Rostoucí incidence karcinomu prostaty je – především s ohledem na snadno vyčerpatelné způsoby terapie při progresi tohoto onemocnění do metastatického kastračně rezistentního stadia – hlavní hnací silou pro rozvoj nových léčebných modalit. Radioligandová terapie je jednou z možností, ke které by bylo možné se uchýlit. Tento způsob léčby využívá ligandy schopné cílit na konkrétní buněčné struktury, a dopravit tak zdroj ionizujícího záření přímo k nádorovým buňkám. Radiofarmakum tak působí radiační poškození přímo nádorovým buňkám při minimálním ozáření zdravé tkáně. Lutecium (177Lu) vipivotid tetraxetan (přípravek Pluvicto) patří mezi radiofarmaka, která je dnes možné použít k radioligandové léčbě, konkrétně k terapii metastatického kastračně rezistentního karcinomu prostaty u pacientů, u kterých jiné léčebné postupy již selhaly. Tento článek si klade za cíl shrnout dosavadní poznatky o výzkumu a možnostech použití tohoto radiofarmaka.

The increasing incidence of prostate cancer, especially with regard to the easily exhausted methods of therapy when once developed to the metastatic castration-resistant stage, is the main driving force for the development of new treatment modalities. Radioligand therapy is one option that one can resort to. This therapy uses ligands capable of targeting specific cell structures, thereby delivering a source of ionizing radiation directly to the tumour cells. The radiopharmaceutical thus causes radiation damage directly to the tumour cells with minimal irradiation of healthy tissue. Lutetium ( 177 Lu) vipivotide tetraxetan (Pluvicto) belongs to the radiopharmaceuticals that can be used today for radioligand therapy, specifically for the treatment of metastatic castration-resistant prostate cancer in cases when other treatments have already failed. This article aims to summarize the current knowledge about research and use of this radiopharmaceutical.

• MeSH
• klinická studie jako téma MeSH
• lidé MeSH
• lutecium * farmakokinetika   farmakologie   terapeutické užití   účinky záření   MeSH
• nádory prostaty rezistentní na kastraci MeSH
• nádory prostaty * terapie   MeSH
• prostatický specifický antigen MeSH
• protokoly protinádorové léčby MeSH
• radiofarmaka aplikace a dávkování   farmakologie   terapeutické užití   MeSH
• radioligandová zkouška metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

We adapted a radioligand receptor binding assay for measuring insulin levels in unknown samples. The assay enables rapid and accurate determination of insulin concentrations in experimental samples, such as from insulin-secreting cells. The principle of the method is based on the binding competition of insulin in a measured sample with a radiolabeled insulin for insulin receptor (IR) in IM-9 cells. Both key components, radiolabeled insulin and IM-9 cells, are commercially available. The IR binding assay was used to determine unknown amounts of insulin secreted by MIN6 β cell line after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. The experimental data obtained by the IR binding assay were compared to the results determined by RIA kits and both methods showed a very good agreement of results. We observed the stimulation of glucose-induced insulin secretion from MIN6 cells by arginine, weaker stimulation by ornithine, but inhibitory effects of dopamine. Serotonin effects were either stimulatory or inhibitory, depending on the concentration of serotonin used. The results will require further investigation. The study also clearly revealed advantages of the IR binding assay that allows the measuring of a higher throughput of measured samples, with a broader range of concentrations than in the case of RIA kits. The IR binding assay can provide an alternative to standard RIA and ELISA assays for the determination of insulin levels in experimental samples and can be especially useful in scientific laboratories studying insulin production and secretion by β cells and searching for new modulators of insulin secretion.

