Application of Magnetic Particles for Fast Determination of Immunoreactive Fraction of 68Ga-Labelled VHH Antibodies to PD-L1.

Quantification of the immunoreactive fraction (IRF) of radioactive isotope-labeled antibodies or their fragments is necessary to assess the specific activity of radiopharmaceuticals. Traditionally, cells expressing the target molecules on their surface are used to determine IRF, but such analysis is time-consuming and has difficulties with standardization. The aim of the study was to develop a fast and reliable method for quantitative determination of IRF by 68Ga-labeled VHH antibodies to PD-L1 based on the use of magnetic particles coated with antigen molecules. Materials and Methods: Commercially available magnetic particles coated with protein A have been used in our study. The antigen conjugated with the Fc fragment (PD-L1-Fc) was immobilized on the particles. The IRF value of 68Ga radionuclide-labeled nanobodies (VHH) against PD-L1 (68Ga-VHH-PD-L1) was determined using magnetic particles coated with antigen molecules and cells expressing the antigen on their surface. When VHH antibodies were conjugated to 68Ga radionuclide, protein molecules were modified using bifunctional chelating agents: tetraazacyclododecanetetraacetic acid (DOTA) or deferoxamine (DFO). The magnitude of IRF was defined as the ratio of radioactivity specifically bound to particles or cells to the total radioactivity added to the sample. Results: The specificity of the 68Ga-VHH-PD-L1 radioimmunoconjugate binding to the antigen-coated magnetic particles has been proved. Some special aspects, which should be taken into consideration when using this method, have been established. The comparison of the IRF estimates using the antigen-expressing cells and magnetic particles has not revealed any significant differences in the results obtained in our study. Nevertheless, the presented method based on magnetic particles with immobilized antigen molecules requires only 15 min to determine the radioimmunoconjugate IRF, which is of fundamental importance for the routine assessment of the specificity of radiopharmaceuticals containing short-lived isotopes.

Metal-Polymer Complexes of Gallium/Gallium-68 with Copolymers of N-Vinylpyrrolidonewith N-Vinylformamideand N-Vinyliminodiacetic Acid: A Hint for Radiolabeling of Water-Soluble Synthetic Flexible Chain Macromolecules.

Copolymer of N-vinylpyrrolidone (VP) with vinylformamide (VFA) and N-vinyliminodiacetic acid (VIDA) was synthesized; its metal-polymer complexes (MPCs) with gallium were obtained. The complexes were characterized by size exclusion chromatography, hydrodynamic and optical methods, scanning electron microscopy, and spectral methods (UV, IR, 1Н NMR spectroscopy). It was demonstrated that in going from polymer to complex, hydrodynamic parameters of macromolecules change only slightly, although the polymer contains intramolecular Ga(VIDA)2 fragments in its structure. A new method for preparation of MPCs with gallium and gallium-68 radionuclide was suggested. The obtained metal-polymer complex is stable over a wide range of pH values as well as in the histidine challenge reaction. In vivo distribution experiments in intact animals showed high primary accumulation of thegallium-68 MPC in blood with subsequent excretion via urinary tract.

[First experience of using positron emission tomography with 11C-choline in prostate cancer (PC)].

Introduction into clinical practice of combined positron emission technology and computer tomography (PET/CT) allows in one study to identify structural and functional abnormalities. The study involves 32 patients who underwent PET/CT with "C-choline, including 5 patients with prostate cancer (PC), 3--with chronic prostatitis and 24--with biochemical PC recurrence. PET/CT with 11C-choline has a high diagnostic efficacy in detection of local recurrence and PC metastases in patients with biochemical PC recurrence. The results of visual analysis do not permit to distinguish PC from benign prostate diseases.

[Biodistribution of the recombinant heat shock protein rhHsp70 in intracranial C6 glioma models in Wistar rats and subcutaneos B16/F10 melanoma in C57BL/6 mice].

