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With low background radiation, tritiate compounds exclusively emit intense beta particles without structural changes. This makes them a useful tool in the drug discovery arsenal. Thanks to the recent rapid progress in tritium chemistry, the preparation and analysis of tritium-labeled compounds are now much easier, simpler, and cheaper. Pharmacokinetics, autoradiography, and protein binding studies have been much more efficient with the employment of tritium-labeled compounds. This review provides a comprehensive overview of tritium-labeled compounds regarding their properties, synthesis strategies, and applications.

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In nucleic acid drug discovery, it is extremely important to develop a technology to understand the distribution in target organs and to trace the degradation process in the body in order to optimize the structure and improve the efficiency of the clinical trial process. Since nucleic acid drugs are essentially metabolically degraded into numerous fragments, labeling at the internal position is preferable to that at the terminus. Due to the high molar specific activity of tritium, various approaches for tritium-labeling have been studied for nucleic acid drugs. Nevertheless, a generally-applicable method for tritium labeling of the internal position of a nucleic acid has not been established. In this study, we have demonstrated a new and efficient method for site-specific tritium labeling of the cytosine base at a predefined internal position in nucleic acid drugs. This method was developed by the chemical modification of the cytosine 4-amino group with the pyridinyl vinyl keto group by the functionality-transfer reaction using the reactive oligodeoxynucleotide (ODN), followed by reduction with NaBT4. Applicability to a variety of chemical structures, such as 5-methyl cytosine, 2'-O-methyl, 2'-fluoro ribose derivatives, Locked/Bridged nucleic acid (LNA/BNA) derivatives, as well as phosphorothioate bonds, has been evidenced using nine oligoribonucleic acid (ORN) substrates. It has been clearly demonstrated that this method is an excellent method for tritium-labeling of nucleic acid with an average conversion efficiency of 74%, an average isolated labeling yield of 60%, and an average specific activity of 61 GBq/mmol. This method is expected to contribute to the preclinical absorption, distribution, metabolism, excretion (ADME) studies of nucleic acid drug candidates.

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[3 H]NAAG, N-acetyl-l-aspartyl-l-glutamic acid, has been widely used as a substrate in glutamate carboxypeptidase II (GCPII) reactions, either to determine the inhibitory constants at 50% inhibition (IC50 ) of novel compounds or to measure GCPII activities in different tissues harvested from various disease models. The importance of [3 H]NAAG, combined with its current commercial unavailability, prompted the development of a reliable eight-step synthetic procedure towards [3 H2 ]NAAG starting from commercially available pyroglutamate. Pure [3 H]NAAG of high molar activity (49.8 Ci/mmol) and desired stereochemistry was isolated in high radiochemical yield (67 mCi) and radiochemical purity (>99%). The identity was confirmed by mass spectrometry and co-injection with unlabeled reference.

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Therapeutic efficiency and toxicity are two of the three critical factors in molecular therapy and pharmaceutical drug development. Specific tumor targeting and rapid renal excretion contribute to improving efficiency and reducing toxicity. We recently found that RNA nanoparticles display rubber-like properties, enabling them to deliver therapeutics to cancer with high efficiency. Off-target RNA nanoparticles were rapidly cleared by renal excretion, resulting in nontoxicity. However, previous biodistribution studies relied mainly on fluorescent markers, which can cause interference from fluorophore quenching and autofluorescence. Thus, the quantification of biodistribution requires further scrutiny. In this study, radionuclide [3H] markers were used for quantitative pharmacokinetic (PK) studies to elucidate the favorable PK profile of RNA nanoparticles. Approximately 5% of [3H]-RNA nanoparticles accumulated in tumors, in contrast to the 0.7% tumor accumulation reported in the literature for other kinds of nanoparticles. The amount of [3H]-RNA nanoparticles accumulated in tumors was higher than that in the liver, heart, lung, spleen, and brain throughout the entire process after IV injection. [3H]-RNA nanoparticles rapidly reached the tumor vasculature within 30 min and remained in tumors for more than 2 days. Nontargeting [3H]-RNA nanoparticles were found in the urine 30 min after IV injection without degradation and processing, and more than 55% of the IV-injected radiolabeled RNA nanoparticles were cleared from the body within 12 h, while the other 45% includes the radiative counts that cannot be recovered due to whole-body distribution and blood dilution after intravenous injection. The high specificity of tumor targeting, fast renal excretion, and low organ accumulation illustrate the high therapeutic potential of RNA nanoparticles in cancer treatment as efficient cancer-targeting carriers with low toxicity and side effects.