• MeSH
• arginin metabolismus   MeSH
• beta-buňky metabolismus   MeSH
• buněčné linie MeSH
• dopamin metabolismus   MeSH
• glukosa metabolismus   MeSH
• inzulin analýza   metabolismus   MeSH
• krysa rodu rattus MeSH
• Langerhansovy ostrůvky metabolismus   MeSH
• lidé MeSH
• myši MeSH
• ornithin metabolismus   MeSH
• potkani Wistar MeSH
• radioimunoanalýza metody   MeSH
• radioligandová zkouška metody   MeSH
• sekrece inzulinu * MeSH
• serotonin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Insulin-like growth factors 2 and 1 (IGF2 and IGF1) and insulin are closely related hormones that are responsible for the regulation of metabolic homeostasis, development and growth of the organism. Physiological functions of insulin and IGF1 are relatively well-studied, but information about the role of IGF2 in the body is still sparse. Recent discoveries called attention to emerging functions of IGF2 in the brain, where it could be involved in processes of learning and memory consolidation. It was also proposed that these functions could be mediated by the receptor for IGF2 (IGF2R). Nevertheless, little is known about the mechanism of signal transduction through this receptor. Here we produced His-tagged domain 11 (D11), an IGF2-binding element of IGF2R; we immobilized it on the solid support through a well-defined sandwich, consisting of neutravidin, biotin and synthetic anti-His-tag antibodies. Next, we prepared specifically radiolabeled [125I]-monoiodotyrosyl-Tyr2-IGF2 and optimized a sensitive and robust competitive radioligand binding assay for determination of the nanomolar binding affinities of hormones for D11 of IGF2. The assay will be helpful for the characterization of new IGF2 mutants to study the functions of IGF2R and the development of new compounds for the treatment of neurological disorders.

• MeSH
• insulinu podobný růstový faktor I metabolismus   MeSH
• insulinu podobný růstový faktor II metabolismus   MeSH
• kompetitivní vazba MeSH
• kultivované buňky MeSH
• lidé MeSH
• radioizotopy jodu MeSH
• radioligandová zkouška metody   MeSH
• receptor IGF typ 2 imunologie   ultrastruktura   MeSH
• signální transdukce MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• autoprotilátky * analýza   izolace a purifikace   MeSH
• beta-buňky patologie   MeSH
• časná diagnóza MeSH
• diabetes mellitus 1. typu * etiologie   MeSH
• diabetes mellitus epidemiologie   klasifikace   MeSH
• ELISA využití   MeSH
• insulinové protilátky analýza   MeSH
• lidé MeSH
• radioligandová zkouška metody   využití   MeSH
• zinkový transportér 8 analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH
• srovnávací studie MeSH

Xanomeline (3-(Hexyloxy)-4-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,5-thiadiazole) is a muscarinic agonist that is considered to be functionally selective for the M1/M4 receptor subtypes. Part of xanomeline binding is resistant to washing. Wash-resistant xanomeline activates muscarinic receptors persistently, except for the M5 subtype. Mutation of leucine 6.46 to isoleucine at M1 or M4 receptors abolished persistent activation by wash-resistant xanomeline. Reciprocal mutation of isoleucine 6.46 to leucine at the M5 receptor made it sensitive to activation by wash-resistant xanomeline. Lowering of membrane cholesterol made M1 and M4 mutants and M5 wild type receptors sensitive to activation by wash-resistant xanomeline. Molecular docking revealed a cholesterol binding site in the groove between transmembrane helices 6 and 7. Molecular dynamics showed that interaction of cholesterol with this binding site attenuates receptor activation. We hypothesize that differences in cholesterol binding to this site between muscarinic receptor subtypes may constitute the basis for xanomeline apparent functional selectivity and may have notable therapeutic implications. Differences in receptor-membrane interactions, rather than in agonist-receptor interactions, represent a novel possibility to achieve pharmacological selectivity. Our findings may be applicable to other G protein coupled receptors.

