RU2043108C1 - Plasma-substituting agent correcting hemodynamic damages - neorondex - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: radiation-modified dextran of the following structural formula where k 370-432; l 5-7; m 10-12 is used as a main component of plasma-substituting agent which corrects hemodynamic damages. Dextran-base plasma-substituting agent shows polyfunctional properties, eliminates damage in central and peripheral hemodynamic, tissue blood current, improves rheological blood properties, shows immunostimulating and antiinflammatory effects. EFFECT: enhanced quality of agent. 7 tbl

Description

 The invention relates to medicine, namely to therapeutic preparations for blood plasma substitutes for the dextran series and can be used to correct conditions associated with impaired central and peripheral hemodynamics and tissue blood flow.

 Known plasma-substituting polysaccharide solutions of nature, used in medical practice, containing acid-hydrolyzed dextran in combination with salts and trace elements. These include polyglucin, polypher, reopoliglyukin and reogluman. All these solutions are used as plasma substituting media, which primarily have a volemic effect.

 Polyglukin is a 6% solution of dextran with an average mol.m. 60,000 ± 10,000 on an isotonic sodium chloride solution. Having high activity in terms of restoring blood pressure and the volume of circulating blood, this drug does not sufficiently normalize the rheological properties of blood. In some cases, its use can cause adverse changes in the state of aggregation of blood and indicators of the hemostasis system: reduce the electrophoretic mobility of red blood cells, increase the adhesion of blood plates, activate thrombinase and thrombin formation.

 The polymer, unlike polyglucin, contains 0.015% iron in the form of an iron-dextran complex. Along with hemodynamic function, it also has the ability to stimulate hematopoiesis, while preserving the adverse effects characteristic of polyglucin.

 Widely used in clinical practice are 10% solutions of low molecular weight dextran mol.m. 40,000 ± 10,000 reopoliglyukin and reogluman. They are characterized by a pronounced rheological effect, which is achieved by lowering blood viscosity and increasing its suspension stability. All solutions based on low molecular weight dextran are less effective in stabilizing blood pressure and circulating blood volume, their use reduces the concentration of fibrinogen, reduces the time of thrombin generation, changes the bleeding time and has a pronounced effect on the functional state of platelets, which makes their use undesirable because of the danger profuse bleeding.

 Among the known hemodynamic plasma substitutes, the closest to the claimed object is a rondex (prototype) plasma substituting solution based on dextran with an average mol.m. 60,000 ± 10,000 on an isotonic sodium chloride solution. This medicine is not manufactured by industry.

 The objective of the invention is to provide a plasma substitute based on dextran with multifunctional properties, the ability to simultaneously and for a long time correct the disturbances of central and peripheral hemodynamics, tissue blood flow, improve the rheological properties of the blood, and provide an immunostimulating and anti-inflammatory effect.

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где k=370-432, l=5-7, m=10-12.The problem is achieved in a plasma-replacing agent based on dextran in that the claimed object contains radiation-modified (radiolysed) dextran, sodium chloride and water in the following ratios:
Dextran Modifi-
cited mol. m. 65000 ± 5000 5.5-6.5 g Sodium chloride 0.85-1.0 g Water for injection Up to 100 ml
The claimed properties of the object are determined by the chemical structure of the polysaccharide

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where k = 370-432, l = 5-7, m = 10-12.

The method of obtaining this compound consists in the fact that in an aqueous solution of native dextran, by adding alkali (NaOH), the pH is adjusted to 9.0-11.0, hydrogen peroxide is added and the solution is irradiated with ⁶⁰ Co gamma rays. The radiation-chemical modification of dextran under the influence of gamma radiation consists in the introduction of carboxyl and carbonyl groups into its macromolecule during radiolysis. The terminal carboxyl groups are represented as gluconic and glucuronic acid lactones.

The determination of the content of carboxyl groups in the macromolecules of radiation-modified dextran is carried out by titration of 0.1 N. barium hydroxide Ba (OH) ₂ } and the method of infrared spectroscopy on the absorption bands at 1615 and 1425 cm ^-1 and establish that on one macromolecule there are on average 10-12 carboxyl groups, two of which are terminal and have the structure of gluconic and glucuronic lactones acids.

