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

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.