The HLDF-6 hexapeptide corresponded to the 41–46 (TGENHR) fragment of the Human Leukemia Differentiation Factor (HLDF) and exhibited a wide spectrum of neuroprotective, normalizing, anxiolytic, nootropic, and antitumor activity. Its N-acetyl C-amide form (Ac-TGENHR-NH2, HLDF-6-AA) was prepared by the solid phase peptide synthesis and served as a basis for a creation of a promising antitumor drug. The hydrolytic stability of this pharmaceutical dosage form of the HLDF-6-AA peptide was studied by the method for an accelerated aging in aqueous solutions at 60°С. The structures of the peptide which were formed after a hydrolysis were analyzed by HPLC and mass spectrometry. The pharmaceutical dosage form of the HLDF-6-AA peptide was found to be practically completely hydrolyzed in solution at 60°С within 30 days. The Asn residue in the Ac-TGENHR-NH2 peptide was hydrolyzed to the Asp residue with the formation of the Ac-TGEDHR-NH2 and Ac-TGED(HR-NH2)-ОН peptides which had α and β bonds between Asp and His, respectively. We concluded on the basis of the results of the accelerated aging that a storage of the peptide dosage form as aqueous solutions did not provide the stability necessary for a pharmaceutical drug. The required stability of the dosage form was achieved during the storage of the HLDF-6-AA peptide as a powder that was lyophilized under sterile conditions.

To clarify the organizing effect of Semax and HLDF-6 peptides on the kinetics of protein synthesis in hepatocytes, in addition to an in vitro study (Brodsky et al., 2019), the effects of these peptides in vivo have been studied. The circahoralian (ultradian) rhythm of protein synthesis, that is, a marker of the direct cell-to-cell communication, was investigated in rats of different ages. Peptides were injected intraperitoneally into young (2–3-month-old) or old (1.5–2-year-old) rats at a 50–100-µg/kg dose. Hepatocytes were isolated and sparse or dense cultures were established. In sparse cultures from young rats that received one or another peptide, the rhythm of protein synthesis was observed; in the cultures from rats of the same age that were injected with saline, no rhythm was found. In dense cultures of old rats after the action of the peptide, the amplitudes of the rhythm of protein synthesis did not differ from the rhythms observed in young rats; after saline injection, the rhythm amplitudes were twice as low. Injection of the peptide into adult rats that had previously received dopamine caused a protein synthesis rhythm characteristic for rats of this age; the administration of dopamine abolished the rhythm. The synchronizing effect of the peptides was maintained for at least 2 days after their single administration to the rat. The use of Semax or HLDF-6 is recommended to compensate for the disturbances of the kinetics of protein synthesis in humans in aging and pathology.

In the search for stable factors regulating direct cell–cell interactions and effects on the properties of cells in aging organisms, the regulatory peptides Semax and HLDF-6 were studied. The circahoralian rhythm of protein synthesis in cells in vitro served as a marker of cell–cell interactions. The peptides normalized the cell–cell interactions, which are greatly weakened during aging. It is shown that the peptides organize the protein-synthesis rhythm in primary rat hepatocyte cultures. The effect of the HLDF-6 peptide was realized via metabotropic glutamate receptors; the blockade of these receptors by the antagonist MCPG abolished the effect of the peptide. The protein kinase inhibitor H7 prevented the effect of the peptides on the protein-synthesis kinetics. Just as for other signaling factors, the activation of protein kinases in the case of the peptides regulates the key process of direct cell–cell interactions. The effect of a single signal of each of the peptides was retained for at least 1 day. Our data allow the peptides to be recommended for improving elderly people’s condition and block the factors that disorganize the protein-synthesis kinetics.

HLDF-6 hexapeptide, which corresponds to the 41 TGENHR 46 fragment of human leukemia differentiation factor (HLDF), shows a wide range of neuroprotective, normalizing, anxiolytic, nootropic and antitumor activities. A promising drug with antitumor activity is being developed based on the N -acetyl, C ‑amide form of HLDF-6 peptide, obtained by the solid phase method. Proteolytic hydrolysis of HLDF-6-AA peptide in blood plasma was studied using its derivatives labeled with hydrogen isotopes. The HLDF-6-AA peptide samples uniformly labeled with tritium and deuterium were obtained by the reaction of high-temperature solid-state catalytic isotope exchange at 170°С. [ 3 H] HLDF-6-AA peptide was obtained with the molar radioactivity of 50 Ci/mmol and the average deuterium incorporation for [ 2 H] HLDF-6-AA peptide was 2.90 atoms per peptide molecule. Proteolytic hydrolysis of HLDF-6-AA peptide in rat blood plasma was studied using radiochromatography. It was established that the main pathway of its proteolytic hydrolysis in rat blood plasma consists in cleavage of His-Arg- NH 2 (HR- NH 2 ) dipeptide from the C -end of the amide with the half-degradation period of 35 min.

