared to muscle, but it did not differ between both prostate tissue compartments. IHC findings of corresponding prostatic histopathological sections indicated weak CB1R expression in locally confined PCa, which was not visualized with 18F-MK-9470 PET. Metastases in the axial skeleton could not be detected while some metastases in the appendicular skeleton showed higher 18F-MK-9470 uptake as compared to the uptake in contralateral normal bone. CONCLUSIONS: 18F-MK-9470 PET could not detect local PCa or bone metastases in the axial skeleton but was able to visualize metastases in the appendicular skeleton. Based on these pilot observations, it seems unlikely that CB1R PET will play a significant role in the evaluation of PCa.

Synthesis and biological evaluation of 68Ga-bis-DOTA-PA as a potential agent for positron emission tomography imaging of necrosis.

INTRODUCTION: Necrosis is a form of cell death that occurs in a variety of pathological conditions but can also be the result of therapy in cancer treatment. A radiotracer that could image necrotic cell death using PET could therefore be a useful tool to provide relevant information on the disease activity or therapeutic efficacy and assist in diagnosis and therapy management of several disorders. Pamoic acid derivatives have previously been reported to show a selective uptake in tissue undergoing cellular death via necrosis. In this study 4,4'-methylene-bis(2-hydroxy-3-naphthoic hydrazide) (pamoic acid bis-hydrazide) was conjugated to the macrocyclic ligand DOTA and labeled with the generator produced positron emitter (68)Ga. The resulting complex ((68)Ga-bis-DOTA-PA; (68)Ga-3) was evaluated as a potential radiotracer for imaging tissues undergoing cellular death via necrosis. METHODS: Bis-DOTA-PA was synthesized and labeled with (68)Ga. Biodistribution of (68)Ga-3 and analysis of plasma were studied in normal NMRI mice. Binding of the complex to necrotic tissue was first evaluated by in vitro autoradiography. Further evaluation of the uptake in necrotic tissue was performed in two different models of necrosis using microPET imaging in correlation with ex vivo autoradiography, biodistribution studies and histochemical staining. A biodistribution study in a mouse model of hepatic apoptosis was performed to study the selectivity of the uptake of (68)Ga-bis-DOTA-PA in necrotic tissue. RESULTS: (68)Ga-3 was obtained with a decay-corrected radiochemical yield of 51.8% ± 5.4% and a specific activity of about 12 GBq/µmol. In normal mice, the complex was slowly cleared from blood, mainly through the renal pathway, and showed high in vivo stability. (68)Ga-bis-DOTA-PA displayed high and selective uptake in necrotic tissue and allowed imaging of necrotic tissue using microPET. CONCLUSION: (68)Ga-3 was synthesized and characterized. In vitro, in vivo and ex vivo studies showed that the complex displays high and selective uptake in tissue undergoing cellular death via necrosis.

Preclinical evaluation of carbon-11 and fluorine-18 sulfonamide derivatives for in vivo radiolabeling of erythrocytes.

BACKGROUND: To date, few PET tracers for in vivo labeling of red blood cells (RBCs) are available. In this study, we report the radiosynthesis and in vitro and in vivo evaluation of 11C and 18F sulfonamide derivatives targeting carbonic anhydrase II (CA II), a metallo-enzyme expressed in RBCs, as potential blood pool tracers. A proof-of-concept in vivo imaging study was performed to demonstrate the feasibility to assess cardiac function and volumes using electrocardiogram (ECG)-gated positron emission tomography (PET) acquisition in comparison with cine magnetic resonance imaging (cMRI) in rats and a pig model of myocardial infarction. METHODS: The inhibition constants (Ki) of CA II were determined in vitro for the different compounds by assaying CA-catalyzed CO2 hydration activity. Binding to human RBCs was estimated after in vitro incubation of the compounds with whole blood. Biodistribution studies were performed to evaluate tracer kinetics in NMRI mice. ECG-gated PET acquisition was performed in Wistar rats at rest and during pharmacological stress by infusing dobutamine at 10 µg/kg/min and in a pig model of myocardial infarction. Left ventricular ejection fraction (LVEF) and volumes were compared with values from cMRI. RESULTS: The Ki of the investigated compounds for human CA II was found to be in the range of 8 to 422 nM. The fraction of radioactivity associated with RBCs was found to be ≥90% at 10- and 60-min incubation of tracers with heparinized human blood at room temperature for all tracers studied. Biodistribution studies in mice indicated that 30% to 67% of the injected dose was retained in the blood pool at 60 min post injection. A rapid and sustained tracer uptake in the heart region with an average standardized uptake value of 2.5 was observed from micro-PET images. The LVEF values obtained after pharmacological stress in rats closely matched between the cMRI and micro-PET values, whereas at rest, a larger variation between LVEF values obtained by both techniques was observed. In the pig model, a good agreement was observed between PET and MRI for quantification of left ventricular volumes and ejection fraction. CONCLUSIONS: The 11C and 18F sulfonamide derivatives can be used for efficient in vivo radiolabeling of RBCs, and proof-of-concept in vivo imaging studies have shown the feasibility and potential of these novel tracers to assess cardiac function.