The content of carbonyl (α-hydroxyketone) groups in the polysaccharide is determined chemically by oximation, as well as by absorption spectra in the infrared region at ν = 1735 cm ^-1 and in the ultraviolet region at λ = 260 nm, and it is established that it is 5-7 groups per molecule .

 Determination of the molecular mass of the claimed object is carried out by light scattering and establish that its value is 65000 ± 5000.

 The composition is prepared as follows.

PRI me R 1. 59.8 g of radiation-modified dextran mol.m. 68200, containing 1.36% glycosidic units with carbonyl (C = O) and 2.72% glycosidic units with carboxyl (COOH) groups, having the above chemical structure, where k = 426, l = 6, m = 12, is dissolved in water for injection, add 9.0 g of pyrogen-free sodium chloride and adjust the pH of the sodium hydroxide solution to 4.8-7.3. Water for injection is adjusted to 1 liter. The solution was filtered through a sterilizing filter with a pore diameter of 0.22 um and autoclaved at ¹²⁰ ° C for 15 min. Plasma replacer is ready to use.

 Comparative analysis with the prototype shows that the claimed object has a chemical structure, ie, the claimed solution meets the criterion of "novelty." It also meets the criterion of "inventive step", since there are no substances among the known plasma substituting solutions that are capable of simultaneously and permanently correcting violations of central and peripheral hemodynamics, tissue blood flow, improving the rheological properties of blood, and providing immunostimulating and anti-inflammatory effects.

 The biological activity of the claimed object is illustrated by the following examples.

 PRI me R 2. In experiments on 8 outbred dogs of both sexes weighing 13.6-26.0 kg studied the therapeutic effect of neorondex in acute fatal blood loss, which was simulated by a single bloodletting from the femoral artery in the amount of 60% of the circulating blood volume in within 8-10 minutes Then they were treated with the claimed plasma substituting agent in a volume corresponding to the volume of blood loss.

In dynamics, according to conventional techniques we investigated the following parameters: mean and maximum blood pressure (BP _{cf., AD.),} Cardiac output and minute volume of blood circulation (with respect to the weight of the animal IA and IC, respectively), heart rate (HR), the ratio index of peripheral vascular resistance to its proper value (IPA / DIPS), half-life of subcutaneously injected radionuclide Na ¹²⁵ I (T 1/2), volumes of circulating blood (BCC) and plasma (CPP).

 The change in recorded indicators was of the following nature (see table. 1). The average blood pressure as a result of the treatment was completely restored and did not differ from the initial values during 7 days of observation of animals.

 The value of cardiac output after blood loss decreased by an average of 80% immediately after the infusion of neorodex normalized and amounted to 55.0-68.0% of the initial level in the first 2 hours 1 day after treatment. By the 4-7th day of the experiment, the values of cardiac output did not statistically significantly differ from the initial values.

 Correction of blood loss with Neorodex not only eliminated a reduced almost three-minute minute volume of blood circulation, but due to the increased heart rate, increased its values by more than 1.5 times at the end of the infusion. For the entire subsequent observation period, the IR values did not go beyond the physiological norm.

After the administration of neorondex, a decrease in the total peripheral vascular resistance occurred, and the half-period of the elimination of the radionuclide Na ¹²⁵ I reflected an acceleration of tissue blood flow and an improvement in the rheological properties of blood, not only immediately after administration of the drug, but also by 1-4 days.

 Neorondex infusions had a pronounced volemic effect, completely eliminating the deficiency of CSP after blood loss, due to which the bcc was also significantly corrected.

 These data confirm the ability of the Neoronedex plasma-replacing agent to simultaneously and continuously normalize disturbances in central and peripheral hemodynamics, tissue blood flow and improve blood rheological properties, and are supplemented in subsequent studies.

 PRI me R 3. In experiments on 9 outbred dogs of both sexes weighing 14.0-21.2 kg studied the therapeutic effect of neorondex in acute blood loss, which was simulated by a single bloodletting from the femoral artery in the amount of 45% of the BCC for 6- 8 minutes Then, Neorondex was treated with a plasma-replacing agent in a volume corresponding to the volume of blood loss.

 In control experiments, polyglucin (analogue, n = 10) and rondex (prototype, n = 9) were injected in the same volume.