Pharmacokinetics of the promising antitumor peptide HLDF-6-AA (Ac-ThrGlyGluAsnHisArg-NH 2 ) was studied using its uniformly tritiated derivative. Experiments were performed on male Wistar rats, Balb/c mice and Chinchilla rabbits. The tritium labeled peptide [ 3 H]HLDF-6-AA with the molar radioactivity of 50 Ci/mmol was obtained by the reaction of high-temperature solid-state catalytic isotope exchange (HSCIE). Under intravenous bolus administration of HLDF-6-AA peptide to rats and rabbits, the characteristics of its pharmacokinetic profile in the blood were obtained and the values of the main pharmacokinetic parameters of HLDF-6-AA peptide, and its active metabolite HisArg-NH 2 were calculated. It was shown that parameters of the retention time in the body and the rate of elimination for peptides HLDF-6-AA and HisArg-NH 2 in rats and rabbits are close and in rats they are about 7 and 21 min, respectively. It was shown that repeated administration of the drug does not lead to a change in its regular cumulation and does not cause a change in its pharmacokinetics compared with a single administration. The linearity of the dependence of pharmacokinetic parameters on the amount of administered peptide in the range of 2–22 mg/kg was proved in experiments in rats. As a result of the study of the distribution of HLDF-6-AA peptide and its metabolite HisArg-NH 2 between the blood and peripheral tissues of mice, it was shown that the maximum concentration of HLDF-6-AA peptide is observed in the kidney tissues and a somewhat smaller concentration in the omentum. It was found that 15–20 min after intraperitoneal administration of HLDF-6-AA to mice, the concentration of HisArg-NH 2 peptide begins to exceed the concentration of HLDF-6-AA peptide, which is caused by its greater resistance to proteolytic hydrolysis. The highest concentration of HisArg-NH 2 peptide is observed in the kidney and liver tissues.

Материалы V съезда фармакологов России «Научные основы поиска и создания новых лекарств» (14-18 мая 2018 года, г. Ярославль)

The novel method for the peptide pharmacokinetics in tissues of laboratory animals was elaborated by the example of the HLDF-6 peptide amide. This method practically completely prevented the proteolytic degradation of peptides in the course of the analysis. The HLDF-6 hexapeptide (TGENHR) is a fragment corresponding to the 41–46 sequence of the human leukocyte differentiation factor (HLDF). It exhibits a wide spectrum of nootropic and neuroprotective activity. Therapeutic agents for prevention and therapy of cerebrovascular and neurodegenerative diseases have been created on the basis of the HLDF-6 amide (TGENHR-NH 2 ). Pharmacokinetics and the molecular mechanism of action of the HLDF-6 peptide amide were studied using its tritium-labeled and deuterium-labeled derivatives. The labeled peptides were prepared with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). The tritiumlabeled [ 3 H]TGENHR-NH 2 peptide was obtained with a molar radioactivity of 230 Ci/mmol. The deuteriumlabeled [ 2 H]TGENHR-NH 2 peptide was prepared with an average deuterium incorporation of 10.5 atoms per the one peptide molecule. The NMR spectroscopy confirmed a uniform distribution of the isotope label throughout the whole peptide molecule. This uniformity allowed a qualitative analysis of both the peptide itself and all the possible fragments of its biodegradation in the organism’s tissues. The main TGENHR-NH 2 metabolites which were formed during its proteolytic cleavage in the blood plasma were quantitatively analyzed and pharmacokinetics of the peptide amide was investigated with the use of its tritium-labeled derivative after intravenous and intranasal administration in mice, rats, and rabbits. Values of the basic pharmacokinetic parameters were calculated, the hypothesis of pharmacokinetic linearity was checked, and metabolism of the peptide was studied on the basis of the obtained pharmacokinetic profiles of TGENHR-NH 2 . The TGENHR-NH 2 peptide was shown to have extremely high bioavailability with its intranasal administration (34% for rats). The peptide was quickly disappeared from blood due to its active proteolytic degradation in organism’s tissues. The TGENHR-NH 2 peptide was shown to be highly stable towards the proteolytic hydrolysis during its incubation with the blood plasma, and a quantitative analysis of the formed metabolites was performed.