Preliminary validation of varicella zoster virus thymidine kinase as a novel reporter gene for PET.

INTRODUCTION: Imaging of gene expression with positron emission tomography (PET) has emerged as a powerful tool for biomedical research during the last decade. The prototypical herpes simplex virus type 1 thymidine kinase (HSV1-TK) PET reporter gene (PRG) is widely used and many other PRGs have also been validated. We investigated varicella zoster virus thymidine kinase (VZV-tk) as new PRG with radiolabeled bicyclic nucleoside analogues (BCNAs) as PET tracers. METHODS: The uptake and washout of four different radiolabeled BCNAs was evaluated in cells expressing VZV-tk after lentiviral vector (LV) transduction and in control cells. Metabolism of the tracers was assayed by high pressure liquid chromatography (HPLC). Mice bearing VZV-TK expressing xenografts were imaged with PET. RESULTS: High uptake in VZV-tk expressing cells was seen for 3 of the 4 tracers tested. The uptake of the tracers could be blocked by the presence of excess thymidine in the incubation solution. Cellular retention was variable, with one tracer showing an acceptable half-life of ~1 hour. The amount of intracellular tracer correlated with the titer of LV used to transduce the cells. VZV-TK dependent conversion into metabolites was shown by HPLC. No specific accumulation was observed in cells expressing a fusion protein containing an HSV1-TK moiety. VZV-tk expression in xenografts resulted in a 60% increase in uptake in vivo as measured with PET. CONCLUSIONS: We have validated the combination of VZV-tk and radiolabeled BCNAs as new PRG/PRP system. Further optimization of the PRPs and the PRG are warranted to increase the signal.

Preclinical evaluation of [11C]NE40, a type 2 cannabinoid receptor PET tracer.

INTRODUCTION: Up-regulation of the type 2 cannabinoid receptor (CB(2)R) has been reported in (neuro)inflammatory diseases. In this study, we report the preclinical evaluation of [(11)C]NE40 as positron emission tomography (PET) radioligand for visualization of the CB(2)R. METHODS: The selectivity of NE40 for CB(2)R and its toxicity and mutagenicity were determined. [(11)C]NE40 was evaluated by biodistribution and autoradiography studies in normal rats and a microPET study in normal mice, rats and a rhesus monkey. Specific in vivo binding of [(11)C]NE40 to human CB(2)R (hCB(2)R) was studied in a rat model with hCB(2)R overexpression. RESULTS: [(11)C]NE40 shows specific CB(2)R binding in the spleen and blood of normal rats and high brain uptake in rhesus monkey. [(11)C]NE40 showed specific and reversible binding to hCB(2)R in vivo in a rat model with local hCB(2)R overexpression. CONCLUSIONS: [(11)C]NE40 shows favorable characteristics as radioligand for in vivo visualization of the CB(2)R and is a promising candidate for hCB(2)R PET imaging.

Synthesis, in vitro and in vivo evaluation of fluorine-18 labelled FE-GW405833 as a PET tracer for type 2 cannabinoid receptor imaging.

The type 2 cannabinoid receptor (CB2R) is part of the endocannabinoid system and is expressed in tissues related to the immune system. As the CB2R has a very low brain expression in non-pathological conditions, but is upregulated in activated microglia, it is an interesting target for visualization of neuroinflammation using positron emission tomography with a suitable radiolabeled CB2R ligand. In this study, we radiolabelled a fluoroethyl derivative of GW405833, a well known CB2R partial agonist, with fluorine-18 (half-life 109.8 min) by alkylation of the phenol precursor with 1-bromo-2-[¹8F]fluoroethane. In vitro studies showed that FE-GW405833 behaved as a selective high affinity (27 nM) inverse agonist for hCB2R. [¹8F]FE-GW405833 showed moderate initial brain uptake in mice and rats, but a slow washout from brain and plasma due to retention of a radiometabolite. Specific binding of the tracer to human CB2R was demonstrated in vivo in a rat model with local CB2R overexpression in the brain. Optimized derivatives of GW405833 that are less susceptible to metabolism will need to be developed in order to provide a useful tracer for CB2R quantification with PET.

