[Proteolysis of simple glyprolines by leucine aminopeptidase and enzymes from nasal slime, brain membranes, and rat blood].

Proteolysis of Pro-Gly-Pro-Leu, Pro-Gly-Pro-Gly and Pro-Gly-Pro were studied comparatively to Met-Glu-His-Phe-Pro-Gly-Pro (semax). It is shown that all three peptides are considerably more stable to proteolysis by N-leucine-aminopeptidase (EC 3.4.11.1, Sigma, type VI, 9.2 units/mg), and by enzymes of nasal slime, brain microsomal fractions, and rat blood. Metabolites of the proteolysis showed that semax derives His-Phe-Pro-Gly-Pro only, Pro-Gly-Pro-Leu forms Gly-Pro-Leu, Pro-Gly-Pro and Gly-Pro, Pro-Gly-Pro-Gly gives Pro-Gly-Pro and Gly-Pro, and Pro-Gly-Pro forms Gly-Pro.

[Proteolysis of semax analogues with different N-terminal amino acids by aminopeptidases].

Proteolysis of semax (Met-Glu-His-Phe-Pro-Gly-Pro, Sem) and its analogues ([Ala1]Sem, [Gly1]Sem, [Thr1]Sem, [Trp1]Sem) that are differ from semax in substitution of N-terminal Met residue were studied. It is shown that such replacement changes the rate of peptides degradation by N-aminopeptidases (EC 3.4.11.2, Sigma, Type VI, 9.2 units. Akt. / mg). [Ala1]Sem, [Gly1]Sem and [Thr1]Sem semax analogues proved to be more stable to proteolysis than semax (Sem), and their initial product of proteolysis is His-Phe-Pro-Gly-Pro (Sem-5). For triptophan analogue both Glu-His-Phe-Pro-Gly-Pro (Sem-6) and Sem-5 product are formed in similar quantities. It is found that all investigated analogues can be used as inhibitors in Sem proteolysis.

[The synthesis of O-substituted 3-oximes of 6alpha-methyl-16alpha,17alpha-cyclohexanopregn-4-ene-3,20-dione tritium-labeled in 1 and 2 positions].

1,2-Tritium-labeled 3-(O-carboxypropyl)- and 3-(O-carbomethoxypropyl)-oximes of 6alpha-methyl-16alpha,17alpha-cyclohexanopregn-4-ene-3,20-diones were obtained by the homogeneous catalytic hydrogenation of 1,2-dehydroprecursors with gaseous tritium and the subsequent separation of the resulting mixtures by HPLC. The specific radioactivities of 50-55 Ci/mmol were prepared using tris-(triphenylphosphine)-rhodium chloride.

[Structural-functional study of glycine-and-proline-containing peptides (glyprolines) as potential neuroprotectors].

A synthetic scheme for preparation of (Gly-Pro)n, (Pro-Gly)n (n = 2, 3), and (Pro-Gly-Pro)n (n = 1, 2) peptides was elaborated. The effect of the synthesized peptides and the Gly-Pro and Pro-Gly dipeptides on survival of cultured cells of PC12 rat pheochromocytoma was studied under the conditions of oxidative stress induced by brief incubation of the cells with hydrogen peroxide. Peptides of the general formula (Gly-Pro)n and the Pro-Gly-Pro peptide at a concentration of 0.2-100 microM were shown to decrease the number of damaged cells. The Gly-Pro peptide was the most active and decreased the number of damaged cells by 49% on average at a concentration of 100 microM.

[Radioreceptor study of [3H]-olanzapine specific binding in the rat frontal cortex and striatum].

