The goal of this study was to examine effects of a novel galanin receptor agonist GalR1-3 [βAla14, His15]-galanine 2-15 (G), obtained by automatic solid-phase peptide synthesis, on the metabolic state of the area at risk and the size of acute myocardial infarction (MI) in rats in vivo and to evaluate its toxicity in BALB/c mice after a single dose administration. Regional ischemia was induced in anesthetized rats by coronary artery occlusion followed by restoration of coronary blood flow. The peptide G was administered intravenously (i.v.) with a bolus after a period of regional ischemia in the dose range of 0.25–3.0 mg/kg. The sizes of MI and activities of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) in blood plasma were evaluated. The effect of administration of the optimal dose of G (1.0 mg/kg) on the myocardial content of adenine nucleotides (AN), phosphocreatine (PCr), creatine (Cr), and lactate was studied. The peptide G toxicity was evaluated after a single intraperitoneal injection of 0.5–3.0% solution of the peptide substance to mice. The i.v. administration of the optimal dose of G to rats (1.0 mg/kg) insignificantly influenced hemodynamic parameters, but reduced the MI size by 40% and decreased plasma LDH and CK-MB activity by the end of reperfusion as compared to control. These effects were accompanied by a significant improvement in the metabolic state of the area at risk (AAR) as evidenced by an increase in the myocardial content of ATP, ΣAN, PCr and ΣCr, and a decrease in the myocardial lactate level compared to control. The absence of signs of intoxication and death of animals after G injection of the maximum possible dose did not allow determining an LD50 dose. The results of this study indicate a therapeutic potential of the peptide G for preventing myocardial ischemia and reperfusion injury and a clear need for further studies of its pharmacological properties and mechanisms of action.

New peptide analogs of galanin corresponding to fragments of the N -terminal sequence were synthesized by an automatic solid-phase method using Fmoc-technology. Incomplete cleavage of the Boc-protection from the Trp indole ring was found during postsynthetic procedures and a method for solving this problem was proposed. Physicochemical properties of a number of natural and synthetic N -terminal fragments of galanin were been studied. A comparative evaluation of cardioprotective action of peptide agonists of the galanin GalR2 receptor was carried out on the model of the regional ischemia and reperfusion of the heart in rats in vivo. The analog that contained the carnosine sequence, H-Trp-Thr-Leu-Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Pro-βAla-His-OH, was found to most effectively protect the heart from ischemic and reperfusion stress.

The P26 peptide corresponding to the 197–222 sequence of the second extracellular loop of the β 1 -adrenoreceptor (β 1 -AR) was synthesized by solid-phase fragment condensation on the Wang polymer. Pentapeptide fragments were prepared on the 2-chlorotrityl resin. The racemization degree of the C-terminal alanine residue of the pentapeptide was experimentally evaluated for the synthetic H-Glu-Ser-Asp-Glu-Ala-Arg-OH hexapeptide β 1 -АR-(202–207) which was prepared by the 5 + 1 fragment condensation with the use of various condensing agents. A content of the diastereoisomeric peptide in the products of the fragment condensation was determined by HPLC on a reversed phase. The D-alanine-containing hexapeptide was specially synthesized and used for a comparison. The minimum racemization degree of the C -terminal alanine residue was observed if complex F was applied to the synthesis of the hexapeptide.

Предложена новая методика получения аффинных сорбентов на основе лигандов – тирамина и триптамина. Для полученных хроматографических материалов были исследованы сорбционные характеристики. Показано, что триптамин-сефароза и тирамин-сефароза эффективно связывают IgG, IgA, липопротеид (а) (Lp(a)) и липопротеиды низкой плотности (LDL) из плазмы крови. Сорбционная емкость (мг/мл геля) составила IgG – 4–9, IgA – 2–4, Lp(a) – 3–5 и LDL – 5–7. Обнаружено, что Lp(a) и IgG могут связываться с сорбентом как независимо, так и в виде комплекса Lp(a) с IgG, существование которого может говорить о наличии у некоторых пациентов аутоантител к Lp(a). Новые сорбенты просты в синтезе, стабильны при использовании и хранении, могут применяться в медицинских и биотехнологических целях для связывания Lp(a), LDL, IgG, IgA.

