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.     

Evenly tritium labeled peptides in study of peptide 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 μg scale of biological samples both in vitro and in vivo.     

Effect of peptide Semax and its C-terminal fragment PGP on Vegfa gene expression during incomplete global cerebral ischemia in rats

Vascular endothelial growth factor (VEGF-A) is hypoxia-inducible signal glycoprotein. VEGF-A induces vascular endothelial cell proliferation, which leads to the reconstitution of the vascular network in brain regions damaged by ischemia. However, this protein is also involved in the processes of inflammation and edema in early stages of ischemia. The synthetic peptide Semax has neuroprotective and anti-inflammatory properties and is actively used in the treatment of cerebral ischemia. We have previously shown that Semax reduces vascular injury and activates the mRNA synthesis of neurotrophins and their receptors during global cerebral ischemia in rats. In this work, we studied the effect of Semax and its C-terminal Pro-Gly-Pro tripeptide on Vegfa mRNA expression in different regions of the rat brain after 0.5, 1, 2, 4, 8, 12 and 24 h, which is the irreversible occlusion of the common carotid arteries. It was shown that ischemia increases the levels of Vegfa mRNA in rat brains (4 h after occlusion in cerebellum, cerebral cortex, and hippocampus; 8 h after occlusion in the cortex and hippocampus; and 24 h after occlusion in the cortex). Treatment with Semax reduces the levels of Vegfa mRNA in the frontal cortex (4, 8 and 12 h after occlusion) and the hippocampus (2 and 4 h after occlusion). The effect of PGP on the Vegfa gene expression was almost negligible. It was shown that Semax prevents the activating effect of hypoxia on the expression of the Vegfa gene at early stages of global cerebral ischemia. In turn, an increase in the level of Vegfa mRNA in the hippocampus 24 h after occlusion and Semax administration apparently reflects the neuroprotective properties of the drug.     

The binding of Semax, ACTH 4-10 heptapeptide, to plasma membranes of the rat forebrain basal nuclei and its biodegradation

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.     

Effect of semax on the temporary dynamics of brain-derived neurotrophic factor and nerve growth factor gene expression in the rat hippocampus and frontal cortex

Semax is a synthetic peptide, which consists of the N-terminal adrenocorticotropic hormone fragment (4-7) (ACTH4-7) and C-terminal Pro-Gly-Pro peptide. Semax promotes neuron survival in hypoxia, increases selective attention and memory storage. It was shown that this synthetic peptide exerted a number of gene expressions, especially brain derived neurotrophic factor gene (Bdnf) and nerve growth factor gene (Ngf). Temporary dynamics of Bdnf and Ngf ex- pression in rat hippocampus and frontal cortex under Semax action (50 mg/kg, single intranasal administration) was studied in this work. It was shown that the studied gene expression levels changed significantly both in the hippocampus and the frontal cortex tissues 20 minutes after the peptide preparation application. The expression levels decreased in the hippocampus and increased in the frontal cortex. Forty minutes after Semax administration both gene expression levels returned to the level typical of control tissues. After that they increased significantly by 90 minutes after experiment start. Bdnf and Ngf expression levels decreased up to the control levels by 8 hours after medicine applying maximum gene expression levels were attained. Thus, Semax administration results in rapid, long-term, and specific activation of Bdnf and Ngf expression changes in different rat brain departments.     

Semax,An ACTH(4-10) Analogue with Nootropic Properties,Activates Dopaminergic and Serotoninergic Brain Systems in Rodents

Corticotrophin (ACTH) and its analogues, particularly Semax (Met-Glu-His-Phe-Pro-Gly-Pro), demonstrate nootropic activity. Close functional and anatomical links have been established between melanocortinergic and monoaminergic brain systems. The aim of present work was to investigate the effects of Semax on neurochemical parameters of dopaminergic- and serotonergic systems in rodents. The tissue content of 5-hydroxyindoleacetic acid (5-HIAA) in the striatum was significantly increased (+25%) 2 h after Semax administration. The extracellular striatal level of 5-HIAA gradually increased up to 180% within 1–4 h after Semax (0.15 mg/kg, ip) administration. This peptide alone failed to alter the tissue and extracellular concentrations of dopamine and its metabolites. Semax injected 20 min prior d-amphetamine dramatically enhanced the effects of the latter on the extracellular level of dopamine and on the locomotor activity of animals. Our results reveal the positive modulatory effect of Semax on the striatal serotonergic system and the ability of Semax to enhance both the striatal release of dopamine and locomotor behavior elicited by d-amphetamine. Special issue dedicated to Dr. Simo S. Oja.     

