Search

Radioligand therapy targeting the prostate-specific membrane antigen (PSMA) is rapidly evolving as a promising treatment for metastatic castration-resistant prostate cancer. The PSMA-targeting ligand p-SCN-Bn-TCMC-PSMA (NG001) labelled with 212 Pb efficiently targets PSMA-positive cells in vitro and in vivo. The aim of this preclinical study was to evaluate the therapeutic potential of 212 Pb-NG001 in multicellular tumour spheroid and mouse models of prostate cancer. The cytotoxic effect of 212 Pb-NG001 was tested in human prostate C4-2 spheroids. Biodistribution at various time points and therapeutic effects of different activities of the radioligand were investigated in male athymic nude mice bearing C4-2 tumours, while long-term toxicity was studied in immunocompetent BALB/c mice. The radioligand induced a selective cytotoxic effect in spheroids at activity concentrations of 3-10 kBq/mL. In mice, the radioligand accumulated rapidly in tumours and was retained over 24 h, while it rapidly cleared from nontargeted tissues. Treatment with 0.25, 0.30 or 0.40 MBq of 212 Pb-NG001 significantly inhibited tumour growth and improved median survival with therapeutic indexes of 1.5, 2.3 and 2.7, respectively. In BALB/c mice, no signs of long-term radiation toxicity were observed at activities of 0.05 and 0.33 MBq. The obtained results warrant clinical studies to evaluate the biodistribution, therapeutic efficacy and toxicity of 212 Pb-NG001.

The nonselective Ca 2+ -permeable transient receptor potential channel subfamily member 5 (TRPC5) belongs to the transient receptor potential canonical (TRPC) superfamily and is widely expressed in the brain. Compelling evidence reveals that TRPC5 plays crucial roles in depression and other psychiatric disorders. To develop a TRPC5 radioligand, following up on our previous effort, we synthesized the iodine compound TZ66127 and its iodine-125-labeled counterpart [ 125 I]TZ66127. The synthesis of TZ66127 was achieved by replacing chloride with iodide in the structure of HC608, and the [ 125 I]TZ66127 was radiosynthesized using its corresponding tributylstannylated precursor. We established a stable human TRPC5-overexpressed HEK293-hTRPC5 cell line and performed Ca 2+ imaging and a cell-binding assay study of TZ66127; these indicated that TZ66127 had good inhibition activity for TRPC5, and the inhibitory efficiency of TZ66127 toward TRPC5 presented in a dose-dependent manner. An in vitro autoradiography and immunohistochemistry study of rat brain sections suggested that [ 125 I]TZ66127 had binding specificity toward TRPC5. Altogether, [ 125 I]TZ66127 has high potential to serve as a radioligand for screening the binding activity of other new compounds toward TRPC5. The availability of [ 125 I]TZ66127 might facilitate the development of therapeutic drugs and PET imaging agents that target TRPC5., (© 2020 Wiley-VCH GmbH.)

Radiosynthesis and evaluation of [ 11 C]GSK1838705A in mice using microPET and determination of specificity in human GBM UG87MR cells are described herein. The radioligand was synthesized by reacting desmethyl-GSK1838705A with [ 11 C]CH 3 I using GE FX2MeI module in ∼5% yield (EOS), >95% radiochemical purity and a specific activity of 2.5±0.5Ci/μmol. MicroPET imaging in mice indicated that [ 11 C]GSK1838705A penetrated blood brain barrier (BBB) and showed retention of radiotracer in brain. The radioligand exhibited high uptake in U87MG cells with >70% specific binding to IGF1R. Our experiments suggest that [ 11 C]GSK-1838705A can be a potential PET radiotracer for the in vivo quantification of IGF1R expression in GBM and other brain tumors., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Selective activation of the M1 muscarinic acetylcholine receptor (mAChR) via a positive allosteric modulator (PAM) is a new approach for the treatment of the cognitive impairments associated with schizophrenia and Alzheimer's disease. Herein, we describe the characterization of an M1 PAM radioligand, 8-((1S,2S)-2-hydroxycyclohexyl)-5-((6-(methyl-t3)pyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one ([(3)H]PT-1284), as a tool for characterizing the M1 allosteric binding site, as well as profiling novel M1 PAMs. 8-((1S,2S)-2-Hydroxycyclohexyl)-5-((6-methylpyridin-3-yl)methyl)-8,9-dihydro-7H-pyrrolo[3,4-hour]quinolin-7-one (PT-1284 ( 1: )) was shown to potentiate acetylcholine (ACh) in an M1 fluorometric imaging plate reader (FLIPR) functional assay (EC50, 36 nM) and carbachol in a hippocampal slice electrophysiology assay (EC50, 165 nM). PT-1284 ( 1: ) also reduced the concentration of ACh required to inhibit [(3)H]N-methylscopolamine ([(3)H]NMS) binding to M1, left-shifting the ACh Ki approximately 19-fold at 10 μM. Saturation analysis of a human M1 mAChR stable cell line showed that [(3)H]PT-1284 bound to M1 mAChR in the presence of 1 mM ACh with Kd, 4.23 nM, and saturable binding capacity (Bmax), 6.38 pmol/mg protein. M1 selective PAMs were shown to inhibit [(3)H]PT-1284 binding in a concentration-responsive manner, whereas M1 allosteric and orthosteric agonists showed weak affinity (>30 μM). A strong positive correlation (R(2) = 0.86) was found to exist between affinity values generated for nineteen M1 PAMs in the [(3)H]PT-1284 binding assay and the EC50 values of these ligands in a FLIPR functional potentiation assay. These data indicate that there is a strong positive correlation between M1 PAM binding affinity and functional activity, and that [(3)H]PT-1284 can serve as a tool for pharmacological investigation of M1 mAChR PAMs., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