 The composition of the determined parameters is identical to that described in example 2.

 Table 2 shows the dynamics of changes in some indicators of central and peripheral hemodynamics, tissue blood flow, characterizing the general condition of animals.

 Withdrawal from the blood circulation system led to a decrease in the compared batch of animals by BCC by 36.9-39.9% and CPP by 31.6-35.3%. A deficit in circulating blood volume caused a decrease in cardiac output by more than three times, while as the value of the coefficient reflecting the ratio of IPA to its proper value and indicating the degree of vasoconstriction increased by 142.8% (P <0.001), tissue blood flow sharply worsened. Although the number of heart contractions was increasing, it was not enough to maintain the IC at the proper level. Infusion of the studied solutions led to the elimination of hypotension, but their effect on the restoration of blood pressure was different. So, while with the introduction of polyglucin, blood pressure continued to remain statistically significantly reduced in the interval 2 hours 1 day after the compensation of blood loss, then rondex and, in particular, neorondex completely stabilized blood pressure, the values of which even slightly exceeded the original values.

 A more favorable effect of the Neorondex plasma replacement agent on the restoration of the main hemodynamic parameters was observed in most of the studied parameters. Thus, if polyglucin and rondex were administered, the UI turned out to be reduced (P <0.05) in comparison with the initial values 2 hours and 1 day after treatment, while in the case of neorondex infusion, the cardiac output in the same time interval did not statistically significantly differ from the initial level , exceeding (P <0.05) the corresponding values in groups of animals with polyglucin and rondex. Characteristic was a tendency to increase UI after neorodex infusion and subsequently, by the 4-7th day of the post-infusion period. When neorondex infusions, statistical differences disappeared by the first day with initial values in terms of heart rate and IPA / DIPS, while with the introduction of polyglucin and rondex, normalization of heart rate and peripheral vascular resistance was “delayed” by 2-3 days.

Particular attention should be paid to the effect of the studied plasma-substituted solutions on the half-life of Na ¹²⁵ I, which integrally reflects the state of nutritive blood flow and blood rheological properties. So, if the compensation for blood loss with polyglucin caused only the normalization of T 1/2 over the entire observation period, then the infusions of rondex and, especially, neorondex, led to a sharp activation of tissue blood flow. Moreover, the intensity of this activation in the case of the introduction of neorodex had the character of a statistically significant pattern with respect to the rendex during the 2 hours of the post-infusion period, as well as in the time interval of 1-4 days.

 Considering the fact that the studied plasma substituting solutions influenced the dynamics of restoration of blood volemic indices (BCC, BCP) of blood almost uniformly (see table 3), the recorded shifts of the main hemodynamic parameters, tissue blood flow and rheological properties of blood after compensation for blood loss by neorodex can be attributed to achieved in the process of obtaining the claimed object, the modification of the main component of the plasma substitute for radiolysed dextran.

 PRI me R 4. For a more detailed study of the effect of the claimed object of a plasma-replacement solution of neoronex on hemorheological blood parameters, a special series of bench experiments with donor blood was performed. We studied the electrophoretic mobility (EFP) of red blood cells and platelet adhesion, after preliminary contact of blood cells with the test drug. As control, intact cells or isotonic sodium chloride solution were used, and the comparison was performed with respect to dextran plasma substitutes for polyglucin and rondex.

 The results of the study of erythrocyte AFP are shown in Table 4. As follows from their analysis, the incubation of erythrocytes with polyglucin caused a statistically significant decrease in EFP cells, while plasma substitutes rondex and neorondex did not negatively affect this important hemorheological parameter.

 In the table. 5 shows data on the effect of neorondex on the adhesive properties of platelets. As it turned out, the strongest anti-adhesive effect was observed in the inventive plasma substituting agent, 2.5 (P <0.001) and 5.2 (P <0.001) times that of rondex and polyglucin, respectively.

 It should be noted that the ability to reduce platelet adhesion is a positive property of any blood substitute, since it prevents the initiation of pathological reactions of intravascular desiminated platelet aggregation. Thus, as a result of radiation-chemical technology for the production of neorodex, its release properties not only did not weaken, but also acquired an even more pronounced character than that of rondex (prototype).

 PRI me R 5. The study of the immunomodulatory properties of the claimed object of neorodex was carried out in reactions of humoral immunity.