The Binding of Semax, ACTH 4–10 Heptapeptide, to Plasma Membranes of the Rat Forebrain Basal Nuclei and Its Biodegradation

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 K _d 2.41 ± 1.02 × 10^–9 M and by the concentration of binding sites B _max 33.5 ± 7.9 × 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.     

Arachidonoyl amino acids and arachidonoyl peptides: Synthesis and properties

N-Arachidonoyl (AA) derivatives of amino acids (glycine, phenylalanine, proline, valine, γ-aminobutyric acid (GABA), dihydroxyphenylalanine, tyrosine, tryptophan, and alanine) and peptides (Semax, MEHFPGP, and PGP) were synthesized in order to study the biological properties of acylamino acids. The mass spectra of all the compounds at atmospheric pressure electrospray ionization display the most intense peaks of protonated molecular ions; the detection limits for these compounds are 10 fmol per sample. AA-Gly showed the highest inhibitory activity toward fatty acid amide hydrolase from rat brain (IC₅₀ 6.5 μM) among all the acylamino acids studied. AA-Phe, AA-Tyr, and AA-GABA exhibited a weak but detectable inhibitory effect (IC₅₀ 55, 60, and 50 μM, respectively). The acylated amino acids themselves, except for AA-Glu, were stable to the hydrolysis by this enzyme. All the arachidonoylamino acids inhibited cabbage phospholipase D to various degrees; AA-GABA and AA-Phe proved to be the most active (IC₅₀ 20 and 27 μM, respectively). Attempts to detect the biosynthesis of AA-Tyr in homogenates of rat liver and nerve tissue in vitro were unsuccessful; however, AA-dopamine and AA-Phe, the products of its metabolism, were found. The highest contents of these metabolites were detected in liver homogenate and in the brain homogenate, respectively. Acylamino acids exert no cytotoxic effect toward the glioma C6 cells. It was shown that N-acylation of Semax with arachidonic acid results in enhancement of its hydrolytic stability and increases its affinity for the sites of specific binding in rat cerebellum membranes.     

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.     

Neuroprotective effects of semax in MPTP-induced disturbances of brain dopamine system

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.     

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.     

Evidence for pituitary-brain transport of a behaviorally potent ACTH analog.

Following intrapituitary injection of ³H-ACTH 4–9 analog, the radioactivity of various brain regions was determined in intact rats and in rats with the pituitary stalk cut one or eight days previously. The regional distribution of radioactivity in the brain was also investigated after intravenous and intrasellar administration. Intrasellar and intrapituitary administration resulted in significantly higher radioactivity levels in the brain than did intravenous injection of an equimolar dose of labeled peptide. Intrapituitary injection resulted in an uptake with clear regional differences and which was highest in the hypothalamus. Twenty four hours after stalk section the uptake of radioactivity in the hypothalamus, but not in other brain regions was markedly depressed. Hypothalamic uptake, however, was restored at eight days after stalk section. The results suggest a significant flow of radioactivity from the pituitary to the brain, particularly to the hypothalamus. Transport to the hypothalamus is presumably partly vascular via the stalk. Transport to other brain areas may occur via the cerebrospinal fluid, but a neural route cannot be excluded.     