Reaction of a high-temperature solid-phase catalytic isotope exchange in peptides and proteins under the action of the catalytically activated spillover hydrogen was studied. The reaction of human recombinant insulin with deuterium and tritium at 120–140°C resulted in an incorporation of 2–6 isotope hydrogen atoms per one insulin molecule. The distribution of the isotopic label by amino acid residues of the tritium-labeled insulin was determined by the oxidation of the protein S-S-bonds by performic acid, separation of polypeptide chains, their subsequent acidic hydrolysis, amino acid analysis, and liquid scintillation counts of tritium in the amino acids. The isotopic label was shown to be incorporated in all the amino acid residues of the protein, but the higher inclusion was observed for the FVNQHLCGSHLVE peptide fragment (B 1–13 ) of the insulin B-chain, and the His5 and His10 residues of this fragment contained approximately 45% of the whole isotopic label of the protein. Reduction of the S-S-bonds by 2-mercaptoethanol, enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius , and HPLC fractionation of the obtained peptides were also used for the analysis of the distribution of the isotopic label in the peptide fragments of the labeled insulin. Peptide fragments which were formed after the hydrolysis of the Glu-Xaa bond of the B-chain were identified by mass spectrometry. The mass spectrometric analysis of the isotopomeric composition of the deuterium-labeled insulin demonstrated that all the protein molecules participated equally in the reaction of the solid-phase hydrogen isotope exchange. The tritium-labeled insulin preserved the complete physiological activity.

The reaction of high temperature solid state catalytic isotope exchange in peptides and proteins under the action of catalyst-activated spillover hydrogen was studied. The reaction of human gene-engineered insulin with deuterium and tritium was conducted at 120-140° C to produce insulin samples containing 2-6 hydrogen isotope atoms. To determine the distribution of the isotope label over tritium-labeled insulin's amino acid residues, oxidation of the S-S bonds of insulin by performic acid was performed and polypeptide chains isolated; then their acid hydrolysis, amino acid analysis and liquid scintillation counts of tritium in the amino acids were conducted. The isotope label was shown to be incorporated in all amino acids of the protein, with the peptide fragment FVNQHLCGSHLVE of the insulin β-chain showing the largest incorporation. About 45% of the total protein isotope label was incorporated in His5 and His10 of this fragment. For the analysis of isotope label distribution in labeled insulin's peptide fragments, the recovery of the S-S bonds by mercaptoethanol, the enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius and HPLC division of the resulting peptides were carried out. Attribution of the peptide fragments formed due to hydrolysis at the Glu-X bond in the β-chain was accomplished by mass spectrometry. Mass spectrometry analysis data of the deuterium-labeled insulin samples' isotopomeric composition showed that the studied solid state isotope exchange reaction equally involved all the protein molecules. Biological studying of tritium-labeled insulin showed its physiological activity to be completely retained.

The reaction of high-temperature solid-state catalytic isotope exchange (HSCIE) between bovine hemoglobin and spillover hydrogen (SH) was studied. It was shown that, in the field of subunit contact, there is a significant decrease in ability for hydrogen exchange by SH. A comparison of the distribution of the isotope label in the hemoglobin α-subunit was carried out for the HSCIE reaction with the hemoglobin complex and with the free α-subunit. To this end, enzymatic hydrolysis of protein under the action of trypsin was carried out. The separation of tritium-labeled tryptic peptides was achieved by HPLC. Changes in availability of polypeptide chain fragments caused by complex formation were calculated using a molecular model. The formation of the protein complex was shown to lead to a decrease in the ability of fragments of α-subunits MFLSFPTTK (A32−40) and VDPVNFK (A93−99) for hydrogen replacement by tritium by almost an order of magnitude; hence, their availability to water (1.4 Å) twice decreased on the average. The decrease in ability to an exchange of hydrogen by spillover tritium on the formation of hemoglobin complex was shown to be connected with a reduction in availability of polypeptide chain fragments participating in spatial interactions of subunits with each other. Thus, the HSCIE reaction can be used not only for the preparative obtaining of tritium-labeled compounds, but also for determining the contact area in the formation of protein complexes.