The direct radioreceptor assay was used to elucidate the interactions of clinically employed atypical antipsychotic drug olanzapine with the specific binding sites in rat brain membranes. Tritium-labeled olanzapine was obtained by isotope exchange method. HPLC and mass spectrometry assays confirmed the identity of [3H]-olanzapine to the initial compound. The specific [3H]-olanzapine binding sites of the two types were identified in frontal cortex by means of the method of Scatchard's plots. High affinity sites showed steady-state dissociation binding constant (KD) of 1.7 nM, which is indicative of olanzapine affinity to 5-HT-2a and histamine receptors. However, according to available literature data, the density of these receptors is several times smaller than that determined in our study (maximum number of binding sites corresponded to B(max) = 1.4 pmol/mg protein), thus allowing an assumption about the existence of yet unknown binding sites of the antipsychotic drug under consideration. Moderate affinity binding sites of olanzapine in frontal cortex (KD= 68.5 nM, B(max) = 10.8 pmol/mg protein) apparently correspond to dopamine, muscarinic, and alpha-1-adrenoceptors. [3H]olanzapine binding sites of the two types were also determined in striatum. The moderate-affinity sites of the receptor interaction had KD = 18.0 nM and B(max) = 13.0 pmol/mg protein. These values in fact correspond to D2 receptors, whose density prevails in this cortical region. The low-affinity binding sites of olanzapine in striatum had KD = 117.0 nM and B(max) = 32.5 pmol/mg protein.

[Synthesis, pharmacokinetics, and metabolism of the C-terminal tripeptide of dermorphin and its diastereomer].

A tritium-labeled C-terminal fragment of dermorphin (H-Tyr-13,4-3HJPro-Ser-NH2) and its isomer (H-Tyr-D-[3,4-3H]Pro-Ser-NH2) with molar radioactivity of 35 Ci/mmol were synthesized, and their pharmacokinetics and metabolism in rat organs were studied after their intramuscular injections. The tripeptides were detected in the blood only for 5 min after the injection, and maximum contents of both compounds (approximately 5% of the total amount of the injected label) were registered in the kidneys after 20 min. Both stereomers were shown to penetrate into the brain. We failed to detect any radioactive metabolite, except proline, due to rapid proteolytic degradation of these peptides.

[Kinetics of Semax penetration into the brain and blood of rats after its intranasal administration].

The radioactive peptide analogue Semax corresponding to the ACTH(4-10) sequence (Met-Glu-His-Phe-Pro-Gly-Pro) with a molar radioactivity of 56 Ci/mmol labeled with tritium at the C-terminal Pro was prepared. The labeled peptide was used for studying the kinetics of Semax penetration into rat brain and blood after its intranasal administration (50 microg/kg, 20 microl of solution) to nonbred white rats of body mass 200-250 g. It was demonstrated that 0.093% of the total introduced radioactivity per gram can be found in the rat brain 2 min after the administration, 80% of this radioactivity belonged to Semax, and the rest, to its metabolites. The peptide undergoes rapid enzymatic degradation, with the tripeptide Pro-Gly-Pro prevailing in biological samples relative to the total content of Semax and its metabolites.

[A synthesis of tritium-labeled olanzapine].

A synthesis of olanzapine, 2-methyl-10-(4-methyl-1-piperazinyl)-4H-thieno[2,3-b][1,5]benzodiazepine was carried out, and the conditions for its tritium labeling were optimized, to get a tritium-labeled olanzapine preparation with molar radioactivity of 12 Ci/mmol.

[An experimental substantiation for using the "Semax" neuroprotector in the treatment of optic-nerve diseases]. / Eksperimental'noe obosnovanie ispol'zovaniia neiroprotektora semaksa v lechenii zabolevanii zritel'nogo nerva.

In order to substantiate the feasibility of using the "Semax" neuroprotector in the treatment of optic-nerve diseases its pharmacokinetics in the intranasal administration was studied in experiments with rats; besides, the physical-and-chemical properties of "Semax" were investigated to define the preparation's stability and mobility in the electric field. A series of experiments, involving a tritium-marked "Semax", showed that the peptide actively penetrates into the brain and eyes of experimental animals after its intranasal administration. It was established, within a study aimed at determining the electrophoretic activity and stability of "Semax" in the electric field, that the preparation does not disintegrate in the electric field, it posses the electrophoretic mobility and moves, in the electric field, from plus to minus; therefore, the "Semax" solution must be fed from anode while making the electrophoresis procedure.