• MeSH
• agonisté muskarinových receptorů farmakokinetika   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• CHO buňky cytologie   MeSH
• cholesterol metabolismus   MeSH
• Cricetulus MeSH
• inositolfosfáty metabolismus   MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární modely MeSH
• průtoková cytometrie MeSH
• pyridiny farmakokinetika   MeSH
• radioligandová zkouška MeSH
• receptory muskarinové genetika   metabolismus   MeSH
• simulace molekulového dockingu MeSH
• thiadiazoly farmakokinetika   MeSH
• tritium farmakokinetika   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Radioimmunoassay belongs to the analytical method enabling highly specific and sensitive quantification of molecules. The verification of the real-time radioimmunoassay technology usefulness for ligand-quality characteristics evaluation such as concentration, influence of radiolabeling on binding affinity and stability was estimated. The anti-epidermal growth factor receptor antibody 131 I-cetuximab was employed as the ligand antibody. The concentration of 131 I-cetuximab was derived from the shape of binding curves coming from the ligand-receptor interaction. The binding curves also allowed the estimation of 131 I-cetuximab binding affinity for different radiolabeling procedures (incubation times 1, 5, and 10 minutes) in stability testing up to 96 hours at 4°C. The stability testing also included comparative analysis by size exclusion high-performance liquid chromatography. The assessment of cetuximab concentrations using real-time method showed acceptable accordance between real and calculated values. The real-time method revealed that 1-minute radiolabeling proved to be the optimal incubation time for direct radioiodination of cetuximab. Stability testing showed the significant change in radioligand affinity by one order at the longest incubation times (72 and 96 hours). Characterization of stability and binding behavior of radiolabeled monoclonal antibodies by the verified real-time method before use in other assays may be employed to eliminate variability and suboptimal antibody performance.

• MeSH
• cetuximab chemie   farmakologie   MeSH
• lidé MeSH
• ligandy MeSH
• nádorové buněčné linie MeSH
• protinádorové látky imunologicky aktivní chemie   farmakologie   MeSH
• radiofarmaka chemie   farmakologie   MeSH
• radioimunoanalýza metody   normy   MeSH
• radioizotopy jodu chemie   farmakologie   MeSH
• radioligandová zkouška metody   normy   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

GHB (γ-hydroxybutyric acid) is a compound endogenous to mammalian brain with high structural resemblance to GABA. GHB possesses nanomolar-micromolar affinity for a unique population of binding sites, but the exact nature of these remains elusive. In this study we utilized the highly selective GHB analogue, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) as a tritiated version (3H-HOCPCA) to radioactively label the specific GHB high-affinity binding site and gain further insight into the density, distribution and developmental profile of this protein. We show that, in low nanomolar concentrations,3H-HOCPCA displays excellent signal-to-noise ratios using rodent brain autoradiography, which makes it a valuable ligand for anatomical quantification of native GHB binding site levels. Our data confirmed that3H-HOCPCA labels only the high-affinity specific GHB binding site, found in high density in cortical and hippocampal regions. The experiments revealed markedly stronger binding at pH 6.0 (Kd73.8 nM) compared to pH 7.4 (Kd2312 nM), as previously reported for other GHB radioligands but similar Bmaxvalues. Using3H-HOCPCA we analyzed the GHB binding protein profile during mouse brain development. Due to the high sensitivity of this radioligand, we were able to detect low levels of specific binding already at E15 in mouse brain, which increased progressively until adulthood. Collectively, we show that3H-HOCPCA is a highly sensitive radioligand, offering advantages over the commonly used radioligand3H-NCS-382, and thus a very suitable in vitro tool for qualitative and quantitative autoradiography of the GHB high-affinity site.