The reference standard MK-1064 {5″-chloro-N-((5,6-dimethoxypyridin-2-yl)methyl)-[2,2':5',3″-terpyridine]-3'-carboxamide} was synthesized from methyl 2-chloro-5-iodonicotinate and 5-(chloropyridin-3-yl)boronic acid in 4 steps with 33% overall chemical yield. The precursor desmethyl-MK-1064 {5″-chloro-N-((5-hydroxy-6-methoxypyridin-2-yl)methyl)-[2,2':5',3″-terpyridine]-3'-carboxamide} for radiolabeling was synthesized from 2-bromopyridin-3-ol and 5″-chloro-[2,2':5',3″-terpyridine]-3'-carboxylic acid in 6 steps with 17% overall chemical yield. The target tracer [(11)C]MK-1064 {5″-chloro-N-((5-[(11)C]methoxy-6-methoxypyridin-2-yl)methyl)-[2,2':5',3″-terpyridine]-3'-carboxamide} was prepared by O-[(11)C]methylation of its corresponding precursor desmethyl-MK-1064 with [(11)C]CH3OTf under basic condition and isolated by a simplified solid-phase extraction (SPE) method in 50-60% decay corrected radiochemical yields based on [(11)C]CO2 at end of bombardment (EOB). The overall synthesis time from EOB was 23min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 185-555GBq/μmol., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Nuclear 9-cis retinoic acid receptors (retinoid X receptors, RXR) are promiscuous dimerization partners for a number of nuclear receptors. In the present study, we established a novel in vitro method for quantitative determination of the nuclear retinoid X receptors in rat liver. One type of high affinity and limited capacity RXR specific binding sites with the Ka value ranging from 1.011 to 1.727×10(9)l/mol and the Bmax value ranging from 0.346 to 0.567pmol/mg, was demonstrated. Maximal 9-cis retinoic acid (9cRA) specific binding to nuclear retinoid X receptors was achieved at 20°C, and the optimal incubation time for the 9cRA-RXR complex formation was 120min. From a number of endocrine disruptors, tributyltins and triphenyltins are known as RXR ligands. Our data confirmed the property of tributyltin chloride or triphenyltin chloride to bind to a high affinity and limited capacity RXR binding sites. Described optimal conditions for ligand binding to RXR molecules enabled us to calculate maximal binding capacity (Bmax) and affinity (Ka) values. This study provides an original RXR radioligand binding assay that can be employed for investigation of novel RXR ligands that comprise both drugs and endocrine disruptors., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The role of 2-methoxyestradiol is becoming a major area of investigation because of its therapeutic utility, though its mechanism is not fully explored. Recent studies have identified the G-protein-coupled receptor 30 (GPR30, GPER) as a high-affinity membrane receptor for 2-methoxyestradiol. However, studies aimed at establishing the binding affinities of steroid compounds for specific targets are difficult, as the tracers are highly lipophilic and often result in nonspecific binding in lipid-rich membrane preparations with low-level target receptor expression. 2-Methoxyestradiol binding studies are essential to elucidate the underlying effects of this novel estrogen metabolite and to validate its targets; therefore, this competitive receptor-binding assay protocol was developed in order to assess the membrane receptor binding and affinity of 2-methyoxyestradiol.

In recent years, mGlu4 has received great research attention because of the potential benefits of mGlu4 activation in treating numerous brain disorders, such as Parkinson's disease (PD). A specific mGlu4 PET radioligand could be an important tool in understanding the role of mGlu4 in both healthy and disease conditions, and also for the development of new drugs. In this study, we synthesized four new N-(methylthiophenyl)picolinamide derivatives 11-14. Of these ligands, 11 and 14 showed high in vitro binding affinity for mGlu4 with IC50 values of 3.4nM and 3.1nM, respectively, and suitable physicochemical parameters. Compound 11 also showed enhanced metabolic stability and good selectivity to other mGluRs. [(11)C]11 and [(11)C]14 were radiolabeled using the [(11)C]methylation of the thiophenol precursors 20a and 20c with [(11)C]CH3I in 19.0% and 34.8% radiochemical yields (RCY), and their specific activities at the end of synthesis (EOS) were 496±138GBq/μmol (n=6) and 463±263GBq/μmol (n=4), respectively. The PET studies showed that [(11)C]11 accumulated fast into the brain and had higher uptake, slower washout and 25% better contrast than [(11)C]2, indicating improved imaging characteristics as PET radiotracer for mGlu4 compared to [(11)C]2. Therefore, [(11)C]11 will be a useful radioligand to investigate mGlu4 in different biological applications., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Thediazabicyclic molecule bispidine named by the chemist Carl Mannich in 1930, is a naturally occurring scaffold with interesting features. Bispidine can form different conformers, has high basicity, can attack dichloromethane, has metal ion coordination properties and interacts with nicotinic acetylcholine receptors. In this review we will discuss important properties, synthetic pathways and biological activities of bispidine and some derivatives. Bispidine can function as a scaffold for compounds with very diverse biological activities, e.g. interacting with ion channels, G-protein coupled receptors, and enzymes, and is even used for the development of new in vivo radiotracers.

Radioligand binding assays on intact cells offer distinct advantages to those on membrane suspensions. Major pharmacological properties like drug affinity and binding kinetics are more physiologically relevant. Complex mechanisms can be studied with a wider choice of experimental approaches and so provide insights into induced-fit type binding, receptor internalisation and even into pharmacomicrokinetic phenomena like drug rebinding and partitioning into the membrane. Hence, intact cell binding constitutes a valuable addition to the pharmacologist's toolbox., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

This study establishes the expression of appreciable populations of sites on mouse lung membranes that exhibit radioligand binding properties and pharmacology consistent with assignment as sigma1 and sigma2 receptors. Specific binding of the sigma1 receptor radioligand [(3)H](+)-pentazocine reached steady state within 6h at 37°C. Saturation studies revealed high affinity binding to a single class of sites (Kd 1.36±0.04nM; Bmax 967±11fmol/mg protein). Inhibition studies showed appropriate sigma1 receptor pharmacology, including higher affinity for (+)-N-allylnormetazocine with respect to the (-)-enantiomer, and positive allosteric modulation of dextromethorphan binding by phenytoin. Using [(3)H]1,3-di(2-tolyl)guanidine in the presence of (+)-pentazocine to assess sigma2 receptor binding, steady state was achieved within 2min at 25°C. Cold saturation studies revealed one high affinity, low capacity binding site (Kd 31.8±8.3nM; Bmax 921±228fmol/mg protein) that displayed sigma2 receptor pharmacology. A very low affinity, high capacity interaction also was observed that represents saturable, but not sigma receptor specific, binding. A panel of ligands showed rank order inhibition of radioligand binding appropriate for the sigma2 receptor, with ifenprodil displaying the highest apparent affinity. In vivo, dextromethorphan inhibited the specific binding of a radioiodinated sigma1 receptor ligand in lung with an ED50 of 1.2μmol/kg, a value near the recommended dosage for the drug as a cough suppressant. Overall, the present work provides a foundation for studies of drug interactions with pulmonary sigma1 and sigma2 receptors in vitro and in vivo., (Published by Elsevier B.V.)