For the first time, the biodistribution of recombinant heat shock protein in rhHsp70 rats with grafted intracranial C6 glioma was evaluated. It was assessed using the fluorescent antibody accumulation chaperone rhHsp70 conjugated with fluorochrome Alexa Fluor 555 in tumor cells by intratumoral or intravenous administration. Assessment of the distribution and accumulation of labeled protein was carried out on the model of subcutaneous B16/F10 melanoma in C57BL/6 mice with the use of single-photon emission computer tomography. After 60 minutes after intravenous administration rhHsp70-I123 (20 MBq, 5 mg chaperone) accumulation of the drug mainly in the liver and tumor tissue was showed. The coefficient of the differential accumulation of the labeled protein KDN(tumor/background) was 3.14. It was turned out that comparing the level of fixation of rhHsp70-I123 in the liver and the tumor KDN(tumor/ liver) = 0.76. After 24 hours from the time of injection of rhHsp70-I123 it was observed increase the level of fixation of the labeled protein in the liver and melanoma: KDN(tumor/background) = 3.43; KDN(tumor/liver = 0.78.

[(18)F-labeled tyrosine derivatives: synthesis and experimental studies on accumulation in tumors and abscesses].

Tyrosine derivatives labeled with a short-living fluorine 18 isotope (T(1/2) 110 min), namely 2-[(18)F]fluoro-L-tyrosine (FTYR) and O-(2'-[(18)F]fluoroethyl)-L-tyrosine (FET), promising radiopharmaceutical products (RPP) for positron emission tomography (PET), were obtained by asymmetric synthesis. Accumulation of FTYR and FET in the rat tumor "35 rat glioma" and in abscesses induced in Vistar mouse muscles was studied and compared with that of a well-known glycolysis radiotracer 2-[(18)F]fluoro-2-deoxy-D-glucose (FDG). It was shown that the relative accumulation indices of amino acid RPP were considerably lower than those of FDG. At the same time, tumor/muscle ratios were high enough (2.9 for FET and 3.9 for FTYR 120 min after injection) for reliable tumor visualization. The data obtained indicated a possibility in principle to use FTYR and FET for differentiated PET diagnostics of brain tumors and inflammation lesions. Of the tyrosine derivatives studied, FET seems to be the most promising agent due to a simple and easily automated method of preparation based on direct nucleophilic substitution of the leaving tosyloxy group of an enantiomerically pure Ni-(S)-BPB-(S)-Tyr(CH2CH2OTs) precursor by an activated [(18)F]fluoride.

Effects of intracisternal administration of insulin on the time dynamics of behavior in rats.

The effects of intracisternal administration of insulin at doses of 2.5, 25, 50, and 200 ng on the behavior of rats in an open field test and elevated plus maze were studied at 45 min, 24 h, and nine days after single doses. On day 1, doses of 2.5 and 25 ng increased the probability of orientational-investigative behavior and locomotion in the open field, while doses of 50 and 200 ng, conversely, produced some tendency to decreases in investigative behavior (mainly at the dose of 50 ng). On day 2 after dosage with insulin, the tendency to increased investigative activity persisted only in rats given a dose of 25 ng of insulin, while on day 9 this was increased in all experimental animals independently of the insulin dose given. In the elevated plus maze, insulin doses of 2.5 and 200 ng decreased anxiety in rats during the first 5 min of testing on day one, while doses of 2.5 and 25 ng reduced anxiety in the second 5 min. On day 2, the level of anxiety increased (at doses of 50 and 200 ng) or tended to increase (at doses of 2.5 and 25 ng); on day 9, anxiety decreased in all experimental rats. Studies of the time dynamics of the behavior of rats showed that single doses of insulin had aftereffects in CNS structures, consisting of weakening of non-associative memory in the open field test on days 2 and 9 and increases in anxiety in the elevated plus maze on day 2, followed by weakening of anxiety on day 9.