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.

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 of 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 delivery of glyprolines 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 high efficacy of action of glyprolines on homeostasis of the gastric mucosa was partially provided by accumulation of these peptides (to high concentrations) in gastric tissues.

Journal of Labelled Compounds and RadiopharmaceuticalsVolume 50, Issue 5-6 p. 483-486 Short Reasearch Article Solid state isotopic exchange of hydrogen in proteins and peptides† Yurii A. Zolotarev, Corresponding Author Yurii A. Zolotarev [email protected] Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, RussiaInstitute of Molecular Genetics, Russian Academy of Sciences, Moscow, RussiaSearch for more papers by this authorAlexander K. Dadayan, Alexander K. Dadayan Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, RussiaSearch for more papers by this authorValerii S. Kozik, Valerii S. Kozik Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, RussiaSearch for more papers by this authorRustam H. Ziganshin, Rustam H. Ziganshin Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, RussiaSearch for more papers by this authorBoris V. Vaskovsky, Boris V. Vaskovsky Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, RussiaSearch for more papers by this authorNikolai F. Myasoedov, Nikolai F. Myasoedov Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, RussiaSearch for more papers by this author Yurii A. Zolotarev, Corresponding Author Yurii A. Zolotarev [email protected] Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, RussiaInstitute of Molecular Genetics, Russian Academy of Sciences, Moscow, RussiaSearch for more papers by this authorAlexander K. Dadayan, Alexander K. Dadayan Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, RussiaSearch for more papers by this authorValerii S. Kozik, Valerii S. Kozik Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, RussiaSearch for more papers by this authorRustam H. Ziganshin, Rustam H. Ziganshin Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, RussiaSearch for more papers by this authorBoris V. Vaskovsky, Boris V. Vaskovsky Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, RussiaSearch for more papers by this authorNikolai F. Myasoedov, Nikolai F. Myasoedov Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, RussiaSearch for more papers by this author First published: 30 July 2007 https://doi.org/10.1002/jlcr.1212Citations: 4 † Proceedings of the Ninth International Symposium on the Synthesis and Applications of Isotopically Labelled Compounds, Edinburgh, 16–20 July 2006. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat REFERENCES 1 Zolotarev YuA, Dadayan AK, Borisov YuA. Rus J Bioorg Chem 2005; 31: 1. 2 Zolotarev YuA, Kozik VS, Zaitsev DA, Dorokhova EM, Myasoedov NF. Dokl Akad Nauk SSSR 1989; 308: 1146. 3 Zolotarev YuA, Kozic VS, Zaitsev DA, Dorokhova EM, Myasoedov NF. J Radioanal Nucl Chem Art 1992; 162: 3. 4 Zolotarev YuA, Tatur VYu, Zaitsev DA, Myasoedov NF. US Patent 5 026 909 1988. 5 Zolotarev YuA, Dorokhova EM, Nezavibatko VN, Borisov YuA, Rosenberg SG, Neumivakin LV, Zverlov VV, Myasoedov NF. Amino Acids 1995; 8: 353. 6 Conner WC, Falconer JL. Chem Rev 1995; 95: 759. 7 Zolotarev YuA, Laskatelev EV, Kozik VS, Dorokhova EM, Rozenberg SG, Borisov YuA, Myasoedov NF. Izv Ross Akad Nauk Ser Khim 1997: 757. 8 Zolotarev YuA, Dadayan AK, Bocharov EV, Borisov YuA, Vaskovsky BV, Dorokhova EM, Myasoedov NF. Amino Acids 2003; 24: 325. 9 Zolotarev YA, Borisov YA, Myasoedov NF. J Phys Chem A 1999; 103: 4861. 10 Zolotarev YuA, Dadayan AK, Borisov YuA, Dorokhova EM, Kozik VS, Vtyurin NN, Bocharov EV, Ziganshin RH, Lunina NA, Kostrov SV, Ovchinnikova TV, Myasoedov NF, Bioorg Chem 2003; 31: 453. Citing Literature Volume50, Issue5-6Special Issue: Proceedings of the Ninth International Symposium on the Synthesis and Applications of Isotopically Labelled Compounds, Edinburgh, 16–20 July 2006.April ‐ May 2007Pages 483-486 ReferencesRelatedInformation