Synthesis and biological evaluation of a 99mTc-labelled sulfonamide conjugate for in vivo visualization of carbonic anhydrase IX expression in tumor hypoxia.

INTRODUCTION: Carbonic anhydrase (CA) IX is a transmembrane protein overexpressed in many frequently occurring tumors associated with tumor hypoxia. Sulfonamides and their bioisosteres are known to inhibit CA IX activity. In this study, 4-(2-aminoethyl)benzenesulfonamide was conjugated to a tridentate ligand, N-2-picolyl-N-acetic acid and labeled with a (99m)Tc(I)-tricarbonyl moiety resulting in [(99m)Tc(CO)(3) (L)] (L=N-(pyridin-2-yl-methyl)-N[2-(4-sulfamoylphenyl)-ethyl]aminoethyl acetate) complex, [(99m)Tc]-5. Similarly the corresponding rhenium congener (Re-4) was synthesized. The in vitro CA IX affinity and inhibitory activity of Re-4 were determined and [(99m)Tc]-5 was evaluated as a tracer for in vivo visualisation of CA IX expression. METHODS: Evaluation of the in vitro affinity (inhibition constant, K(i)) of Re-4 for CA isozymes I, II, IX and XII was carried out by assaying the CA catalyzed CO(2) hydration activity and efficacy studies were performed in HT 29 cell lines expressing CA IX under normoxia or hypoxia. Biodistribution studies of [(99m)Tc]-5 were performed in xenograft mice bearing CA IX expressing tumors. RESULTS: The in vitro affinity of Re-4 for CA IX was 58 nM and CA IX induced acidification of extracellular medium was efficiently reduced (P<.05) in the presence of 1 mM Re-4. Biodistribution studies indicated a maximal tumor uptake of [(99m)Tc]-5 of 0.1% ID/g at 30 min post injection. CONCLUSION: [(99m)Tc]-5 and its rhenium congener were synthesized and characterized. In vitro studies showed that the rhenium compound has a high affinity for CA IX and effectively inhibits CA IX activity. In vivo studies revealed a limited tracer accumulation in a CA IX expressing tumor but with increasing tumor-to-blood activity ratios as a function of time.

Non-invasive imaging of the type 2 cannabinoid receptor, focus on positron emission tomography.

The type 2 cannabinoid receptor (CB(2)R) is a relatively new target for drug development, as the receptor was only discovered in 1993. The CB(2)R is mainly expressed in tissues and organs related to the immune system. However, in pathological conditions, mostly inflammatory, a strong upregulation has been observed. Because of its expression in activated microglia, the type 2 cannabinoid receptor might play an important role in pathologies with a neuroinflammatory component. Positron emission tomography provides a sensitive non-invasive imaging technique to study and quantify receptor expression. In this review, the importance of CB(2)R imaging, the current status and the future possibilities for the development of CB(2)R PET radioligands are discussed.

Synthesis and biological evaluation of carbon-11- and fluorine-18-labeled 2-oxoquinoline derivatives for type 2 cannabinoid receptor positron emission tomography imaging.

INTRODUCTION: The type 2 cannabinoid (CB(2)) receptor is part of the endocannabinoid system and has been suggested as a mediator of several central and peripheral inflammatory processes. Imaging of the CB(2) receptor has been unsuccessful so far. We synthesized and evaluated a carbon-11- and a fluorine-18-labeled 2-oxoquinoline derivative as new PET tracers with high specificity and affinity for the CB(2) receptor. METHODS: Two 2-oxoquinoline derivatives were synthesized and radiolabeled with either carbon-11 or fluorine-18. Their affinity and selectivity for the human CB(2) receptor were determined. Biological evaluation was done by biodistribution, radiometabolite and autoradiography studies in mice. RESULTS: In vitro studies showed that both compounds are high affinity CB(2)-specific inverse agonists. Biodistribution study of the tracers in mice showed a high in vivo initial brain uptake and fast brain washout, in accordance with the low CB(2) receptor expression levels in normal brain. A persistently high in vivo binding to the spleen was observed, which was inhibited by pretreatment with two structurally unrelated CB(2) selective inverse agonists. In vitro autoradiography studies with the radioligands confirmed CB(2)-specific binding to the mouse spleen. CONCLUSION: We synthesized two novel CB(2) receptor PET tracers that show high affinity/selectivity for CB(2) receptors. Both tracers show favourable characteristics as radioligands for central and peripheral in vivo visualization of the CB(2) receptor and are promising candidates for primate and human CB(2) PET imaging.