GAD autoantibodies (GADAs) are sensitive markers of islet autoimmunity and type 1 diabetes. They form the basis of robust prediction models and are widely used for the recruitment of subjects at high risk of type 1 diabetes to prevention trials. However, GADAs are also found in many individuals at low risk of diabetes progression. To identify the sources of diabetes-irrelevant GADA reactivity, we analyzed data from the 2009 and 2010 Diabetes Autoantibody Standardization Program GADA workshop and found that binding of healthy control sera varied according to assay type. The characterization of control sera found positive by radiobinding assay (RBA), but negative by ELISA, showed that many of these sera reacted to epitopes in the N-terminal region of the molecule. This finding prompted development of an N-terminally truncated GAD65 radiolabel, (35)S-GAD65(96-585), which improved the performance of most GADA RBAs participating in an Islet Autoantibody Standardization Program GADA substudy. These detailed workshop comparisons have identified a source of disease-irrelevant signals in GADA RBAs and suggest that N-terminally truncated GAD labels will enable more specific measurement of GADAs in type 1 diabetes., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Aim: The simplified reference tissue model (SRTM) is used for estimation of receptor occupancy assuming that the non-displaceable binding in the reference region is identical to the brain regions of interest. The aim of this work was to extend the SRTM to also account for inter-regional differences in non-displaceable concentrations, and to investigate if this model allowed estimation of receptor occupancy using white matter as reference. It was also investigated if an apparent higher affinity in caudate compared with other brain regions, could be better explained by a difference in the extent of non-displaceable binding., Methods: The analysis was based on a PET study in six healthy volunteers using the 5-HT1B receptor radioligand [(11)C]-AZ10419369. The radioligand was given intravenously as a tracer dose alone and following different oral doses of the 5-HT1B receptor antagonist AZD3783. Non-linear mixed effects models were developed where differences between regions in non-specific concentrations were accounted for. The properties of the models were also evaluated by means of simulation studies., Results: The estimate (95% CI) of Ki(PL) was 10.2 ng ml(-1) (5.4, 15) and 10.4 ng ml(-1) (8.1, 13.6) based on the extended SRTM with white matter as reference and based on the SRTM using cerebellum as reference, respectively. The estimate (95% CI) of Ki(PL) for caudate relative to other brain regions was 55% (48, 62%)., Conclusions: The extended SRTM allows consideration of white matter as reference region when no suitable grey matter region exists. AZD3783 affinity appears to be higher in the caudate compared with other brain regions., (© 2014 The British Pharmacological Society.)

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a major messenger for Ca(2+) mobilization in cells. NAADP-binding proteins are highly selective and have a strong affinity for NAADP. This is the basis of the radioreceptor binding assay, which is used to measure NAADP levels in cells and tissues and to identify cellular stimuli that use NAADP as an intracellular messenger. In the radioreceptor binding assay, radiolabeled NAADP ([(32)P]NAADP) competes with endogenous NAADP present in samples for binding to their receptors. Here, we describe the synthesis of [(32)P]NAADP for use in the radioreceptor binding assay., (© 2014 Cold Spring Harbor Laboratory Press.)

Many neuropsychiatric drugs interact with more than one molecular target, and therapeutic indices might be improved by prospectively designing compounds with profiles optimized against a combination of targets. The dibenzo-epine scaffold is considered a privileged structure, and this scaffold has been explored rigorously in the search for potential novel neuropharmacologic treatments. Members of this chemical class are known to interact with many receptors and transporters, particularly those of the biogenic amine class. In this study, four points of diversity within a dibenzo-epine scaffold were varied systematically and the pharmacologic profiles of the compounds were assessed across 14 receptors and transporters thought to be important to clinical profiles of efficacy and safety. The resulting data were analyzed using a modified forward selection linear regression procedure, thus revealing potential pharmacophoric relationships of the assessed targets within this chemical class. The results highlight a strong covariance across numerous targets. Moreover, the outcome quantifies the innately problematic issue of prospectively designing compounds with defined affinities across multiple targets. Finally, an exploration of the correspondence of binding affinities to in vitro functional activity reveals an additional layer of complexity central to prospectively designing compounds to engage multiple targets. The apparent relatedness of the 5-HT(2a) and D₂ activities suggests that the structural pharmacophores of these receptors overlap more closely with each other than with members of their respective families.

Neuroinflammation is a well-orchestrated, dynamic, multicellular process playing a major role in neurodegenerative disorders. The microglia which make up the innate immune system of the central nervous system are key cellular mediators of neuroinflammatory processes. In normal condition they exert a protective function, providing tissue repair by releasing anti-inflammatory cytokines and neurotrophic factors. Upon neuronal injury or infection, they become overactivated, thereby releasing neurotoxic substances, amplifying neuroinflammation leading to neurodegeneration. Positron emission tomography (PET) provides a sensitive non-invasive imaging technique to study and quantify receptor and enzyme expression. A radiolabeled tracer for a protein (over)expressed in neuroinflammation and more specifically for the overactivated microglia would be useful as a diagnostic tool in the follow-up of neuroinflammation progression and to study the efficacy of anti-inflammatory therapy over time. In this manuscript, an overview of potential PET tracer targets upregulated during neuroinflammation is provided together with the current radiotracers used to image these targets. In addition, lead structures to develop radiotracers for new targets are suggested.

G protein-coupled receptors (GPCRs) are a large group of receptors of great biological and clinical relevance. Despite this, the tools for a detailed analysis of ligand-GPCR interactions are limited. The aim of this paper was to demonstrate how ligand binding to GPCRs can be followed in real-time on living cells. This was conducted using two model systems, the radiolabeled porcine peptide YY (pPYY) interacting with transfected human Y2 receptor (hY2R) and the bombesin antagonist RM26 binding to the naturally expressed gastrin-releasing peptide receptor (GRPR). By following the interaction over time, the affinity and kinetic properties such as association and dissociation rate were obtained. Additionally, data were analyzed using the Interaction Map method, which can evaluate a real-time binding curve and present the number of parallel interactions contributing to the curve. It was found that pPYY binds very slowly with an estimated time to equilibrium of approximately 12h. This may be problematic in standard end-point assays where equilibrium is required. The RM26 binding showed signs of heterogeneity, observed as two parallel interactions with unique kinetic properties. In conclusion, measuring binding in real-time using living cells opens up for a better understanding of ligand interactions with GPCRs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In this study, positron emission tomography (PET) imaging with a radioligand to adenosine A2A receptors (A2AR)-a potent regulator of inflammation-was used to gain insight into the molecular alterations in normal-appearing white matter (NAWM) and gray matter (GM) in secondary progressive multiple sclerosis (SPMS). Normal-appearing white matter and GM, despite seeming normal in conventional magnetic resonance imaging (MRI), are important loci of widespread inflammation, neuronal damage, and source of progressive disability in multiple sclerosis (MS). Dynamic PET imaging using A2AR-specific [(11)C]TMSX and brain MRI with diffusion tensor imaging were performed to eight SPMS patients and seven healthy controls. Distribution volumes (VT) of [(11)C]TMSX were analyzed from 13 regions of interest using Logan plot with arterial plasma input. The SPMS patients had significantly increased [(11)C]TMSX-VT in NAWM compared with controls (mean (s.d.): 0.55 (±0.08) vs. 0.45 (±0.05); P=0.036). Both the increased VT and the decreased fractional anisotropy (FA) in NAWM were associated with higher expanded disability status scale (EDSS) scores (P=0.030 and P=0.012, respectively), whereas the T2-lesion load of SPMS patients did not correlate with EDSS. This study shows, that A2ARs are increased in the brain of SPMS patients, and that [(11)C]TMSX-PET provides a novel approach to learn about central nervous system pathology in SPMS in vivo.