A new procedure for the preparation of affinity sorbents based on tyramine and tryptamine ligands has been proposed. Sorption characteristics for the developed chromatographic materials were investigated. It was shown that tryptamine-sepharose and tyramine-sepharose efficiently bind IgG, IgA, lipoprotein (a) (Lp(a)) and low density lipoproteins (LDL) from blood plasma. The sorption capacity was 4–9 mg of IgG, 2–4 mg of IgA, 3–5 mg of Lp(a), and 5–7 mg of LDL per mL of gel. It was found that Lp(a) and IgG can bind to the sorbent either as themselves or as a complex of Lp(a) with IgG, the existence of which may indicate the presence of autoantibodies to Lp(a) in some patients. New sorbents are easy in synthesis, stable during use and storage, and they can be used for medical and biotechnological purposes to bind Lp(a), LDL, IgG, and IgA.

Получены гемосовместимые аффинные сорбенты на основе полисахаридной матрицы и лигандов – пептидов WY, WTY, WNY, связывающиe иммуноглобулины G человека (IgG). Проведено сравнение характеристик сорбентов по связыванию как общего IgG так и подклассов IgG. Обнаружено, что все новые сорбенты имеют хорошие характеристики по удалению IgG из плазмы крови, но сорбент на основе WNY в полтора раза эффективней остальных связывает IgG подкласса 3 (IgG3). Определены физико-химические характеристики процесса связывания IgG. Для сорбентов на основе пептидов WY, WTY, WNY константы десорбции IgG составили 10 ± 3, 28 ± 4 и 13 ± 3 мкМ. Максимальная расчетная сорбционная емкость по IgG составила 43 ± 2, 45 ± 3 и 46 ± 3 мг IgG на 1 мл сорбента. Показана гемосовместимость сорбентов, пригодность для медицинского использования.

The automated Fmoc solid-phase technique was used to synthesize Cys-containing linear peptide fragments of monocyte chemoattractant protein-1, chemokine domain of fractalkine, and their analogues, with the Cys residue being either modified or replaced with Ser. Chimeric symmetric and asymmetric disulfides were also prepared from the linear precursors. A SAR study of a set of the newly synthesized peptides showed that the capacity to stimulate migration of monocytes and influence cell motility in vitro critically depends, in general, on the presence of free Cys thiol group in the molecule. Notably, all analogues, including chimeric disulfides, containing no SH groups demonstrated the lack of chemokinetic properties.

Твердофазным методом с использованием Fmoc-технологии проведен синтез ряда цистеинсодержащих линейных фрагментов моноцитарного хемотаксического белка-1 и хемокинового домена фракталкина и их аналогов, в которых остаток цистеина был модифицирован или заменен остатком серина. На основе линейных предшественников получены химерные молекулы – симметричные или несимметричные дисульфиды. Изучено влияние синтезированных соединений на клеточную подвижность in vitro. Показано, что пептиды, стимулирующие миграцию моноцитов, содержат в своем составе остаток цистеина со свободной тиольной группой. Пептиды с блокированной SH-группой цистеина, с заменой остатка цистеина на серин, а также дисульфидные химерные молекулы хемокинетическими свойствами не обладают.

Fragments corresponding to the 83–98 sequence of the first extracellular loop and to the 168–192 and 171–182 sequences of the second extracellular loop of the M2-muscarinic receptor (antibodies to this receptor could be markers of early symptoms of heart disorders) were synthesized by solid phase method using the Fmoc-SPPS strategy. A new conformational antigen with the natural location of the disulfide bridge was prepared by selective formation of disulfide bond between the corresponding cysteine residues in the synthe-sized peptides and characterized. The comparative analysis of reactivity of the synthesized peptides towards sera from patients which had no organic heart disease was performed. A new conformational antigen was effectively bound to the sera from patients with idiopathic arrhythmias, but without symptoms of organic heart disease.

Effects of apelin-12 H-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-OH (A12) and its modified analogue H-(NMe)Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-OH (I) on activity of antioxidant enzymes, formation of malonic dialdehyde (MDA) and generation of reactive oxygen species (ROS) were studied in ex vivo and in vivo models of myocardial ischemia and reperfusion (I/R) injury in Wistar rats. Preischemic infusion of peptide A12 or AI enhanced cardiac function recovery of isolated perfused heart and was accompanied by a marked attenuation of ROS generation detected by electron paramagnetic resonance (EPR) technique in myocardial effluent at early reperfusion compared with control. Intravenous administration (i.v.) of peptides in narcotized rats with regional myocardial ischemia limited infarct size and reduced activity of lactate dehydrogenase and MB-fraction of creatine kinase in plasma at the end of reperfusion. Treatment with peptide A12 prevented reduction or augmented activity of myocardial u/Zn superoxide dismutase, catalase and glutathione peroxidase by the end of reperfusion in both I/R models compared with control. Increased MDA content in the area at risk of rat heart in situ at the end of reperfusion was reduced to the initial value under the effect of i.v. A12 administration. Therefore, cardioprotective action of natural apelin-12 and its structural analog AI involve reduction of short-lived ROS generation and improvement of the antioxidant state of ischemic heart during reperfusion.

Apelin-12 and a number of its analogues (Nle 10 -, MeArg 1 , Nle 10 , MeArg 1 , Nle 10 , Phe 12 -NH 2 -, Arg 1 (NO 2 ), Nle 10 , Phe 12 -NH 2 -), resistant to the degradation of proteases, were synthesized by the Fmocmethod of SPPS. By-products of synthesis were examined. It was found that the serine hydroxyl group was sulfating during the final deprotection of apelin-12 and its analogues. The sulfate moiety of the Arg-protecting group transfers into the hydroxyl group of Ser. The amount of by-product depends on the water presence in cleavage mixture. Furthermore, the final deprotection of amide analogues of apelin-12 was accompanied by the formation of a by-product — 4-hydroxybenzylamide; its amount ranged from 20% to 8% in the reaction mixture (according to HPLC data) and also depended on the composition of the cleavage mixture. Effects of the synthesized peptides on recovery of the cardiac function after ischemia were examined in a model of isolated perfused rat heart. Infusions of any of the peptides (I–V) before ischemia resulted in a significant improvement of contractile and pump function recovery compared with the control. Cardioprotective efficacy of the peptides increased in the following rank ( I ) < ( II ) = ( III ) < ( IV ) = ( V ).

Novel peptides originating from the peptide inhibitor of myosin light chain kinase (MLCK), L-PIK (Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys), have been studied for their ability to attenuate the thrombin-induced hyperpermeability of an endothelial cell monolayer in culture. Peptides [NαMeArg1]-Lys-Lys-Tyr-Lys-Tyr-Arg-(D)Arg8-Lys and H-Arg(NO2)Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 (designated PIK2 and PIK4, respectively) appeared to be the most effective inhibitors of endothelial cell monolayer hyperpermeability, and surpassed other known peptide inhibitors of MLCK derived from original L-PIK. Our results validate PIK2 and PIK4 as the leading molecules for the development of novel drugs intended to counteract pathological hyperpermeability of vascular endothelium.

Chromatographic material based on tryptophil-threonyl-tyrosine and efficiently binding human, sheep, goat, and bovine immunoglobulin G was obtained. High selectivity of the sorbent for extraction of immunoglobulins from blood plasma has been demonstrated. Effective sorption capacity is 15–25 mg of immunoglobulin G per 1 ml of the matrix. Optimization of the method of triptophyl-threonyl-tyrosine covalent binding to the polysaccharide matrix allowed the achievement of high sorbent stability in conditions of use and storage. This sorbent may be used in medicine and biotechnology.

На оcнове пептидного ингибитоpа киназы легкиx цепей миозина L-ПИК (Arg-Lys-Lys-Tyr- Lys-Tyr-Arg-Arg-Lys) pазpаботаны новые модифициpованные пептидные ингибитоpы. Иccле- довано иx влияние на индуциpованное тpомбином повышение пpоницаемоcти cоcудиcтого эндотелия в культуpе. Уcтановлено, что cоединения ПИК2 ([NαMeArg1]-Lys-Lys-Tyr-Lys-Tyr- Arg-(D)Arg8-Lys) и ПИК4 (H-Arg(NO2)Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2) обладают наи- более выcокой cпоcобноcтью подавлять гипеpпpоницаемоcть cоcудиcтого эндотелия, пpевоcxодя вcе извеcтные аналоги, cозданные на оcнове пептидного ингибитоpа L-ПИК. На оcновании полученныx pезультатов ПИК2 и ПИК4 могут pаccматpиватьcя как лидиpующие молекулы пpи pазpаботке новыx пептидныx лекаpcтвенныx cpедcтв для боpьбы c патологичеcким по- вышением пpоницаемоcти cоcудиcтого эндотелия.