• MeSH
• autoradiografie metody   MeSH
• cyklopentany farmakologie   MeSH
• hlodavci MeSH
• hydroxybutyráty farmakologie   MeSH
• kompetitivní vazba MeSH
• kyseliny karboxylové farmakologie   MeSH
• mozek účinky léků   metabolismus   MeSH
• myši MeSH
• radioligandová zkouška metody   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cell lines are common model systems in the development of therapeutic proteins and in the research on cellular functions and dysfunctions. In this field, the protein interaction assay is a frequently used tool for assessing the adequacy of a protein for diagnostic and therapeutic purposes. In this study, we investigated the extent to which the interaction characteristics depend on the choice of cell line for HER-family receptors. The interaction characteristics of two therapeutic antibodies (trastuzumab and cetuximab) and one Affibody molecule (ZHER2:342), interacting with the intended receptor were characterized with high precision using an automated real-time interaction method, in different cell lines (HaCaT, A431, HEP-G2, SKOV3, PC3, DU-145). Clear differences in binding affinity and kinetics, up to one order of magnitude, were found for the interaction of the same protein binding to the same receptor on different cells for all three proteins. For HER-family receptors, it is therefore important to refer to the measured affinity for a protein-receptor interaction together with the hosting cell line. The ability to accurately measure affinity and kinetics of a protein-receptor interaction on cell lines of different origins may increase the understanding of underlying receptor biology, and impact the selection of candidates in the development of therapeutic or diagnostic agents.

• MeSH
• afinita protilátek MeSH
• buňky Hep G2 MeSH
• erbB receptory metabolismus   MeSH
• humanizované monoklonální protilátky metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• kinetika MeSH
• lidé MeSH
• mapování interakce mezi proteiny MeSH
• monoklonální protilátky metabolismus   MeSH
• radioizotopy jodu MeSH
• radioligandová zkouška MeSH
• receptor erbB-2 metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

BACKGROUND AND PURPOSE: Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. EXPERIMENTAL APPROACH: Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor. KEY RESULTS: Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins. CONCLUSIONS AND IMPLICATIONS: These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding.

• MeSH
• agonisté muskarinových receptorů metabolismus   MeSH
• alosterická regulace MeSH
• antagonisté muskarinových receptorů metabolismus   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• guanosin 5'-O-(3-thiotrifosfát) metabolismus   MeSH
• guanosindifosfát metabolismus   MeSH
• kinetika MeSH
• křečci praví MeSH
• lidé MeSH
• N-methylskopolamin metabolismus   MeSH
• proteiny vázající GTP klasifikace   metabolismus   MeSH
• radioligandová zkouška MeSH
• receptor muskarinový M2 genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• transfekce MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

OBJECTIVES: Our previous study showed that administration of nicotine is capable to protect the neurons of hippocampus against the kainic acid induced damage. Here we tested the hypotheses that multiple nicotine administration would prevent the effects of kainic acid on neuronal nicotinic receptor subtypes densities (α-bungarotoxin sensitive and α-bungarotoxin insentive) and on hippocampal cell degeneration. METHODS: Radioligand binding study was used to detect the particular nAChR subtypes densities. Two histochemical methods (bis-benzimide staining and Fluoro-Jade B dye) were used to detect and evaluate neuronal degeneration. RESULTS: Our study shows that: a) kainic acid single administration increased the number of α-bungarotoxin insentive nicotinic receptors, b) nicotine was able to prevent such changes, c) repeated nicotine administration is capable to attenuate the damage of CA1 and CA3 areas of the hippocampus. No effect on α-bungarotoxin sentive nicotinic receptors was observed. Our data therefore reveal the importance of α-bungarotoxin insentive nicotinic receptors in the response to kainite and the ability of nicotine to prevent such changes both in the cell degeneration and in number of receptors. CONCLUSION: Nicotine administration influences α-bungarotoxin insensitive receptors and repeated administration is capable to protect against toxicity caused by kainic acid in hippocampal area.

• MeSH
• agonisté excitačních aminokyselin farmakologie   MeSH
• bungarotoxiny metabolismus   MeSH
• hipokampus cytologie   MeSH
• krysa rodu rattus MeSH
• kyselina kainová farmakologie   MeSH
• neurony cytologie   účinky léků   patologie   MeSH
• neuroprotektivní látky farmakologie   MeSH
• nikotin farmakologie   MeSH
• nikotinové receptory metabolismus   MeSH
• nikotinoví agonisté farmakologie   MeSH
• potkani Wistar MeSH
• protein - isoformy metabolismus   MeSH
• radioligandová zkouška MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH