[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.

[A comparative analysis of distribution of glyprolines administered by various routes].

The distribution of the glyprolines Pro-Gly-Pro and Thr-Lys-Pro-Arg-Pro-Gly-Pro (Selanc) was analyzed and compared in tissues of rat organs after different ways of their administration using the peptides uniformly labeled with tritium. Comparative data on changes in concentrations of the peptides in the rat organs after their intraperitoneal, intranasal, intragastric, and intravenous administration are given. The intranasal administration of both peptides was shown to be optimal for the delivery of glyproline molecules in the CNS. A high affinity of the studied glyprolines for gastric tissues was found for all the ways of their administration. We suggest that a high efficiency of action of glyprolines on homeostasis of the gastric mucous tunic was partially provided by accumulation of these peptides (to high concentrations) in gastric tissues.

[Analgesic activity of dipeptide Tyr-Pro].

The effect of dipeptide Tyr-Pro, present in representatives of most families of opioid peptides, and two its analogs, Tyr-Pro-NH2 and Tyr-Pro-OMe, on analgesic activity was studied in different tests (tail-flick test, tail pinch (Haffner's) test, formalin test, and acetic acid writing test) describing different organization levels of pain sensitivity. Intraperitoneal administration of the dipeptide decreased the pain threshold in all above-mentioned tests. Coadministration of the dipeptide and naloxone or naloxone methiodide insignificantly decreased the dipeptide analgesic effect in the tail-flick and acetic acid writing tests. Its analogs Tyr-Pro-NH2 and Tyr-Pro-OMe demonstrated a similar analgesic activity in the tail-flick test and a higher activity in the acetic acid writing test. Administration of individual amino acids (Tyr or Pro) or their mixture had no effect on the pain threshold.

[Effects of chronic Semax administration on exploratory activity and emotional reaction in white rats].

Effects of chronic intranasal administration of ACTH(4-10) analog Semax (MEHFPGP) on exploratory activity, anxiety level, and depression-like behaviour were studied in white rats. The peptide was injected daily in dose 0.05 mg/kg during 10 or 14 days. It was shown that chronic Semax administration at 1-2 weeks induced anxiolytic and antidepressant effects but did not influenced the exploratory activity in non-stressogenic environment. The Semax effects may be the results of activation of the brain serotoninergic system as well as increased BDNF expression in the rat hippocampus.

[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.

[The relationship between the structure of dermorphines and their thermoregulatory activity].

The effect of structural modifications in the molecule of dermorphin on the functional state of thermoregulation of rats under cold, neutral, and hot environment has been studied. Thermoregulatory and vasomotor activities of 43 peptides (fragments and analogs) were tested in comparison with dermorphin. The role of amino acid residues in the second, third, fourth, and sixth positions was studied. Different fragments of dermorphin molecule proved to underlie different types of thermoregulatory activity (the hypothermic effect was observed in the N-terminal tripeptide, while the N-terminal hexapeptide had the vasodilator activity), and targeted modification of dermorphin molecule makes it possible to separate the hypothermic, vasodilatory, and vasoconstrictive activities. It was proposed that dermorphin is the ligand not only for micro-opioid receptors.

[Study of the relationship between analgesic activity and structure of synthetic melanocortin analogs].

Melanocortins, peptides of the family of adrenocorticotropic (ACTH) and melanocyte-stimulating (MSH) hormones, have a wide range of physiological activity. Heptapeptide semax (MEHFPGP) is an analog of the ACTH4-10 fragment. Previously, pronounced nootropic and neuroprotective activities were shown for this peptide; in addition, it decreases pain sensitivity in animals. In this work, the relationship between analgesic activity of the peptide and its structure was studied. The following analogs of the N-terminal ACTH fragments were used: rMEHFPGP, where r is glucuronic acid, KEHFPGP, GEHFPGP, EHFPGP, HFPGP, and ERP. The peptides were administered intraperitoneally at doses of 0.015, 0.05 and 0.5 mg/kg. Rat pain sensitivity was assessed using the paw-withdrawal test. Truncations of the N-terminal residues eliminated the analgesic activity. The peptide analgesic effect was observed after the replacement of the N-terminal methionine with lysine or after the attachment of glucuronic acid to the methionine, while the peptide with glycine instead of the methionine had no effect on pain sensitivity. Hence, the amino acid at position 1 of semax analogs plays a key role in mediating the analgesic effects of the peptides.

[Evenly tritium-labeled peptides and their in vivo and in vitro biodegradation].

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50-150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium-labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a tg scale of biological samples both in vitro and in vivo.

[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.

[Effect of modification of the N-terminal region of molecule on the expression of neotropic effect of semax analogues].

A comparative study of neotropic activity of semax (MEHFPGP), an analogue of the ACTH(4-10), and some of its derivatives in which the N-terminal methionine was modified or substituted with other amino acid residues was performed. The effect of these peptides on learning of albino rats in tests with positive (alimentary) and negative (pain) reinforcement was studied. In the case of modification of methionine by attachment of the gluconic-acid residue or substitution of methionine with lysine, the neotropic effect of the peptide was retained. The substitution of methionine with tryptophan or serine resulted in a decrease in the neotropic activity. The substitution of methionine with glycine, threonine, or alanine caused a complete loss of the neotropic activity of the peptide. Therefore, the amino acid residue located at position 1 of the heptapeptide analogue semax, plays a key role in retaining the neotropic effects of the peptide and determines the degree of their expression.

[Comparative study of the physical dependence on proline analog of dermorphin and morphine].

On the model of acute physical dependence, naloxone treatment of animals dependent on D-Pro6 peptide ([D-Pro6]DM) (a demorphin analog) led to tremor, shaking, convulsions, and rare jumps typical of morphine-dependent animals. The variety and intensity of reactions depended on the naloxone dose and the interval between naloxone and peptide injections. In the state of reject syndrome upon peptide and morphine subchronic treatments according to identical schedules, the symptoms in [D-Pro6]DM dependent animals were much less pronounced than in morphine-dependent ones.

[Analgesic activity of dermorphin and its proline analogs].

Dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2; DM) and its analogs obtained via stereochemical transformation of L-Pro6 to D-Pro6 peptide in DM ([Dpro6]DM) and via dehydration of L-Pro6 peptide ([dHPro6]DM) were characterized with respect to the analgesic activity in rats tested under conditions corresponding to various levels of pain sensitivity organization. The drugs were introduced by intraperitoneal injections in various doses (0.1, 1.0, and 10 mg/kg). In the case of acetic-acid induced convulsions (writhing), DM and [Dpro6]DM produced analgesic action in the minimum dose, while the analogous effect of [dHPro6]DM was observed only in greater doses. In the tail-clamp (Haffner) test, DM and [Dpro6]DM also inhibited nociception while the latter compound was ineffective. In the grid-shock test, [Dpro6]DM showed a higher activity than [dHPro6]DM, whereas DM did not produce analgesic action. Thus, all the studied peptides exhibit pronounced analgesic activity, and the replacement of L-Pro6 in DM by its stereomer D-Pro is more effective than the substitution of dHPro6.

[Dependence of long-lasting effects of the ACTH(4-10) analogue semax on the time of its neonatal administration].

Long-lasting behavioural effects of chronic administration of synthetic ACT(4-10) analogue Semax (MEHFPGP) during early neonatal life were studied. The peptide was injected daily intraperitoneally in dose 0.05 mg/kg during the first, second or second-third weeks of postnatal development. It was shown that the peptide injections during the first week lead to a decrease and during second or second-third weeks--to an increase of exploratory activity in 4-8-week aged rats. Furthermore, the peptide adminictration at all times diminished anxiety and improved learning ability of adult rats. The data obtained show that Semax neonatal administration during the first three weeks of life modulates development of brain structures involved in regulation of exploration, anxiety and learning.

[Study of thermoregulatory activity of a fragment of natural dermorphin precursor Arg-dermorphin and its analogs].

We studied the effect of a fragment of natural dermorphin (DM) precursor Arg-DM and its analogs (Pro-DM, 4Amino-Pro-DM, Mike et-DM, Kre-DM, Arg-[DArg2]-DM, and Arg-[DAla4]-DM after intraperitoneal administration on the functional status of the thermoregulation system in rats. The obtained data demonstrated that the hypothermic and vasomotor effects of Arg-DM were temperature-dependent and had the same pattern as DM (Emel'yanova et al., 1996). In the thermoneutral and room environment, the peptide induced a two-phase vascular response. The first phase was vasodilation; it was twice as strong as for DM and was not removed by naloxone pretreatment. The second phase was vasoconstriction; it was blocked by naloxone. Replacement of Arg with 4Amino-Pro, Met, and Kre as well as DAla2 to DArg2 or Gly4 to DAla4 replacements in the Arg-DM molecule affected the thermoregulatory activity of the peptide. For instance, only the vasodilation response was observed for Arg-[DAla2]-DM and Arg-[DAla4]-DM while only the vasoconstriction response was observed for 4Amino-Pro-DM.

[Thermoregulatory activity of dermorphin fragments]. / Termoreguliatornaia aktivnost' dermofinov.

We studied the effect of C-terminal truncation of the dermorphin (DM) molecule and analogs of its N-terminal tetrapeptide, [DOrn2]-DM(1-4), [DArg2]-DM(1-4), [DAla4]-DM(1-4), [DArg2, DAla4]-DM(1-4), Arg-DM(1-4), Arg-[DArg2]-DM(1-4), Arg-[DAla4]-DM(1-4), and Arg-[DArg2, DAla4]-DM(1-4), on the functional status of the thermoregulation system in rats at different ambient temperatures. For the first time, we demonstrate that the N-terminal tetrapeptide is the minimal fragment with the hypothermic effect. Only the N-terminal octapeptide exerted the vasomotor effect. Amino acid substitutions in the tetrapeptide affected its hypothermic effect. [DArg2]-DM(1-4) and [DArg2, DAla4]-DM(1-4) had the greatest effect. Addition of Arg to the N-terminus of DM(1-4) analogs changed their thermoregulatory activity. The greatest thermoregulatory effect was observed for Arg-[DArg2]-DM(1-4) and Arg-[DArg2, DAla4]-DM(1-4).

[The binding of Semax, ACTH 4-10 heptapeptide, to plasma membranes of the rat forebrain basal nuclei and its biodegradation]. / Sviazyvanie analoga ACTH-(4-10)-geptapeptida Semaks s plazmaticheskimi membranami bazal'nykh iader perednego mozga krysy i ego biodegradatsiia.

The binding characteristics of the peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) to plasma membranes of basal nuclei of the rat forebrain and the dynamics of its degradation during its incubation with these membranes were studied. Binding of the homogeneously labeled [G-3H]Semax was shown to be time-dependent, specific, and reversible. Specific binding of the heptapeptide depended on calcium ions and was characterized by the dissociation constant of the ligand-receptor complex Kd = 2.41 +/- 1.02 x 10(-9) M and by the concentration of binding sites Bmax = 33.5 +/- 7.9 x 10(-15) mol/mg of protein. A method of studying Semax biodegradation in the presence of plasma membranes of rat brain was developed. It is based on the use of the peptide homogeneously labeled with tritium and on an HPLC analysis with UV detection at 220 and 254 nm of the peptide fragments formed. The half-life of Semax in the presence of the plasma membranes was demonstrated to be longer than 1 h. Dipeptidylaminopeptidases are considered to be the main enzymes responsible for its biodegradation; they successively cleave Semax to the HFPGP pentapeptide and the PGP tripeptide. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

[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.

[Effect of structural changes of Pro6 in dermorphin molecule on its antinociceptive activity]. / Vliianie strukturnykh izmenenii Pro6 v molekule dermorfina na anal'geticheskuiu aktivnost'.

We studied effect of dermorphin (H-Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-NH2) and its analogs with modified amino acid residue proline in position 6, H-Tyr-DAla-Phe-Gly-Tyr-[DPro]-Ser-NH2, H-Tyr-DAla-Phe-Gly-Tyr-[dehydro-Pro]-Ser-NH2, and H-Tyr-DAla-Phe-Gly-Tyr-[D-dehydro-Pro]-Ser-NH2, on nociception in the tail-flick and hot plate tests after intraperitoneal injection. Replacement of LPro with the stereoisomer DPro as well as Pro dehydration (LdHPro) was shown to increase antinociceptive activity. Replacement of LdHPro with DdHPro cancelled the activity in the tail-flick test. All three dermorphin analogs retained antinociceptive activity in the hot plate test; however, the effect of dermorphin was more pronounced.

[Effect of dermorphin analogs on thermoregulation of rats under various thermal conditions]. / Vliianie analogov dermorfina na termoreguliatsiiu krys v razlichnykh temperaturnykh rezhimakh.

We studied the influence of dermorphin (dermorphin) analogs with stereochemical modification of the amino acid residue proline in position 6 (Pro6), Tyr-D-Ala-Phe-Gly-Tyr-Hyp-Ser-NH2, Tyr-D-Ala-Phe-Gly-Tyr-[D-Pro]-Ser-NH2, Tyr-D-Ala-Phe-Gly-Tyr-[dehydro-Pro]-Ser-NH2, and Tyr-D-Ala-Phe-Gly-Tyr-[D-dehydro-Pro]-Ser-NH2, after their intraperitoneal injection at 0.5 mg/kg dose in the cold (4-7 degrees C), thermoneutral (27-28 degrees C), and hot (31-33 degrees C) environment. Stereochemical modifications of amino acid residue Pro6 proved to induce specific changes in the thermoregulatory effect of the peptide. Substitution of DPro6 for Pro6 has the most dramatic consequences: it considerably attenuates the thermoregulatory effect of dermorphin in the cold environment, cancels it in the hot environment, and inverts the dermorphin-specific thermoregulatory response in thermoneutral conditions. The data obtained indicate the important role of Pro6 residue in realization of this physiological activity of dermorphins.

[Neuroprotective effects of semax in MPTP-induced disturbances of brain dopamine system]. / Neiroprotektornye éffekty semaksa na fone MFTP-vyzvannykh narushenii dofaminergicheskoi sistemy mozga.

Effects of an ACTH (4-10) analogue Semax (MEHFPGP) on behaviour of white rats with MPTP-induced disturbances of brain DA-system have been studied. It was shown that MPTP administration (25 mg/kg) reduced motor activity and auhmented the anxiety level in rats. Semax administration (daily intranasal 0.2 mg/kg) attenuated behaviour disturbances induced by neurotoxin. The observed protective action of Semax in rats with MFTP-induced DA system disturbances may be due to both its modulating influence on the brain DA system and peptide neuroprotective effects.