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Many natural substances exhibit anti-inflammatory activity and considerable potential in prophylaxis and treatment of allergies. Knowing exact molecular targets, which is required for developing these as medicinal products, is often challenging for multicomponent compositions. In the present study we examined novel polyphenolic substance, a water-soluble fraction of wood lignin (laboratory code BP-Cx-1). In our previous study, a number of polyphenolic components of BP-Cx-1 (flavonoids, sapogenins, phenanthrenes etc.) were identified as the major carriers of biological activity of BP-Cx drug family, and several molecular targets involved in cancer and/or inflammation signaling pathways were proposed based on the results of the in vitro and in silico screening studies. In the present study, half maximal inhibitory concentration (IC50) of BP-Cx-1 was established with a radioligand method and a range of IC50 values between 22.8 and 40.3 µg/ml were obtained for adenosine receptors A1, A2A and prostaglandin receptors EP2, IP (PGI2). IC50 for serotonin 5-HT1 and for glucocorticoid GR receptors were 3.0 µg/ml and 12.6 µg/ml, respectively, both being within the range of BP-Cx-1 concentrations achievable in in vivo models. Further, distribution of [3H] labelled BP-Cx-1 in NIH3T3 murine fibroblasts and MCF7/R carcinoma cells was studied with autoradiography. [3H]-BP-Cx-1 (visualized as silver grains produced by tritium beta particles) was mainly localized along the cell membrane, in the perinuclear region and in the nucleus, suggesting ability of BP-Cx-1 to enter cells and bind to membrane or cytosol receptors. In our experiment, we observed the effect of BP-Cx-1 on maturation of dendritic cells (DCs): downregulation of expression of the lipid-presentation molecule CD1a, co-stimulatory molecules CD80, CD83 and CD 40, decreased production of pro-inflammatory cytokines IL-4 and TNF-α and increased production of anti-inflammatory cytokine IL-10. It is hypothesized that [3H]-BP-Cx-1 detectable in the nucleus is part of the activated GR complex, known to be involved in regulation of transcription of genes responsible for the anti-inflammatory response. Based on IC50, cell distribution data and results of the experiment with DCs it is suggested that the in vivo effects of BP-Cx-1 are mediated via GR and 5-HT1 receptors thus promoting development of tolerogenic effector function in dendritic cells.

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Radiolabelled azidophenyl analogues can make powerful photoaffinity probes for the identification of molecular targets. We describe our efforts to prepare tritiated azidophenyl analogues of the taxols cabazitaxel and docetaxel. Late-stage tritiation by isotope exchange with diiodo precursors resulted in reduction of the azide moiety, which could only be overcome by addition of high excess of a sacrificial azide. Iodine-deuterium exchange experiments on a model system established that deiodination with concomitant azide reduction is a general problem when performing such isotope-exchange reactions on azide-containing aryl iodides.

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Positron emission tomography (PET) imaging of P-glycoprotein (P-gp) in the blood-brain barrier can be important in neurological diseases where P-gp is affected, such as Alzheimer´s disease. Radiotracers used in the imaging studies are present at very small, nanomolar, concentration, whereas in vitro assays where these tracers are characterized, are usually performed at micromolar concentration, causing often discrepant in vivo and in vitro data. We had in vivo rodent PET data of [11C]verapamil, (R)-N-[18F]fluoroethylverapamil, (R)-O-[18F]fluoroethyl-norverapamil, [18F]MC225 and [18F]MC224 and we included also two new molecules [18F]MC198 and [18F]KE64 in this study. To improve the predictive value of in vitro assays, we labeled all the tracers with tritium and performed bidirectional substrate transport assay in MDCKII-MDR1 cells at three different concentrations (0.01, 1 and 50 µM) and also inhibition assay with P-gp inhibitors. As a comparison, we used non-radioactive molecules in transport assay in Caco-2 cells at a concentration of 10 µM and in calcein-AM inhibition assay in MDCKII-MDR1 cells. All the P-gp substrates were transported dose-dependently. At the highest concentration (50 µM), P-gp was saturated in a similar way as after treatment with P-gp inhibitors. Best in vivo correlation was obtained with the bidirectional transport assay at a concentration of 0.01 µM. One micromolar concentration in a transport assay or calcein-AM assay alone is not sufficient for correct in vivo prediction of substrate P-gp PET ligands.