Regionaland Interspecies Differences in Brain Progesterone Metabolism

Metabolites of [³H]progesterone were studied in slices prepared from different brain regions of male rat, mouse, and monkey. The major metabolites were 5α-dihydroprogesterone (5α-DHP) and 3α,5α-tetrahydroprogesterone (3α,5α-THP) in rat brain slices, 5α-DHP and 20α- dihydroprogesterone (20α-DHP) in mouse brain slices, and 20α-DHP in monkey brain slices. In rat olfactory bulb slices, 5α-DHP represented 25.2 ± 3.3% of total radioactivity and 3α,5α-THP 17.5 ± 2.8%, whereas in rat medulla oblongata slices, 5α-DHP was 31.3 ± 3.5% and 3α,5α- THP 5.4 ± 1.5% of total radioactivity. In slices from other rat brain regions, both metabolites represented 12–20% of total radioactivity.-The highest metabolite content in mouse brain was also detected in olfactory bulb slices, where 5α-DHP represented 16.6 ± 4.6% and 20α-DHP 9.5 ± 2.3% of total radioactivity. In cortical and corpus callosum slices of monkey brain, 26.8 ± 4.4% and 2.4 ± 0.5% of total radioactivity, respectively, were converted to 20α-DHP, and less than 3% of total radioactivity could be attributed to any of the other metabolites detected. The 3α,α-THP content in both rat and monkey brain was below 1 nM, but increased in rat brain to 6.7 ± 2.5 nM after electroshock. Endogenous 3α,5α-THP might play an important role in the regulation of rat behavior through the modulation of GABA action on the GABA_A receptor. The significant interspecies differences in the brain progesterone metabolism should be considered in evaluating the functional role of neurosteroids in various species.     

Stress-protective activity of the CH<Subscript>3</Subscript>CO-Lys-Lys-Arg-Arg-NH<Subscript>2</Subscript> synthetic peptide (protectin)

The CH₃CO-Lys-Lys-Arg-Arg-NH₂ peptide (the author has named it protectin) was synthesized, and its activity was studied during different stress actions. Protectin was found to normalize the content of corticosterone and adrenalin in adrenal glands and blood after its intranasal administration to rats one day before a cold or heat shock, or hypobaric hypoxia at doses of 1–10 μg/animal and after its intravenous administration just after acute hemorrhage at doses of 0.5–2 μg/animal. The intranasal administration of protectin at doses of 1–10 μg/rat one day before the heat or cold shock was also shown to prevent a change in the content of free histamine and the activity of diamine oxidase in myocardium, which was induced by the dramatic change in the activity of the enzyme after the temperature actions.     

Glycerol incorporation into brain lipids in rat pups born to ethanol-intoxicated dams

The incorporation of labeled glycerol into glycerolipid of rat brain was influenced by the age of the animal (2-month-oldvs. newborn); indeed, 12 min after the administration, diglyceride was the most heavily labeled glycerolipid in the newborn brain, whereas the labeling of glycerophospholipid was highest in the adult. Various amounts of ethanol (0% to 36% of total energy intake) were administered to pregnant female rats, and the brains of their pups were examined for the ability to incorporate labeled glycerol into glycerolipid. The radioactivity incorporated into lipid diminished with increasing the amounts of alcohol consumed. The labeling pattern of lipid classes was also influenced; indeed, the radioactivity of diglyceride decreased markedly, whereas that of triglyceride and glycerophospholipid was affected to a lower degree. The distribution of radioactivity among different phospholipids also varied with age; on a percent basis, phosphatidylcholine was labeled less and phosphatidylinositol was labeled more in the newborn than in the adult. Ethanol influenced the pattern of glycerophospholipid labeling, increasing the radioactivity of phosphatidylserine and decreasing that of phosphatidylinositol.     

Nootropic and analgesic effects of Semax following different routes of administration

Heptapeptide Semax (MEHFPGP) is the fragment of ACTH(4-10) analogue with prolonged neurotropic activity. The aim of the present work was to study the Semax effects on learning capability and pain sensitivity in white rats following intraperitoneal and intranasal administration in different doses. Semax nootropic effects were studied in the test of acquisition of passive avoidance task. Pain sensitivity was estimated in Randall-Selitto paw-withdrawal test. It was shown that Semax exerts nootropic and analgesic activities following intraperitoneal administration. Analysis of dependence of these effects on dose resulted in different dose-response curves. Following intranasal administration, Semax was more potent in learning improvement compared to intraperitoneal administration. The peptide failed to affect the animal pain sensitivity following intranasal administration as opposed to intraperitoneal administration. The data obtained suggest different mechanisms and brain structures involved in realization of the nootropic and analgesic effects of Semax.     

Effect of various drugs producing convulsive seizures on rat brain glycerolipid metabolism

Convulsive seizures were elicited in the rat by the injection of several different drugs (pyridoxal phosphate, bicuculline, penicillin and ouabain). Glycerolipid metabolism was studied after the intraventricular injection of [2-³H]glycerol, which was incorporated into rat brain glycerides. The percentage of total lipid label found in each lipid class (phosphatidylethanolamine, PE; phosphatidylcholine, PC; phosphatidylserine, PS; phosphatidic acid, PA; phosphatidylinositol, PI; diacylglycerol (+ monoacylglycerol), DG and triacylglycerol, TG) depended on the time elapsed from the injection of the labeled precursor. The percent of total lipid radioactivity as PE and PC increased with time (3–60 min), whereas the opposite was true for the radioactivity of DG and PA. The radioactivity of other lipid classes did not appreciabily vary between 3 and 60 min from the injection of the labeled glycerol. The intraventricular administration of pyridoxal phosphate together with labeled glycerol decreased the percent of lipid radioactivity as PE and increased that as DG. This lipid effect was detected also after the administration of other convulsants, such as ouabain and penicillin. The intraperitoneal administration of bicuculline affected lipid metabolism in cerebellum.     

Synacton and individual activity of synthetic and natural corticotropins

Short endogenous peptides represent one of the most important constituents of the mammalian body's general regulatory system. Some synthesized analogs and modified natural peptides (eg, corticotropins) also show high biological activity. Nevertheless, the mechanism of action of regulatory peptides remains unclear. To explain the effects of peptides of intermolecular processes, the hypothesis that a synactonal mechanism underlies the action of regulatory peptides, exemplified by the heptapeptide Semax, has been proposed. Thus, in the total pool of Semax metabolites, which includes the cleavage products of the parental molecule, we can distinguish the functional core, represented by the major metabolic products—peptides HFPGP and PGP. These peptides have their own binding sites with similar although differing characteristics. Together with Semax, they constitute a single complex of bioregulators acting in a certain sequence and in interaction, ie, synacton. It can be assumed that the diverse clinically significant effects of the drug Semax are determined by its synacton. Specific interactions between some tritium‐labeled peptides (basic constituents of the Semax synacton) and plasma membranes of neurons have been characterized. Only a few peptides of the Semax synacton showed competitive activity for the Semax binding sites. Fragments comprising 5 amino acid residues (EHFPG and HFPGP) showed the highest competitive activity. We also characterized the processes of specific ligand‐receptor interactions of some tritium‐labeled corticotropins ([³H‐Pro]MEHFPGP, [³H‐Pro]HFPGP, and [³H‐Pro]PGP) by applying mathematical discriminative models (Scatchard, Hill, Bjerrum, and Lineweaver‐Burk plots). So the intermolecular interactions of these peptides with plasma membranes of neuronal brain targets are probably not limited by specific binding at orthosteric sites. The effect of peptides that act in the synacton considerably extends the regulatory potential of the initial molecule.     

Metabolism of labeled carnitine in the rat

In two series of rats, the concentration of carnitine in plasma was 39.9 and 37.8 μmol/ liter, in skeletal muscle tissue 2.97 and 3.26 μmol/g dry wt and the urinary excretion 3.2 and 2.4 μmol/24 h. The renal clearance of carnitine was calculated to 88 and 76 ml/24 h. L-[Me-¹⁴C]Carnitine and DL-[Me-¹⁴C]carnitine have been administered to rats. Only labeled l-carnitine has been found on chromatographic analysis of plasma, urine, and muscle tissue. The specific radioactivity of carnitine in plasma, urine, and muscle tissue has been followed for up to 16 days. A two-compartment metabolic model has been used to interpret the result of the experiment with labeled l-carnitine and the rate constants and compartment sizes have been calculated. The total body content of carnitine was 57 μmol (about 35 μmol/100 g body wt) and the daily turnover was about 7% of the body pool. The daily synthesis of carnitine in the rat is estimated to about 2 μmol/100 g body wt.