The effect of neorondex on the humoral immune response was assessed by the change in the accumulation of antibody-forming cells (AOK) in the spleen after immunization of mice with highly responsive (CBA) and low responsive (C ₅₇ B1 / 6) lines of sheep erythrocytes (EB) taken in a suboptimal dose (2 × 10 ⁷ cells /mouse).

 In the experimental series (n = 10), the study drug was administered to mice intraperitoneally at a dose of 100 mg / kg of body weight (calculated on dry dextran) in an isotonic sodium chloride solution simultaneously with EB. In the control series, isotonic sodium chloride solution (n = 10) in the same dose or EB (n = 10) was introduced instead of neorondex.

The dynamics of the AOK content after the administration of neorondex (see table 6) indicates that the claimed object effectively stimulates the formation of AOK to EB, and the immunostimulating activity of the drug was manifested in both high and low mouse EB-responsive lines. The number of AOKs in a series of experiments on CBA animals increased by 40% relative to the control (P <0.05), and on C ₅₇ B1 / 6 animals by 112% (P <0.05), respectively.

 The results obtained indicate the pronounced ability of neorondex to stimulate humoral immunity.

 PRI me R 6. The study of the anti-inflammatory effect of neorondex was carried out on 40 outbred white mice males weighing 18-20 g in order to determine the effect of the inventive plasma substituting agent on the activation of release factors of inflammatory mediators. The method is based on the ability of plant lectins (concanavalin A) to release inflammatory mediators (including histamine).

 Mice of 3 experimental groups were injected intravenously with a neorodex solution at a dose of 5.7 ml / kg (n = 10), 28.6 ml / kg (n = 10) and 50 ml / kg (n = 10). These doses correspond to the minimum and maximum therapeutic doses (400 ml to 2000 ml), and also take into account the effect on the body of an excessive dose (3.5-4.0 l). Animals of the control group (n = 10) were injected with isotonic sodium chloride solution. Then, after 1 h, mice of the experimental and control groups were subplanarly injected with concanavalin A at a concentration of 5 mg / ml, in a volume of 20 μl, at a dose of 100 μg / mouse, and isotonic sodium chloride solution in the same volume. After 1 h, the mice were switched off from the experiment by decapitation and the reaction index in animals of the experimental and control groups was determined by the difference in mass of the right and left limbs.

 As follows from the data obtained (see table 7), neorondex suppressed the release of inflammatory mediators. The administration of the drug in therapeutic doses of 5.7 ml / kg and 28.6 ml / kg suppressed the inflammation reaction by 28.3 and 23.1%, respectively. Infusions of neorondex in an excessive dose of 50 ml / kg caused an even more significant suppression of the reaction (more than 2 times).

 The results obtained indicate the presence of a neorondex anti-inflammatory property in a plasma substitute, which was not detected in other plasma substitutes for the dextran series (polyglucin, reopoliglukin, rondex).

So, as a result of studies of the biological effectiveness of the plasma-replacing neorondex, the following conclusions are made:
in acute massive blood loss, the neorondex plasma-replacing agent has a pronounced effect on the restoration and stabilization of central and peripheral hemodynamics, improvement of tissue blood flow and rheological properties of blood; the positive biological effect of the drug on the macro- and microhemodynamics system is manifested simultaneously and can be traced for a long time, which distinguishes neorondex as a corrector of hemodynamic disturbances from polyglucin (analogue) and rondex (prototype);
the claimed agent has a stronger anti-adhesive effect on platelets in comparison with rondex and, especially, polyglucin and significantly increases the electrophoretic mobility of red blood cells, which indicates the presence of pronounced properties to improve microhemorheology;
Neorondex has an immunostimulating and anti-inflammatory effect, which is an advantage compared to blood substitutes of the dextran series, for which these properties are not shown.

Claims (1)

Plasma replacement agent that corrects hemodynamic disturbances, having an immunostimulating and anti-inflammatory effect, consisting in the fact that it is a traditionally modified dextran with mol.m. 65,000 ± 5,000, having the following structural formula:

where K 370 432, 1 5 7;
m 10 12,
when the content of 1 molecule of dextran 10 12 carboxyl and 5 7 carbonyl groups.

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