[The influence of intracysternal insulin administration on the rat temporal behaviour dynamics].

Rat behaviour in the open field and elevated plus-maze was analyzed in rats after intracysternal administration of 2.5, 25, 50 and 200 ng of insulin in 45 min, 24 hrs and on the 9th day after single injection. Dose-dependent changes in 45 min occurred in both behavioural tests: insulin in low doses (2.5 and 25 ng) increased probability of locomotion and investigative activity in open field, while insulin in high doses (50 and 200 ng) did not alter locomotor activity and showed tendency to weakening of the investigative behavior (especially in the dose of 50 ng). Tendency was found in 24 hrs to increase probability of investigative behavior in open field after injection of 25 ng of insulin, although on the 9th day after insulin administration this behaviour increased in all experimental groups for all used doses. Insulin in the doses 2.5 and 200 ng decreased anxiety in elevated plus-maze in 45 min during the first five min; the doses 2.5 and 25 ng at the second five min exerted the same effect. In 24 hrs, the anxiety level increased for the doses 50 and 200 ng, and there was a tendency for an increase in the doses 2.5 and 25 ng; anxiety was decreased on the 9th day for all used doses of insulin. Thus, single insulin administration induced weakness of non-associative memory in open field on the day 2 and day 9 as well as increase of anxiety level on the day 2 and decrease of anxiety level on the day 9 in elevated plus-maze.

[Dose-dependent effects of intracisternally administered insulin on rat's behavior and glucose level].

Rat behavior in the open field and elevated plus-maze as well as glycaemia level were analyzed in rats after intracisternal administration of 2.5, 25, 50 and 200 ng of insulin. Dose-dependent changes were found in both behavioral tests: insulin in low doses (2.5 and 25 ng) increased probability of locomotion and investigative activity in open field, while insulin in high doses (50 and 200 ng) did not alter locomotor activity and showed tendency to weakening of the investigative behavior (especially in the dose of 50 ng). Significant decrease of rat anxiety level during the first 5 minutes of testing was found after administration of 2.5 and 200 ng of insulin and during the next 5 minutes after administration of 2.5 and 25 ng of insulin in elevated plus-maze. The glucose level in rats was increased in 1-2 hours after insulin administration, though glycaemia level did not exceed normal values. Thus revealed alterations of behavior are supposed to be the result of direct insulin influence on central mechanisms of activation and/or suppression of underlying behavioral characteristics of animals.

Catalytic enantioselective synthesis of 18F-fluorinated alpha-amino acids under phase-transfer conditions using (S)-NOBIN.

We describe a new method for the asymmetric synthesis of [(18)F]fluorinated aromatic alpha-amino acids (FAA) under phase transfer conditions using achiral glycine derivative NiPBPGly and (S)-NOBIN as a novel substrate/catalyst pair. The key alkylation step proceeds under mild conditions. Substituted [(18)F]fluorobenzylbromides were prepared using nucleophilic [(18)F]fluoride and were used as alkylation agents. Two important FAA, 2-[(18)F]fluoro-L-tyrosine (2-FTYR) and 6-[(18)F]fluoro-L-3,4-dihydroxyphenylalanine (6-FDOPA), were synthesized with an ee of 92 and 96%, respectively. The total synthesis time was 110-120 min and radiochemical yields (d.c.) were 25+/-6% for 2-FTYR and 16+/-5% for 6-FDOPA.

[Chromatographic analysis of low molecular weight fraction of cerebrospinal fluid in children with acute neuroinfections]. / Khromatograficheskoe opredelenie sostava nizkomolekuliarnoi fraktsii tserebrospinal'noi zhidkosti pri ostrykh neiroinfektsiiakh u detei.