This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to purified MHC molecules. This unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. An alternate protocol describes alterations in the basic protocol that are necessary when performing direct binding assays, which are required for (1) selecting appropriate high-affinity, assay-specific, radiolabeled ligands, and (2) determining the amount of MHC necessary to yield assays with the highest sensitivity. After a predetermined incubation period, dependent upon the allele under examination, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Three methods for separation are described, two utilizing size-exclusion gel-filtration chromatography and a third using monoclonal antibody capture of MHC. Data analysis for each method is also explained., (© 2013 by John Wiley & Sons, Inc.)

G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors as key targets for pharmacological manipulation. G proteins have long been recognized as allosteric modulators of GPCR ligand binding. More recently, small molecule allosteric modulators have now been widely characterized for a number of GPCRs, and some are now used clinically. Many studies have also underscored the importance of GPCR dimerization or higher-order oligomerization in the control of the physiological responses they modulate. Thus, allosterism can also, between monomer equivalents in the context of a dimer, oligomer, or receptor mosaic, influence signaling pathways downstream. It therefore becomes essential to characterize both small molecule allosteric ligands and allosteric interactions between receptors modulated by canonical orthosteric ligands, in a pathway-specific manner. Here, we describe a simple, radioligand-binding method, which is designed to probe for allosteric modulation mediated by any GPCR interactor, from small molecules to interacting proteins. It can also detect allosteric asymmetries within a GPCR heterodimer, via orthosteric or allosteric ligands. This assay measures time-dependent ligand occupancy of radiolabeled orthosteric or (with adaptations) allosteric ligands as modulated by either small molecules or receptor dimer partners bound or unbound with their own ligands., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Tritiated opioid ligands are essential tools for the identification of opioid receptors. This review deals with the syntheses of tritiated opioid peptide derivatives, including enkephalins, dynorphins, dermorphins, deltorphins and endomorphins, and also discusses tritium-labeled nonpeptide opioids. It additionally focuses on the relevance of tritium-labeled opioid compounds as research tools for investigating opioid receptor pharmacology. Agonists and antagonists are used for the characterization of new opioid ligands by means of radioreceptor binding assays. Further topics covered in this review are the distribution of the endogenous peptides in the central nervous system and peripheral tissues, and degradation studies of opioids in brain membrane preparations and the blood.

Human α(2)-adrenoceptors (α(2)-ARs) are rhodopsin-like G-protein coupled receptors, and potential drug targets. The three different human α(2)-AR subtypes α(2A), α(2B) and α(2C) are widely distributed in tissues, but so far only a few subtype-selective ligands have been identified. In this project, we set off to conduct a large chemical screen for activity on the human α(2B)-AR and studied the selectivity of the active compounds towards the human α(2A)- and α(2C)-AR subtypes. We employed a radioligand competition binding assay that was optimized and miniaturized into a robotic environment. Membrane fractions containing recombinant human receptor subtypes were prepared from stably transfected Chinese hamster ovary (CHO) cell lines. Initially identified hits were followed up and characterized, and chemoinformatics tools were applied to gain better understanding of the relevance of the results. After a primary screen against α(2B)-AR, 176 compounds of the 17,952 included in the library were declared as active at 10 μM, of which 89 compounds were further selected for potency and affinity determinations using the three human α(2)-AR subtypes. One of the identified positive hits was 2″,2″″-Bisepigallocatechin digallate, which was found to have high affinity at all three human α(2)-AR subtypes. This represents the first non-protonable molecule identified as able to interact with these receptors. Additionally, results obtained with a functional assay (agonist-induced stimulation of [(35)S]GTPγS binding) supported the identification of another positive hit, lysergol, as a partial agonist of the human α(2)-AR subtypes. The dataset of confirmed active chemical species represents a readily available, high quality source for follow-up studies. Altogether, these results provide novel research approaches for drug discovery of modulators of the α(2)-AR subtypes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The development of assays in single-addition mode is of great interest for screening purposes given the multiple advantages of minimizing the number of intervention steps. Binding assays seem to be more prone to this attractive format because no functional biological activity is taking place but instead a biophysical process, whose dynamics seem easier to control without introducing significant alterations, is happening. Therefore, single-addition assays based on the displacement of prebound labeled ligands can be conceived, but careful kinetic considerations must still be taken to maximize the sensitivity of the assay and to avoid jeopardizing the identification of compounds with slow-binding kinetics. This article shows the development of a single-addition, displacement-based binding assay intended to identify modulators that act by binding to the gabapentin site of the ion channel regulatory protein α2δ1. After studying the kinetics of gabapentin binding and the influence they might have on the assay sensitivity, the best conditions were identified, and the sensitivity was compared with that of the more classical two-additions competition-based assay. Although the present study focuses on α2δ1 and its interaction with gabapentin, the rationale and the methodology followed are of broad purpose and can be applied to virtually every binding assay.

Introduction: In many situations, optimal drug therapy requires continuing high levels of target occupancy and this notion has led pharmacologists to focus their attention on the rate by which drug candidates dissociate from their target. To this end, radioligand dissociation experiments are often carried out on in vitro models, such as intact cells and the membranes thereof, but the interpretation of the collected data is sometimes ambiguous., Areas Covered: Pharmacodynamics is concerned about what the drug does to the target and, in this respect, allosteric modulation constitutes a quite novel, very promising research topic. The ability of unlabeled drugs to accelerate radioligand dissociation is often advocated to be a hallmark of such mechanism. Yet, the present computerized simulations reveal that competitive drugs produce the same effect by preventing hindered diffusion- and "forced proximity"-related rebinding of the radioligand. Herein, the authors provide hints to discern among those mechanisms., Expert Opinion: A critical, but constructive appraisal of radioligand dissociation binding data leads to the viewpoint that, from a physiological perspective, dissociation from confluent target-expressing plated cells, when in a naïve medium, is likely to provide the most pertinent insight in that ligand's in vivo residence time.

Patients with autoimmune polyendocrine syndrome type I (APS I) or acquired thymoma-associated myasthenia gravis (MG) surprisingly share several common features, including defective expression of the transcription factor AIRE and autoantibodies against type I interferons. Here, we have adapted and validated the radioligand-binding assay we recently developed against (35)S-Met-interferon-ω, for rapid and specific screening for autoantibodies against interferons-α2 and -α8. We then investigated their potential for diagnosis and for predicting clinical manifestations in patients with APS I and different subgroups of MG. Autoantibodies against interferons-ω, -α2, and -α8 occurred more often in patients with APS I (100%) and MG with thymoma (73%) than in late-onset MG (39%) and early-onset MG (5%). These autoantibodies showed preferences for interferon-ω in APS I and for the interferon-αs in MG, hinting at thymic aberrations in both groups. The exact profile of type I interferon antibodies may indicate MG subtype and may hint at thymoma recurrence.