[Synthesis of O-substituted 6-oximes of 16alpha,17alpha-cyclohexanopregn-4-en-3,6,20-triones containing a tritium label at the 1,2-position]. / Sintez O-zameshchennykh 6-oksimov 16alpha,17alpha-tsiklogeksanopregn-4-en-3,6,20-triona, soderzhashchikh tritievuiu metku v polozhenii 1,2.

6-O-(3-Methoxycarbonylpropyl)- and 6-O-(3-carboxypropyl)oximes of 16 alpha,17 alpha-cyclohexanopregn-4-ene-3,6,20-trione labeled by tritium in position 1,2 were synthesized. When using homogenous catalysts, the molar radioactivity of the resulting preparations was 1.5-1.7 PBq/mol.

[Species and tissue distribution specificity of proteins binding 16alpha,17alpha-cycloalkane derivatives of progesterone]. / Vidovye i tkanevye osobennosti raspredeleniia belkov, sviazyvaiushchikh 16al'fa,17-al'fa-tsikoalkanovye proizvodnye progesterona.

The binding of [3H]progesterone and [3H] 16 alpha,17 alpha-cycloalkanoprogesterones to proteins from rat, rabbit, and human uteri and other organs was studied. We found that 16 alpha,17 alpha-cycloalkanoprogesterone derivatives display affinities for the uterine progesterone receptors comparable with that of the natural hormone and no substantial species differences in the affinity. Rabbit uterus was found to have no proteins distinct from the progesterone receptor that specifically bind [3H] 16 alpha,17 alpha-cycloalkanoprogesterones. At the same time, in the human uterus, we found another protein that binds some of these progesterone derivatives; it turned out to be similar to the protein from rat uterus. A similar protein with the same selectivity and affinity for steroids was also found in rat and human kidneys. Blood serum, liver, lung, and a number of other tissues were found to contain a protein of the third type that binds the same 16 alpha,17 alpha-cycloalkanoprogesterones and exhibits submicromolar Kd values for these steroids and a very low affinity for progesterone. We speculated that the introduction of a bulky substituent adjacently to the 17 beta-side chain of progesterone could result in a change in the general biodynamics of the derivative including its transport, uptake, and accumulation in tissues, which may determine the selectivity of its effect.

[Synthesis of tritium-labelled prostaglandin E1 and its binding to human platelets]. / Sintez mechennogo tritiem prostaglandina e1 i ego sviazyvanie s trombotsitami cheloveka.

A method has been developed that makes it possible to obtain [5,6-3H2]PGE1 with a yield of 35% and a molar radioactivity of 1.7-1.8 TBq/mmol. The binding of [5,6-3H2]PGE1 to native platelets proved to be specific, saturating and reversible. It is characterized by low values (approximately 10(-9) M) of dissociation constants for high-affinity sites, correlates with the inhibition of ADP-induced aggregation of platelets and can be considered as receptor binding. Specific binding of 10 +/- 2 molecules of PGE1 with one platelet was found to cause 50% inhibition of the ADP-induced aggregation.

[Directed biosynthesis of prostaglandin F2 alpha multiple labeled with tritium]. / Napravlennyi biosintez kratnomechennogo tritiem prostaglandina F2 alpha.

By selecting specific activators of PGF reductase and determining their optimal ratio, we have performed the directed biosynthesis of PGF2 alpha with a 45-50% yield. With the use of [5,6,8,9,11,12,14,15-3H8]arachidonic acid, multiple-labelled PGF2 alpha with molar radioactivity 6.1 TBe/mmol has been synthesised.