Developing new molecular imaging probes for PET.

Positron emission tomography (PET) is a fully translational molecular imaging technique that requires specific probes radiolabelled with short-lived positron emitting radionuclides. This review discusses relevant methods which are applied throughout the different steps in the development of new PET probes for in vivo visualization of specific molecular targets related to diagnosis or important for drug development.

Labelling and biological evaluation of [(11)C]methoxy-Sch225336: a radioligand for the cannabinoid-type 2 receptor.

INTRODUCTION: The cannabinoid type 2 receptor (CB(2) receptor) is part of the endocannabinoid system and has been suggested as mediator of a number of central and peripheral inflammatory processes. In the present study, we have synthesized N-[(1s)-1-[4-[[4-methoxy-2-[(4-[(11)C]methoxyphenyl)sulfonyl)-phenyl]sulfonyl] phenyl]ethyl]methanesulfonamide ([(11)C]methoxy-Sch225336) and evaluated this new tracer agent as a potential positron emission tomography radioligand for the in vivo visualization of CB(2) receptors. METHODS: Sch225336 was demethylated and the resulting phenol precursor was radiolabelled with a carbon-11 methyl group by methylation using [(11)C]methyl iodide, followed by purification by high-performance liquid chromatography. The log P of [(11)C]methoxy-Sch225336 and its biodistribution in normal mice were determined. Enhancement of brain uptake by inhibition of blood-brain barrier (BBB) efflux transporters was studied. Mouse plasma was analysed to quantify the formation of radiometabolites. The affinity of Sch225336 for the human cannabinoid type 1 and type 2 receptor was determined. RESULTS: [(11)C]methoxy-Sch225336 was obtained with a decay corrected radiochemical yield of about 30% and a specific activity of 88.8 GBq/mumol (end of synthesis). After intravenous injection in mice, the compound is rapidly cleared from the blood through the hepatobiliary pathway and does not show particular retention in any of the major organs. Polar metabolites were found in mouse plasma. Brain uptake was low despite the favourable log P value of 2.15, which is partly due to efflux by BBB pumps. CONCLUSION: [(11)C]methoxy-Sch225336 is a good candidate for in vivo imaging of the CB(2) receptor, although the low blood-brain barrier penetration limits its potential for central nervous system imaging.

Efficient purification and metabolite analysis of radiotracers using high-performance liquid chromatography and on-line solid-phase extraction.

This study describes an efficient method using on-line solid-phase extraction (SPE) (Oasis HLB) for preparative HPLC purification of short-lived radiotracers for positron emission tomography (PET) and for HPLC analysis of radiotracers and their metabolites in cell homogenates, plasma and urine samples. The radiochemical purity of tracers (fluorine-18 labeled) purified using this method (Oasis column) was >99% compared to 90% when no Oasis column was used. Radiometabolites of several fluorine-18 and carbon-11-labeled tracers and one technetium-99m tracer were quantified in cell homogenates, plasma and urine samples. Samples were analyzed using Oasis column and analytical HPLC system without prior precipitation of proteins or removal of other biological matrices. The metabolites observed for the evaluated tracers were all polar relative to the unchanged tracer. The extraction repeatability was found to be good (RSD 2.2%) and recoveries of Oasis column/HPLC-injected radioactivity (plasma) were found to be high (mean recovery >91%). The same Oasis column was used for several times without back pressure build-up or decrease of the HPLC separation characteristics.

A p-[18F]fluoroethoxyphenyl bicyclic nucleoside analogue as a potential positron emission tomography imaging agent for varicella-zoster virus thymidine kinase gene expression.

We recently reported a new positron emission tomography (PET) reporter gene, namely, varicella-zoster virus thymidine kinase (VZV-tk) in combination, with carbon-11 or fluorine-18 labeled m-alkoxyphenyl bicyclic nucleoside analogues (BCNAs) as PET reporter probes. We now report the synthesis and evaluation of p-alkoxyphenyl-BCNA tracers ([11C]-4 and [18F]-5), which are found to be superior to the m-alkoxyphenyl-BCNA tracers. In particular, the fluorine-18 labeled tracer ([18F]-5, IC50 of 5 is 4.2 microM) shows a higher accumulation in VZV-tk expressing cells than the previously reported m-methoxyphenyl BCNA. [11C]-4 and [18F]-5 were synthesized by heating the phenol precursor 3 with 11CH 3I and 18FCH 2CH 2Br, respectively, as alkylating agents. In vitro evaluation of [11C]-4 and [18F]-5 in 293T cells showed about 14- and 54-fold higher uptake, respectively, into VZV-tk gene-transduced cells compared to control cells. LC-MS analysis confirmed the formation of monophosphate derivative of 5 upon catalysis by VZV TK. In vivo studies of this new reporter gene/probe system are in progress.