Background: Tissue transglutaminase (tTG) autoantibodies are serological markers for celiac disease. The aim was to study the efficacy of the pTnT-rhtTG plasmid and subsequent diagnostic accuracy of tTG autoantibodies for childhood celiac disease using radioligand binding assays., Methods: Coupled in vitro transcription and translation of tTG were performed by pTnT-rhtTG as well as by the pGEMt Easy-rhtTG vectors using the TNT SP6 Coupled Reticulocyte Lysate System in the presence of [³⁵S] methionine. Sera from 190 celiac disease children and 74 controls were measured for tTG autoantibodies in two separate radioligand binding assays using anti-human IgA agarose and protein A sepharose beads for the detection of IgA-tTG and IgG-tTG, respectively., Results: Median incorporation of [³⁵S] methionine into the pTnT-rhtTG was 26% compared to 16% for the pGEMt Easy-rhtTG plasmid (p = 0.0016). Using pTnT-rhtTG (as compared to pGEMt Easy-rhtTG), sensitivities were IgA-tTG = 96.3% (95.7%) and IgG-tTG = 95.8% (97.3%) and specificities were IgA-tTG = 91.9% (90.5%) and IgG-tTG = 94.6% (98.4%). According to receiver operator characteristics for the pTnT (pGEMt Easy) assays, area under the curves were IgA-tTG = 98.4% (98.4%) and IgG-tTG = 97.7% (97.2%), respectively., Conclusion: The pTnT-rhtTG plasmid increased the efficacy of tTG antigen usage without reducing the diagnostic accuracy of IgA-tTG and IgG-tTG for childhood celiac disease. The pTnT-rhtTG plasmid is therefore recommended over the pGEMt Easy-rhtTG for the assessment of IgA-tTG and IgG-tTG using radioligand binding assays.

Objective: To characterize pharmacologically relevant muscarinic receptors in the human bladder mucosa and detrusor muscle using radioligand binding assays with [N-methyl-3H]scopolamine methyl chloride ([3H]NMS) and 4-DAMP mustard., Methods: Muscarinic receptors in homogenates of bladder mucosa, detrusor muscle, and parotid gland were measured using the radioligand [3H]NMS. 4-DAMP mustard was used to inactivate M3 receptors irreversibly., Results: Specific [3H]NMS binding in the homogenates of the mucosa and detrusor muscle was saturable and of high affinity as shown by dissociation constants (Kd) of 260 ±82 and 237 ±49 pM, respectively. Antimuscarinic agents (oxybutynin, propiverine, tolterodine, and darifenacin) and their active metabolites competed with [3H]NMS for the binding sites in the human mucosa in a concentration-dependent manner. These agents exhibited similar affinity in the detrusor muscle. The Bmax. values of [3H]NMS in the detrusor, bladder mucosa, and parotid gland were significantly decreased by pretreatment with 4-DAMP mustard (36%, 41% and 63%, respectively)., Conclusion: The density and binding affinity profile of the muscarinic receptor population in the human bladder mucosa was shown to be similar to that of the detrusor muscle. The density of the M3 subtype in the mucosa was similar to that in the detrusor muscle but lower than that in the parotid gland., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

N-acetylglucosaminyltransferase V (GnT-V) is one of the most relevant glycosyltransferases to tumor invasion and metastasis. Based on previous findings of molecular recognition between GnT-V and synthetic substrates, we designed and synthesized a p-iodophenyl-derivatized trisaccharide, 2-(4-iodophenyl)ethyl 6-O-[2-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-α-d-mannopyranosyl]-β-D-glucopyranoside (IPGMG, 1) and its radiolabeled form, [(125)I]IPGMG ([(125)I]1), for use in assays of GnT-V activity in vitro. The tributyltin derivative, 2-[4-(n-tributylstannyl)phenyl]ethyl 6-O-[2-O-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl]-2,3,4-tri-O-acetyl-β-D-glucopyranoside (21), was synthesized as a precursor for the preparation of [(125)I]1. The iododestannylation of 21 using hydrogen peroxide as an oxidant followed by deacetylation yielded [(125)I]1. When [(125)I]1 was incubated in GnT-V-expressing cells with a UDP-GlcNAc donor, the production of β1-6GlcNAc-bearing IPGMG (IPGGMG, 2) was confirmed by radio-HPLC. In kinetic analysis, 1 was found to be a good substrate with a K(m) of 23.7 μM and a V(max) of 159 pmol/h. μg protein. [(125)I]1 would therefore be a useful synthetic substrate for the quantitative determination of GnT-V activity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Tritiated opioid radioligands have proven valuable in exploring opioid binding sites. However, tritium has many limitations. Its low specific activity and limited counting efficiency makes it difficult to examine low abundant, high affinity sites and its disposal is problematic due to the need to use organic scintillants and its relatively long half-life. To overcome these issues, we have synthesized both unlabeled and carrier-free radioiodinated iodobenzoyl derivatives of 6β-naltrexamine ((125)I-BNtxA, 18), 6β-naloxamine ((125)I-BNalA, 19) and 6β-oxymorphamine ((125)I-BOxyA, 20) with specific activities of 2100Ci/mmol. To optimize the utility of the radioligand, we designed a synthesis in which the radiolabel is incorporated in the last synthetic step, which required the selective iodination of the benzoyl moiety without incorporation into the phenolic A ring. Competition studies demonstrated high affinity of the unlabelled compounds for opioid receptors in transfected cell lines, as did the direct binding of the (125)I-ligands to the opioid receptors. The radioligand displayed very high sensitivity, enabling a marked reduction in tissue, as well as excellent signal/noise characteristics. These new (125)I-radioligands should prove valuable in future studies of opioid binding sites., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The drive to produce safer and more receptor subtype selective drugs has sparked a renewed interest in allosteric modulators of G protein-coupled receptors. The increasing use of functional assays has shown that allosteric ligands are capable of modulating both orthosteric agonist affinity and efficacy, as well as mediating receptor activation in their own right. Such a complex range of behaviours can be difficult to discern from single datasets; this chapter seeks to explain how to use radioligand binding and functional assay datasets in concert to elucidate allosteric modulator mechanism of action.

The radioligand binding assay is a relatively simple but powerful tool for studying G protein-coupled receptors. There are three basic types of radioligand binding experiments: (1) saturation experiments from which the affinity of the radioligand for the receptor and the binding site density can be determined; (2) inhibition experiments from which the affinity of a competing, unlabeled compound for the receptor can be determined; and (3) kinetic experiments from which the forward and reverse rate constants for radioligand binding can be determined. Detailed methods for typical radioligand binding assays for G protein-coupled receptors in membranes and intact cells are presented for these types of experiments. Detailed procedures for analysis of the data obtained from these experiments are also given.