Low molecular-weight (oligopeptide) fraction of the cerebrospinal fluid was analyzed by high-performance reversed phase liquid chromatography in 30 children with bacterial and viral neuroinfections. The incidence and height of chromathoraphic peaks in bacterial meningitis depended on the disease etiology, stage, and severity. Qualitative and quantitative composition of low molecular-weight fraction of the liquor varied in patients with viral neuroinfections, depending on the severity of the cerebral parenchyma involvement. Differences in chromatographic profiles in complicated and uneventful course of neuroinfections indicate a possible damaging, protective, or regulatory effect of the liquor peptides. These data focus the attention on the role of oligopeptides in the genesis of neuroinfectious process, significance of search for peptide markers, their further isolation, identification, and development of test systems available for clinical application.

[The effect on rat behavior of phenamine contained in a cannula chronically implanted in the neostriatum]. / Vliianie na povedenie krys fenamina, soderzhashchegosia v khronicheski vzhivlennoi v neostriatum kaniule.

Avoidance conditioning in a shuttle-box was studied in rats under conditions of daily three-week amphetamine microinjections (45 mcg) into the rostral striatum and in rats with chronically implanted intrastriatal amphetamine-containing cannulae (45 mgg/0.75 mcl). Amphetamine stimulatory behavioural effects were recorded in both groups of animals (improvement of avoidance conditioning, stereotyped hyperactivity etc.). The dopaminomimetic effects in rats with implanted cannulae were less expressed than in those with daily injections. High chemical stability of cannulated amphetamine was demonstrated by means of HPLC technique. Neurodegenerative changes were described in the loci of amphetamine administration similar to those in its long-lasting systemic injections. The methodological problems are discussed which are essential for neuropharmacological studies of behaviour.

[Neuropeptide factors of pathogenesis of brain-related motor disorders]. / Neiropeptidnye faktory patogeneza tserebral'nykh dvigatel'nykh rasstroistv.

Overall 136 patients with cerebral tumors, sequels of craniocerebral injury and brain strokes with motor disorders of varying intensity were examined. Factors of the peptide nature, provoking postural asymmetry of the homolateral hind limb in an experimental animal were detected in the blood and CSF of the overwhelming majority of the patients. Provided the compensatory process runs a favorable course (recovery of motor functions), the neuropeptide factors are inactivated with inactivating substances. The data obtained attest to the advisability of further studies into the activity and nature of the factors of postural asymmetry in patients with cerebral motor disorders with a purpose of elaborating new methods of treatment and rehabilitation.

[The activity of postural asymmetry factors in symmetrical sections of the rat spinal cord]. / Aktivnost' faktorov poznoi asimmetrii v simmetrichnykh otdelakh spinnogo mozga krysy.

The distribution of the low-molecular weight and high-molecular weight postural asymmetry factors (FPA) activity in the left and right parts of the lumbal region of the rat spinal cord was studied. Low-molecular weight FPA induces flexion of the hind limb ipsilateral to the half of the spinal cord from which FPA was isolated, while high-molecular weight FPA induces contralateral flexion. The activities of the low- and high-molecular weight FPAs in each half of the spinal cord are comparable in normal rat. After the suction lesion of the motor areas in the left hemisphere the increase of the low-molecular weight FPA activity in the right half of the lumbal region of the spinal cord was observed.

[The structural specificity of factors in the chemical regulation of muscle tonus at the spinal cord level]. / Strukturospetsifichnost' faktorov khimicheskoi reguliatsii myshechnogo tonusa na urovne spinnogo mozga.

The chemical factors of the postural asymmetry (FPA) were studied on the recipients without hemispheres. The structural specificity of the fore and hind limb spinal centers regulation in normal and damaged CNS was observed. In the normal CNS this structural specificity was displayed in the selective activation of the cross-situated hemicenters in the cervical and lumbal regions (for example in the left part of the cervical and in the right part of the lumbal region). In the case of the unilateral lesion of the central motor systems the normal pattern of chemical structural specificity was modified by activation of FPA selectively acting on the partly denervated regions of the spinal cord.