Synthesis and biological evaluation of an 123I-labeled bicyclic nucleoside analogue (BCNA) as potential SPECT tracer for VZV-tk reporter gene imaging.

An iodine-123 labeled bicyclic nucleoside analogue ([(123)I]-4) has been synthesized and evaluated as a potential single photon emission tomography (SPECT) reporter probe for the non-invasive imaging of expression of the varicella zoster virus thymidine kinase (VZV-tk) reporter gene. In vitro enzymatic assays revealed that the non-radioactive mono-iodo derivative 4 has good affinity for VZV-TK (IC(50): 4.2 microM). Biodistribution of [(123)I]-4 was examined in normal mice. Evaluation of [(123)I]-4 in HEK-293T cells showed 1.74-fold higher accumulation in VZV-TK-expressing cells compared to control cells.

Synthesis and preliminary evaluation of 18F- or 11C-labeled bicyclic nucleoside analogues as potential probes for imaging varicella-zoster virus thymidine kinase gene expression using positron emission tomography.

Two radiolabeled bicyclic nucleoside analogues (BCNAs) were synthesized, namely 3-(2'-deoxy-beta-d-ribofuranosyl)-6-(3-[18F]fluoroethoxyphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one ([18F]-2) and 3-(2'-deoxy-beta-d-ribofuranosyl)-6-(3-[11C]methoxyphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one ([11C]-3), and evaluated as PET reporter probes for varicella-zoster virus thymidine kinase (VZV-tk) gene expression imaging in brain. [18F]-2 and [11C]-3 were synthesized starting from phenol precursor 1. The phenol precursor 1 was converted to stable as well as to radiolabeled compounds 2 and 3 using (19/18)FCH(2)CH(2)Br or (12/11)CH(3)I as alkylating agent. In vitro evaluation of [18F]-2 and [11C]-3 in 293T cells showed a 4.5 and 53-fold higher uptake, respectively, into VZV-tk gene-transduced cells compared to control cells. However, biodistribution studies in mice demonstrated low uptake of these tracers in the brain. RP-HPLC analysis of plasma and urine samples of mice injected with [11C]-3 revealed that this tracer is very stable in vivo. These data warrant further evaluation of these tracers as noninvasive imaging agents for VZV infection and VZV-tk reporter gene expression in vivo.

Evaluation of (99m)Tc-labeled tropanes with alkyl substituents on the 3beta-phenyl ring as potential dopamine transporter tracers.

Technetium(V)-oxo-3beta-(4-chlorophenyl)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl[N-(2-mercaptoethyl), N-(N'-(2-mercaptoethyl)-2-aminoethyl)]-aminomethyl ((99m)Tc-TRODAT-1) and three derivatives with one or two substituents on the 3beta-phenyl ring (4-methylphenyl, 4-ethylphenyl and 2,4-dimethylphenyl) were prepared and evaluated as potential imaging agents for the central nervous dopamine transporter (DAT). Labeling of the ligands with (99m)Tc yielded for each of them a mixture of two radiolabeled species, which were purified and isolated using reversed-phase high-performance liquid chromatography. Employing radio-LC-MS, we found both species to have the same molecular mass suggesting diastereoisomers. After intravenous injection in mice and rats, the compounds were stable in vivo and no important metabolites were found in plasma or urine. Replacement of the 4-chloro atom on the 3beta-phenyl ring by a methyl group causes no loss of affinity for the DAT system. However, substitution of an ethyl group for the 4-chloro atom or introduction of a second methyl group in the 2-position of the phenyl ring results in a serious reduction of the affinity for the DAT transporter. Ex vivo autoradiography on mice brain slices and biodistribution studies in rats showed specific uptake of (99m)Tc-TRODAT-1 and the 4-methylphenyl derivative in striatum and putamen. Although the 4-ethylphenyl and 2,4-dimethylphenyl derivatives show brain uptake in rats and mice, no specific uptake in striatum was found. In addition, differences in biological behavior between the different diastereomers were observed. In conclusion, small changes to (99m)Tc-TRODAT-1 at the phenyl ring in the 3beta position of the tropane moiety significantly change the biological behavior of the studied compounds.