Some G protein-coupled receptors (GPCRs) have functional links to cancer biology, yet the manifestation of GPCRs in tumor types is little studied to date. Using a battery of radioligand binding assays, we sought to characterize GPCR recognition binding sites on HeLaS3 tumor cells. High levels of binding of the selective serotonin 5-HT(1A) receptor agonist [3H]8-OH-DPAT were observed in these cells. Saturation and homologous competition experiments indicated that [3H]8-OH-DPAT bound different populations of high- and low-affinity sites. In competition experiments, several serotonergic compounds displaced [3H]8-OH-DPAT binding with low potency from its high-affinity binding sites, suggesting that low-affinity binding is the predominant mode of binding. A variety of drugs targeting different classes of receptors did not affect [3H]8-OH-DPAT binding. These observations may help elucidate the pathophysiological and functional relevance of 5-HT receptors in tumor cells and link GPCRs and tumorigenic mechanisms to pharmacological and chemotherapeutic paradigms., (2009 Elsevier Inc. All rights reserved.)

Over the last years, ligands for angiotensin II subtype 1 receptor (AT1R) have been developed as an alternative to the use of angiotensin-converting enzyme (ACE) inhibitors for controlling high blood pressure. Radiolabeled versions of these ligands have proven vital to the development of more potent and specific drugs for the treatment of hypertension. Imaging studies using radiolabeled AT1R ligands have also elucidated the role these receptors play in angiogenesis as well as in various disease states beyond hypertension.

Autoantibodies to islet antigen 2 (IA-2A) are important markers for predicting diabetes in children and young adults. Harmonization of IA-2A assay measurement is essential if results from different laboratories are to be compared. We investigated whether sodium azide, a bacteriostatic agent added to some assays, could affect IA-2A binding and thereby contribute to differences in IA-2A measurement between laboratories. Addition of 0.1% azide to assay buffer was found to reduce median IA-2A binding of 18 selected sera from IA-2A positive patients with type 1 diabetes and their relatives by 41% (range, 78 to -33%, p<0.001). The effect on binding was epitope specific; median IA-2A binding by 14 sera with antibodies to the protein tyrosine phosphatase region of IA-2 was reduced by 48% (range, 11 to 78%, p<0.001), while binding by 4 sera with antibodies specific to only the juxtamembrane region of IA-2 showed no change (median increase 16% (range 6 to 33%, p=0.125). When the Tween-20 concentration was reduced from 1% to 0.15% the median reduction in IA-2A binding with azide by the 18 sera was only 10% (range, -12 to 41%, p<0.001). Tween-20 also exerted an independent effect, since median IA-2A binding increased by 23% (range 3% to 86%, p<0.001) when Tween-20 concentration was reduced from 1% to 0.15% in the absence of azide. We conclude that common assay reagents such as azide and Tween-20 can strongly influence IA-2A binding in an epitope-related manner, and their use may explain some of the differences between laboratories in IA-2A measurement.

Species differences occur in the brain concentrations of drugs, but the reasons for these differences are not yet apparent. This study was designed to compare brain uptake of three radiolabeled P-glycoprotein (P-gp) substrates across species using positron emission tomography. Brain concentrations and brain-to-plasma ratios were compared; [(11)C]verapamil in rats, guinea pigs, and monkeys; [(11)C](S)-(2-methoxy-5-(5-trifluoromethyltetrazol-1-yl)-phenylmethylamino)-2(S)-phenylpiperidine (GR205171) in rats, guinea pigs, monkeys, and humans; and [(18)F]altanserin in rats, minipigs, and humans. The fraction of the unbound radioligand in plasma was studied along with its metabolism. The effect of P-gp inhibition was investigated by administering cyclosporin A (CsA). Pronounced species differences were found in the brain and brain-to-plasma concentrations of [(11)C]verapamil, [(11)C]GR205171, and [(18)F]altanserin with higher brain distribution in humans, monkeys, and minipigs than in rats and guinea pigs. For example, the brain-to-plasma ratio of [(11)C]GR205171 was almost 9-fold higher in humans compared with rats. The species differences were still present after P-gp inhibition, although the increase in brain concentrations after P-gp inhibition was somewhat greater in rats than in the other species. Differences in plasma protein binding and metabolism did not explain the species-related differences. The findings are important for interpretation of brain drug delivery when extrapolating preclinical data to humans. Compounds found to be P-gp substrates in rodents are likely to also be substrates in higher species, but sufficient blood-brain barrier permeability may be retained in humans to allow the compound to act at intracerebral targets.

We introduce a novel experimental method to determine both the extent of ex vivo receptor occupancy of administered compound and its dissociation rate constant (k4). [Here, we reference k4 as the rate of offset of unlabeled ligand in convention with Motulsky and Mahan (1)]. We derived a kinetic rate equation based on the dissociation rate constant for an unlabeled compound competing for the same site as a labeled compound and describe a model to simulate fractional occupancy. To validate our model, we performed in vitro kinetics and ex vivo occupancy experiments in rat cortex with varying concentrations of (R)-dimethindene, a sedating antihistamine. Brain tissue was removed at various times post oral administration, and histamine H1 receptor ligand [3H]-doxepin binding to homogenates from drug-treated or vehicle-treated rats was measured at multiple time points at room temperature. Fractional occupancy and k4 for (R)-dimethindene binding to H1 receptors were calculated by using our proposed model. Rats dosed with 30 and 60 mg/kg (R)-dimethindene showed 42% and 67% occupancy of central H1 receptors, respectively. These results were comparable to occupancy data determined by equilibrium radioligand binding. In addition, drug k4 rate determined by using our ex vivo method was equivalent to k4 determined by in vitro competition kinetics (dissociation half-life t(1/2) approximately 30 min). The outlined method can be used to assess, by simulation and experiment, occupancy for compounds based on dissociation rate constants and contributes to current efforts in drug optimization to profile antagonist efficacy in terms of its kinetic drug-target binding parameters. Data described by the method may be analyzed with commercially available software. Suggested fitting procedures are given in the appendix.

Medullary thyroid carcinoma (MTC) expresses CCK-2 receptors. (111)In-labeled DOTA-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH(2) (DOTA-MG11), DOTA-DAsp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH(2) (DOTA-CCK), and (99m)Tc-labeled N(4)-Gly-DGlu-(Glu)(5)-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH(2) ((99m)Tc-Demogastrin 2) are analogs developed for CCK-2 receptor-targeted scintigraphy. All 3 radiolabeled analogs were selected on the basis of their high CCK-2 receptor affinity and their good in vitro serum stability, with in vitro serum t(1/2) values of several hours. Radiolabeling of DOTA-peptides with (111)In requires a heating procedure, typically in the range of 80 degrees -100 degrees C up to 30 min. Following this procedure with DOTA-MG11 resulted in a >98 % incorporation of (111)In, however, with a radiochemical purity (RCP) of <50 %. The decrease in RCP was found to be due to oxidation of the methionine residue in the molecule. Moreover, this oxidized compound lost its CCK-2 receptor affinity. Therefore, conditions during radiolabeling were optimised: labeling of DOTA-MG11 and DOTA-CCK with (111)In involved 5 min heating at 80 degrees C and led to an incorporation of (111)In of >98 %. In addition, all analogs were radiolabeled in the presence of quenchers to prevent radiolysis and oxidation resulting in a RCP of >90 %. All 3 radiolabeled analogs were i.v. administered to 6 MTC patients: radioactivity cleared rapidly by the kidneys, with no significant differences in the excretion pattern of the 3 radiotracers. All 3 radiolabeled analogs exhibited a low in vivo stability in patients, as revealed during analysis of blood samples, with the respective t(1/2) found in the order of minutes. In patient blood, the rank of radiopeptide in vivo stability was: (99m)Tc-Demogastrin 2 (t(1/2) 10-15 min)>(111)In-DOTA-CCK (t(1/2) approximately 5-10 min)>(111)In-DOTA-MG11 (t(1/2)<5 min).