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To make a detailed characterization of the mechanism of inhibition and selectivity of a novel fatty acid amide hydrolase inhibitor PF-622, 3 tritium isotopomers were prepared. [3 H]PF-622a labeled at the piperazine ring B and [3 H]PF-622b labeled at both the ring B and phenyl ring A were synthesized via catalytic H(hydrogen)-T(tritium) exchange, utilizing 1 equiv and excess of Crabtree's catalyst, respectively. The preparation of [3 H]PF-622c labeled only at the phenyl ring A was achieved via tritiodebromination of the bromide precursor, using Pd(PPh3 )4 as a catalyst. The observations from these tritiation reactions might open a new perspective in the labeling for the targets having a similar moiety.

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A regiospecific and enantiospecific synthesis of tritium-labeled 28-homocastasterone is reported. Appropriate chlorocarbonate, efficiently synthesized from the starting 28-homocastasterone in an overall yield of 46%, undergoes catalytic tritium dechlorination by the T2 /Pd[0]/Et3 N system, providing 28-[3ß-3 H]homocastasterone, in a good yield, radiochemical purity (>97%), and with a high specific activity (5.8 Ci/mmol).

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The orally active, α-hemoglobin derived hemopressin (PVNFKFLSH, Hp(1-9)) and its truncated (PVNFKFL, Hp(1-7) and PVNFKF, Hp(1-6)) and extended ((R)VDPVNFKFLSH, VD-Hp(1-9) and RVD-Hp(1-9)) derivatives have been postulated to be the endogenous peptide ligands of the cannabinoid receptor type 1 (CB1). In an attempt to create a versatile peptidic research tool for the direct study of the CB1 receptor-peptide ligand interactions, Hp(1-7) was radiolabeled and in vitro characterized in rat and CB1 knockout mouse brain membrane homogenates. In saturation and competition radioligand binding studies, [(3)H]Hp(1-7) labeled membrane receptors with high densities and displayed specific binding to a receptor protein, but seemingly not to the cannabinoid type 1, in comparison the results with the prototypic JWH-018, AM251, rimonabant, Hp(1-9) and RVD-Hp(1-9) (pepcan 12) ligands in both rat brain and CB1 knockout mouse brain homogenates. Furthermore, functional [(35)S]GTP γS binding studies revealed that Hp(1-7) and Hp(1-9) only weakly activated G-proteins in both brain membrane homogenates. Based on our findings and the latest literature data, we assume that the Hp(1-7) peptide fragment may be an allosteric ligand or indirect regulator of the endocannabinoid system rather than an endogenous ligand of the CB1 receptor.

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AK-toxin I, a host-specific toxin to Japanese pear (Pyrus serotina), was synthesized as its methyl ester from three precursor fragments: conjugated diene-carboxylic acid, chiral epoxyalcohol and ß-methylphenylalanine. The epoxyalcohol fragment was derived from D-fructose, in which effective homologation of the hemiacetal carbon to alkyne by using dimethyl 1-diazo-2-oxopropylphosphonate was the key reaction. The diene-carboxylic acid fragment was prepared by repeated Wittig reactions, and was combined with the epoxyalcohol fragment by the Stille reaction. Esterification of the combined product with the stereochemically-pure ß-methylphenylalanine fragment afforded the target compound. This method was used to prepare the methyl ester of tritium-labeled AK-toxin I with a specific radioactivity of 213 GBq/mmol.