About oral application of a thrombolytic based on immobilized subtilisins for thrombosis treatment: 615.015.33

Abstract

Summary. Background. Thrombolytic therapy with plasminogen activators is quite effective, but it is associated with the risk of hemorrhagic complications and is implemented only by intravenous administration. Need for oral thrombolytic therapy exists. Among the possible options for effective oral fibrinolysis/thrombolysis, attention should be paid to serine proteinases that do not cause site-specific hydrolysis of blood coagulation factors. It is necessary to study their enteral bioavailability and thrombolytic activity. Objectives: to study the route of entry of serine proteinase subtilisin into the systemic circulation after oral administra- tion, and also to compare the thrombolytic activity of the proteolytic enzymes trypsin, chymotrypsin, subtilisin. Materials/Methods. We carried out an open comparative experimental study in parallel groups of rats to determine the thrombolytic activity of the studied drugs in vitro. Four groups were formed: 3 groups to study subtilisin, trypsin, chymotrypsin and 1 control group. In each group, fragmented thrombi were incubated with the putative thrombolytic agent. To study the absorption of immobilized subtil- isin (IS), 30 rats after midline laparotomy were intubated with a jejunum fragment for infusion of solutions. For IS labelled with fluorescein-5-isothiocyanate (FITC), the features of absorption from the jejunum into the blood-lymph system were determined. The labelled IS was extracted from the interstitial fluid adjacent to the intubation area, the mucous layer of which was removed, and from the parenchyma of the lymph nodes (sentinel and second-tier nodes). Confocal microscopy was used to detect visually the IS absorption on the jejunal epithelium. The thrombolytic activity of IS, trypsin, and chymotrypsin was studied on a frag- mented thrombus. Results. It was found in situ that IS 60 min after the start of incubation in the jejunum was detected in both blood plasma and lymph (p<0.01). Plasma drug concentration from the mesenteric vein (223.8 8±39.8 mU/ml) is more than 10 times higher than that in the lymph from the thoracic duct cistern (19.27 ± 2.33 mU/ml). Reducing drug concentration in the caudal vena (143.7 ± 24.4 mU/ml) compared to the mesenteric vein (p < 0.01) is explained by primary hepatic metabolism and subsequent renal clearance. Confocal microscopy clearly shows that IS is actively absorbed at the mucous membrane of the small intestine with accumulation in the crypt and entering the vasculature. In vitro experiments have shown that under conditions of the normal or compensated acid-base blood state, the IS has significantly the highest thrombolytic activity (p<0.05 compared to control, trypsin and chymotrypsin). After 30 minutes of incubation, IS reduces the thrombus area by more than 20%. Conclusions. IS enteral bioavailability is provided due to active absorption at the mucous membrane of the small intestine, direct entry into the vessels of the portal system and accumulation in the lymphatic bed with further drainage into the systemic circulation. IS thrombolytic effect is significantly superior to trypsin and chymotrypsin, which makes the development of oral thrombolytics based on trypsin and chymotrypsin unpromising.

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