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.

Analysis of dopamine receptors (DR) in lymphocytes of the human peripheral blood mononuclear cell (PBMC) fraction is an attractive tool for evaluation of functional properties of dopaminergic function underlying variation in complex psychological/psychopathological traits. Receptor binding assays (RBAs) with selective radioligands, which are widely used in CNS studies, have not produced consistent results when applied to isolated PBMC. We tested the assay conditions that could be essential for detection of DR in human PBMC and their membrane preparations. Using [(3)H]SCH23390, a dopamine D1-like receptor antagonist, we demonstrated the presence of two binding sites in PBMC-derived membrane fraction. One of them is characterized by the K(d) value consistent with that reported for D5 dopamine receptors in human lymphocytes, whereas the other K(d) value possibly corresponds to serotonin receptor(s). Although D5 receptor binding sites in PBMC membranes could be characterized by binding assays, the low protein expression and the large volume of blood needed for membrane preparation render the binding method impracticable for individual phenotyping. In contrast, real-time RT-PCR may be used for this purpose, contingent on the relationship between DR expression in the brain and in lymphocytes. The expression of the DRD2-DRD5 genes, as detected by this method, varied widely among samples, whereas the DRD1 expression was not detected. The expression levels were comparable with those in the brain for DRD3 and DRD4, and were significantly lower for DRD2 and DRD5.

The synthesis and investigation of 5-[125I]iodoindol-3-yl-beta-d-galactopyranoside ([125I]IBDG) as a radioligand for single-photon emission computed tomography (SPECT) imaging of b-galactosidase expression are described. No-carrier-added [125I]IBDG was synthesized by a radioiododestannylation approach in > 75% overall radiochemical yield and > 99% radiochemical purity. [125I]IBDG was evaluated as a substrate using beta-galactosidase-expressing (D54L) and nonexpressing (D54) human glioma cell lines. A 24-hour incubation of this substrate with cultured cells revealed a 6.5-fold greater intracellular trapping of radioactivity in D54L cells compared with D54 cells. Systemic delivery of [125I]IBDG in nude mice bearing D54L tumors failed to show significant trapping of radioactivity within these tumors by SPECT imaging. In contrast, intratumoral injection of the substrate resulted in efficient trapping of radioactivity in D54L tumors but not D54 tumors, resulting in clear SPECT visualization of the former tumor. Based on dynamic SPECT imaging and blood metabolite analysis, we conclude that although [125I]IBDG is an efficient in vivo substrate for beta-galactosidase, its rapid renal clearance hampers its intratumoral availability on systemic administration.

The ability of the sigma(1) receptor to interact with a huge range of drug structural classes coupled with its wide distribution in the body has contributed to it being implicated as a possible therapeutic target for a broad array of disorders ranging from substance abuse to depression to Alzheimer's disease. Surprisingly, the reported affinity values for some sigma(1) receptor ligands vary more than 50-fold. The potential of the sigma(1) receptor as a pharmacotherapeutic target prompted us to develop an unambiguous assay system for measuring the affinity of ligands to the cloned human sigma(1) receptor. In the course of characterizing this system and determining the true affinity values for almost three dozen compounds, it was discovered that some dopamine D(4) receptor selective compounds bind sigma(1) receptors with high affinity. A systematic analysis of haloperidol-like compounds revealed a clear structure-affinity relationship amongst clinically relevant butyrophenones. The antidepressant fluvoxamine, the drug of abuse methamphetamine, and the neurosteroid progesterone were amongst the many ligands whose interactions with the sigma(1) receptor were confirmed with our screening assay.

Unlabelled: Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9,10-dimethoxy-1,3,4,6,7-hexahydro-11bH-benzo[a]-quinolizine, DTBZ) has become the ideal radioligand for the presynaptic vesicular monoamine transporter VMAT2 based on its high binding affinity and optimal lipophilicity., Objective: To develop an automatic procedure for labelling DTBZ with carbon-11, which has been shown to be a highly effective marker for in vivo studies of neuronal losses in animal models with Parkinson's disease using positron emission tomography (PET)., Materials and Methods: We have developed a new fully automated synthesis procedure to obtain 11C-(+)DTBZ quickly and simply through labelling the precursor -(+)desmethyldihy-drotetrabenazine- at room temperature in the presence of dimethyl sulfoxide (DMSO) and potassium hydroxide (KOH), using 11CH3I as primary precursor. The final purification was carried out by solid phase extraction using commercially available cartridges and the residual solvents (DMSO and ethyl ether) were eliminated by evaporation., Results: The whole procedure was automated, and after 54 syntheses, an average production of 1.94 GBq of sterile, pyrogen-free 11C-(+)DTBZ with a radiochemical purity > 99 % was obtained with 5 minutes irradiation and 6 minutes of synthesis after 11CH3I production. 11C-(+)DTBZ binding to presynaptic dopamine nerve terminals has been demonstrated by MicroPET studies in Wistar rats and M. Fascicularis monkeys., Conclusions: This new synthesis procedure is quick and simple, due to optimised techniques, which have allowed elimination of residual solvents based on their polarity for the final purification. It is also applicable to other automatic syntheses for obtaining compounds labelled by methylation reactions.

Benzamide derivatives as radiotracers have played an important role in diagnosing malfunction in dopaminergic neurotransmission. A variety of halogenated and two unsubstituted benzamide derivatives were synthesised and their in vitro affinities to dopaminergic, serotonergic and adrenergic receptors and their lipophilicities were determined. As references IBZM (3), raclopride (4) and FLB457 (5) were tested as well. The two iodinated compounds NAE (27) and NADE (28) displayed K(i) values of 0.68 and 14 nM for the D(2) receptor. The well-established radiotracers FP (1) and DMFP (2) showed affinities in the same range as did the brominated compounds NABrE (29) and NABrDE (30). The log D(7.4) values of 2.91 for NAE (27) and of 2.81 for NADE (28) are in the range of those found for IBZM (3), FP (1) and DMFP (2). These facts allow to expect good properties for the two iodinated compounds NAE (27) and NADE (28) regarding in vivo imaging with SPECT.

