Fibrinspecific liposomes as a potential method of delivery of the thrombolytic preparation streptokinase.

The use of streptokinase (SK) in the clinic is limited by the lack of fibrin-specificity and the short half-life of the drug. We have developed a new dosage form of streptokinase (immunoliposome), which consists of "free" native streptokinase and "bound" encapsulated in liposomes conjugated through carboxylated dextran with fibrin-specific monoclonal antibodies FnI-3C (IgG2 class), in a ratio of 60 and 40%, respectively, and studied their physicochemical properties, pharmacokinetic parameters, and the ability of fibrin-specific liposomes with SK for targeted delivery to fibrin in an in vivo experiment. The obtained immunoliposomes had a hydrodynamic diameter of ~ 140 nm, a zeta potential of - 19.6 mV, and entrapment efficiency of 14.1%. Fluorescent labels bound to immunoliposomes with streptokinase selectively accumulated in model rat vein thrombi at sites containing fibrin in 30 min after injection. Studies of pharmacokinetic parameters showed that the administration of immunoliposomes with streptokinase to rats was accompanied by an increase in the half-life from 1.8 to 24.1 min, the time to reach the maximum concentration from 15 to 30 min, and a decrease in the elimination constant by about 13 times compared with the native streptokinase preparation. Further studies are needed to evaluate the thrombolytic efficacy a new dosage form of streptokinase in experiment in vivo.

[Extraction effect of ultrasound during plasma clot disruption].

The products of plasma clot destruction by the low-frequency ultrasound (US) were analyzed by the combination of SDS gel-electrophoresis, gel filtration chromatography and scanning electron microscopy. It was found that US (27 kHz) did not cause activation of the plasmin system or covalent bonds cleavage in the fibrin molecules. With the US intensities less than 21.6 W/cm2 the extraction of blood serum proteins, which are located in the pores of the fibrin network was occurred. An increase of the intensity of ultrasonic action led to protofibril dissociation, which was accompanied by further release into the solution of the blood serum proteins located inside the fibrin fibers. Being extracted from aggregated plasma clot proteins free protofibrils formed insoluble fibrin particles.

Effect of ultrasound on activation of serine proteases precursors.

The effect of ultrasound (US) (26.4 kHz, 26 W/cm2) on the activation process of a mixture of chymotrypsinogen and trypsinogen was studied. US led to a significant decrease in proteolytic activity, as well as inhibition of the activation process in general. It was shown that inhibition of proteinase activity under US influence was a consequence of inhibition of chymotrypsinogen-chymotrypsin transformation and the complete proteolytic trypsin degradation in the proenzymes mixture.

[Damage to erythrocyte membranes induced by high-intensity ultrasound]. / Povrezhdenie membran éritrotsitov pri ul'trazvukovom vozdeistvii vysokoi intensivnosti.

The effect of high-intensity ultrasound (11.2-54.2 W/cm2, frequency 36 kHz) on the structural and functional state of erythrocytes was investigated. It was shown that, at short-term action of the ultrasound (up to 1 min), the dose-dependent hemolysis of erythrocytes occurs. It was found that the exposure to ultrasound of high intensity (54.2 W/cm2) leads to the disruption of the structural state of erythrocyte membranes, which manifests itself in a change of microviscosity of the lipid bilayer of membranes and inhibition of the activity of the lipid-dependent membrane-bound enzyme acetylcholinesterase.

Effect of ultrasound on structure and functional properties of antithrombin III and proteins of PPSB complex.

A combination of gel-permeation HPLC, affinity chromatography on heparin-Sepharose, gel electrophoresis, and estimation of inhibitory activity showed that effect of low-frequency ultrasound (26 W/cm(2), 37 degrees C, pH 7.4) on homogeneous antithrombin III was accompanied by formation of aggregates and a latent form of serpin. Heparin and pentosan polysulfate stabilized antithrombin III; this resulted in decrease in ultrasonic-induced formation of the aggregate and latent forms. The influence of ultrasound was not accompanied by significant changes in the contents of non-activated blood coagulation factors in the PPSB complex.

Polydisulfides of substituted phenols as effective protectors of peroxidase against inactivation by ultrasonic cavitation.

Kinetics of inactivation of horseradish peroxidase (HP) induced by low-frequency ultrasonic (US) treatment (27 kHz) with the specific power of 60 W/cm2 were studied in phosphate (pH 7.4) and acetate (pH 5.2) buffers within the temperature range of 36.0 to 50.0 degrees C and characterized by effective first-order rate constants of US inactivation k(in)(us) in min(-1). Values of k(in)(us) depend on the specific ultrasonic power within the range of 20-60 W/cm2, on the concentration of HP, and on pH and temperature of the solutions. The activation energy of US inactivation of HP is 9.4 kcal/mole. Scavengers of HO* radicals, mannitol and dimethylformamide, significantly inhibit the US inactivation of HP at 36.0 degrees C, whereas micromolar concentrations of polydisulfide of gallic acid (poly(DSG)) and of poly(2-aminodisulfide-4-nitrophenol) (poly(ADSNP)) virtually completely suppress the US inactivation of peroxidase at the ultrasonic power of 60 W/cm2 on the sonication of the enzyme solutions for more than 1 h at pH 5.2. Various complexes of poly(DSG) with human serum albumin effectively protect HP against the US inactivation in phosphate buffer (pH 7.4). The findings unambiguously confirm a free radical mechanism of the US inactivation of HP in aqueous solutions. Polydisulfides of substituted phenols are very effective protectors of peroxidase against inactivation caused by US cavitation.

Ultrasound fibrin clot destruction in vitro in the presence of fibrinolytic agent.

Ultrasound fibrin clot destruction was investigated in vitro using electron microscopy and by monitoring the changes in the light transmission of clot debris suspension. It has been established that in the course of a combined action of ultrasound and fibrinolytic agent at high ultrasound intensities and short sonification periods, fibrin clot is disrupted mainly due to sonomechanical treatment, while fermentative lysis takes place in parallel and at a significantly lower rate. However, the streptokinase action prevails after ultrasound switching off and results in the prevention of clot debris conglomeration.

[Comprehensive evaluations of the action of galvanic current dosages used in physiotherapy on the intact myocardium in an experiment]. / Kompleksnaia otsenka deistviia ispol'zuemykh v fizioterapii dozirovok gal'vanicheskogo toka na intaktnyi miokard v éksperimente.

The most common physiotherapeutically used intensity (2.4 and 8.0 mA) and duration (15, 30, and 45 min) of galvanic current were examined for their effects on the intact myocardium in laboratory rats. These effects were found to be alterative (damaging) and directly proportional to the dosage: the greater the current intensity and duration were, the higher myocytic lesion is. However, these changes were transient and myocardial microstructural normalization was observed 24 hours later.

[Effect of a galvanic current on ischemic damage to the myocardium]. / Vliianie gal'vanicheskogo toka na ishemicheskoe povrezhdenie miokarda.

Galvanization of precardial region in rats with experimental infarction elicits considerable cardioprotective effect manifested in a decrease of the mass of necrotic tissue and in partial normalization of ion content both in the necrotic focus and outside it.