The qualitative analysis of the amide of the HLDF-6 peptide and its metabolites in tissues of laboratory animals with the use of tritium-labeled and deuterium-labeled derivatives

Abstract

The goal of the study was to elaborate the pharmacokinetics methods of the amide derivative of peptide HLDF-6 (TGENHR-NH2) and its range of nootropic and neuroprotective activity is wide. The hexapeptide 41TGENHR46 is a fragment of the HDLF differentiation factor. It forms the basis for the development of preventive and therapeutic preparations for treating cerebrovascular and neurodegenerative conditions. Pharmacokinetic and molecular mechanisms of the action of the HLDF-6 peptide were studied using tritium- and deuterium-labeled derivatives of this peptide, produced with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). This reaction was employed to produce the tritium-labeled peptide [3H]TGENHR-NH2 with a molar radioactivity of 230 Ci/mmol and the deuterium-labeled peptide [2H]TGENHR-NH2 with an average deuterium incorporation equal to 10.5 atoms. It was shown by the NMR spectroscopy that the isotope label distribution over the labeled peptide's molecule was uniform, which allowed qualitative analysis ofboth the peptide itself and its fragments in the organism's tissues to be conducted. The newly developed pharmacokinetics method makes it possible to avoid almost completely losses of the peptides under study due to biodegradation during the analysis of tissues. These labeled peptides were used in mice, rats and rabbits to study the pharmacokinetics of the peptide and to calculate the values of its principal pharmacokinetic parameters. Characteristics of its pharmacokinetic profile in the blood were obtained, the hypothesis of pharmacokinetics linearity tested, its metabolism analyzed and its bioavailability value, 34%, calculated. It has been shown that the studied TGENHR-NH2 peptide shows high resistance to hydrolysis in the blood plasma, with dipeptidyl aminopeptidases making the largest contribution to its hydrolysis.

Anxiolytic activity of the neuroprotective peptide HLDF-6 and its effects on brain neurotransmitter systems in BALB/c and C57BL/6 mice

“…It should be mentioned that the behavioural studies were followed by an analysis of the morphological conditions of the animals’ internal organs. It showed that the chronic introduction of HLDF-6-amide brought about no pathological changes, neither when the dose was equivalent to the estimated therapeutic dose (300 μg/kg) nor when it was raised to five times as much (Zolotarev et al, 2015).…”

“…Generally, the lifetime of regulatory neuropeptides is only several minutes but their introduction brings about changes in principal neurochemical systems of the brain that are observed after many hours. We have shown that the half-life of HLDF-6-amide in BALB/c line mouse blood is 2.8 min (Zolotarev et al, 2015), and the specific anxiolytic response to its introduction can be observed for many hours. Thus, the anxiolytic and nootropic action of the neuroprotective peptide HLDF-6-amide is equally witnessed even when the organism is already free from it.…”

“…Peptides are characterized by postponed effects owing to their regulatory (trigger) function. We have shown earlier that intranasal administration of the HLDF-6 peptide provides its high bioavailability after its traverse into the brain (Zolotarev et al, 2015). The peptide has a normalizing regulatory effect on the brain’s main neuromodulator systems according to the trigger mechanism; it recovers pathology-caused disorders.…”

“…Another study showed that the activity of HLDF-6-amide exceeded those of reference compounds and its effect was achieved with considerably smaller doses (Bogachouk et al, 2016). Tritium- and deuterium-labelled derivatives of the peptide HLDF-6-amide were used in order to study its pharmacokinetics and molecular mechanism of action (Zolotarev et al, 2015). This study showed that the half-life time of HLDF-6-amide in the blood of BALB/c mice and Wistar rats after intravenous and intranasal administration was 3–4 min.…”

Pharmacokinetics and Molecular Modeling Indicate nAChRα4-Derived Peptide HAEE Goes through the Blood–Brain Barrier

“…Frozen and weighed tissue samples in plastic tubes were freeze-dried for 48 h. The freeze-dried tissue samples were heated at 65 °C for 30 min, after which they were homogenized, and the peptide fraction was extracted with 90% aqueous acetonitrile containing 1% TFA [ 13 ]. To facilitate HPLC analysis of radioactively labeled HAEE and normalize the loss of the peptide during extraction, 10 μg of HAEE was added to the extraction solution for each sample.…”