Background and Purpose: Agonist apparent affinities (pK(I)') in histamine H(3)-receptor binding assays were higher than expected from apparent affinity values (pK(app)) estimated in bioassay. Here, we investigate whether the degree of pK(I)' overestimation is related to agonist intrinsic efficacy, by studying the effect of buffer composition on the pK(I)' of ligands with varying intrinsic activity., Experimental Approach: In the guinea-pig ileum bioassay, intrinsic activity (alpha) was determined from the maximal inhibition of the contraction produced by increasing agonist concentration. pK(app) values were estimated using the method of Furchgott. The pK(L) of [(3)H]clobenpropit in guinea-pig cerebral cortex was estimated by saturation analysis in 20 mM HEPES-NaOH buffer (buffer B(0,0,0)), or buffer B(0,0,0) containing 70 mM CaCl(2), 100 mM NaCl and 100 mM KCl (buffer B(0.07,0.1,0.1)). PK(I) values were determined in competition studies in both buffers., Key Results: [(3)H]clobenpropit saturation isotherms had n (H) values of unity in both buffers. In buffer B(0.07,0.1,0.1), agonist pK(I)' values were closer to pK(app) values than in buffer B(0,0,0) but were associated with n (H) values <1. A two-site analysis of agonist data in buffer B(0.07, 0.1, 0.1) provided a better fit than a one-site fit and low affinity values (pK(IL)) were comparable to pK(app). Differences between the pK(I)' in buffer B(0,0,0) and pK(IL) values in buffer B(0.07,0.1,0.1) (DeltapK) were correlated with alpha., Conclusions and Implications: H(3)-receptor binding assays conducted in buffer B(0,0,0) and buffer B(0.07,0.1,0.1) can provide a measure of ligand affinity (pK(app)) and intrinsic efficacy. The assay predicts that some ligands previously classified as H(3)-receptor antagonists may possess residual intrinsic efficacy.

Purpose: Silodosin (KMD-3213 or [(-)-1-(3-hydroxypropyl)-5-[(2R)-2-({2-[2-(2,2,2trifluoroethoxy)phenoxy]ethyl}amino)propyl]-2,3-dihydro-1H-indole-7-carboxamide]) (Kissei Pharmaceutical Co., Ltd., Matsumoto, Japan) is a selective antagonist for alpha-1A and alpha-1L adrenoceptors. Using this tritiated ligand the 2 alpha-1 adrenoceptors were examined in binding studies with intact tissue segments and membrane preparations of human prostate, and compared with functionally identified alpha-1 adrenoceptor., Materials and Methods: Binding assays with tissue segments and membrane preparations of human prostate samples were performed using [3H]-silodosin and binding affinities for various drugs were estimated. In functional experiments antagonist affinities were evaluated from the inhibitory potency against the contractile response to noradrenaline., Results: [3H]-silodosin bound to intact segments and membrane preparations of human prostate with subnanomolar affinity. [3H]-silodosin binding sites in intact segments were divided into 2 distinct components with different affinities for prazosin and RS-17053 (N-[2(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha, alpha-dimethyl1H-indole-3-ethanamine hydrochloride) (Research Biochemicals International, Natick, Massachusetts), while binding in membrane preparations showed single high affinity for these drugs. [3H]-silodosin binding sites also showed high affinity for silodosin and tamsulosin but low sensitivity to BMY 7378 (8-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4.5)decane-7,9-dione) (Research Biochemicals International) in intact segments and in membrane preparations. In functional experiments silodosin and tamsulosin potently inhibited the contractile response to noradrenaline but prazosin, RS-17053 and BMY 7378 showed low antagonistic affinity., Conclusions: The current binding studies in human prostate samples clearly show that alpha-1L and alpha-1A adrenoceptors coexist as pharmacologically distinct entities in intact tissues but not in crude membrane preparations. Also, alpha-1 adrenoceptors involved in the contractile response to noradrenaline are the alpha-1L subtype.

[(18)F]FCWAY and [(18)F]FPWAY, analogues of the high affinity 5-HT(1A) receptor (5-HT(1A)R) antagonist WAY100635, were evaluated in rodents as potential radiopharmaceuticals for determining 5-HT(1A)R density and changes in receptor occupancy due to changes in endogenous serotonin (5-HT) levels. The in vivo hippocampus specific binding ratio [(hippocampus(uptake)/cerebellum(uptake))-1] of [(18)F]FPWAY was decreased to 32% of the ratio of [(18)F]FCWAY, indicating that [(18)F]FPWAY has lower affinity than [(18)F]FCWAY. The 5-HT(1A)R selectivity of [(18)F]FPWAY was confirmed using ex vivo autoradiography studies with 5-HT(1A)R knockout, heterozygous, and wildtype mice.Pre- or post-treatment of awake rodents in tissue dissection studies with paroxetine had no effect on hippocampal binding of [(18)F]FCWAY or [(18)F]FPWAY compared to controls, indicating neither tracer was sensitive to changes in endogenous 5-HT. In mouse ex vivo autoradiography studies in which awake mice were treated with fenfluramine following the [(18)F]FPWAY, a significant decrease was not observed in the hippocampus specific binding ratios. In rat dissection studies with fenfluramine administered following [(18)F]FPWAY or [(18)F]FBWAY ([(18)F]-MPPF) in awake or urethane-anesthetized rats, no significant differences in the specific binding ratios of the hippocampus were observed compared to their respective controls. [(18)F]FPWAY and [(18)F]FBWAY uptakes in all brain regions were increased variably in the anesthetized group (with the greatest increase in the hippocampus) vs. the awake group, but were decreased in the fenfluramine-treated anesthetized group vs. the anesthetized group. These data are best explained by changes in blood flow caused by urethane and fenfluramine, which varies from region to region in the brain.

The binding and functional properties of adenosine receptor ligands were compared in Chinese hamster ovary cells transfected with human adenosine A(3) receptors. Inhibition of [(125)I]-aminobenzyl-5'-N-methylcarboamidoadenosine ([(125)I]-AB-MECA) binding by adenosine receptor ligands was examined in membrane preparations. Inhibition of forskolin-induced cAMP accumulation by agonists was measured using a cAMP enzyme immunoassay. The rank order of agonist potency for both assays was N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > (-)-N(6)-[(R)-phenylisopropyl] adenosine (R-PIA) > 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA) > N(6)-cyclopentyl adenosine (CPA) > adenosine. The radioligand binding rank order of antagonist potency was N-[9-chloro-2-(2-furanyl)[1,2,4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide (MRS1220) > 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) > 8-phenyltheophylline (8-PT) > 8-(p-sulfophenyl)-theophylline (8-SPT). MRS1220 competitively inhibited the effect of IB-MECA on cAMP production, with a K(B) value of 0.35 nm. These data are characteristic of adenosine A(3) receptors. The absence of Mg(2+) and presence of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) significantly reduced agonist binding inhibition potency, indicating binding to high- and low-affinity states. The IB-MECA, NECA and R-PIA IC(50) values were greater for the cAMP assay than for radioligand binding, suggesting an efficient stimulus-response transduction pathway.