Technical Field

The disclosure relates to hemostatic compositions and, more particularly to hemostatic first aid products for external use.

Background Art

The bleeding is a threatening and most dangerous consequence of many wounds and injuries and diseases, and is one of the largest causes of death in people aged 1 to 34 years worldwide.

In accordance with generally accepted classifications, hemorrhages are divided by source of hemorrhage (arterial, venous, capillary, and mixed), by direction (internal and external) and by time of occurrence (primary and secondary). Primary hemorrhages occur at the moment of injury when the integrity of anatomical integument and vessels is damaged. Secondary hemorrhages occur at a certain time after the pathological impact (for example, lysis of thrombus or erosion of blood vessel wall due to purulent wound process). By mechanism of their occurrence, hemorrhages are divided into mechanical (wounds and injuries, as well as, in case of vessel lysis due to inflammatory, tumorous or other pathological process), and neurotrophical. The latter include hemorrhages arising from metabolic disorders in liver diseases (decompensated cirrhosis), blood diseases (hemophilia, Werlhof's disease etc.), sepsis and some infectious diseases (hemorrhagic fever group etc.).

In human body there are protective opportunistic mechanisms to prevent blood loss. Hemostasis (haimatos - blood, and stasis - stopping) is a complex process preventing or arresting blood flow from the vessel lumen, ensuring formation of blood clot necessary to stop the hemorrhages, and finally eliminating fibrin when the need for it disappears.

At the first stage of the process, platelets collect into conglomeration, thus plugging the injury in the vessel wall. At that, blood coagulation factors are released (Ca ²⁺ , ADP, thromboxane A2, serotonin, and von Willebrand factor), which in turn induce further aggregation of blood plates, vasoconstriction takes place, and blood coagulation factors are cascade activated, and first fibrin strands precipitate. The essence of coagulation lays in successive activation of blood coagulation factors resulting in fibrinogen cleaving and its transformation into insoluble fibrin. Mechanism of coagulation is triggered as follows: blood coagulation factors come into contact with collagen from injured vessel in the place of injury (internal mechanism), which in combination with activation of tissue factors (external mechanism) leads to prothrombin transformation into thrombin, which catalyzes transformation of fibrinogen to fibrin.

Undoubtedly, besides the formation of primary thrombus, following factors participate in stopping hemorrhage: decrease of arterial pressure, tone increase and spasm of peripheral vessels with the slowing down of the blood flow, secretion arrest, and decrease in urination. If the bleeding is not very rapid (small-caliber vessel, compression of vessel by swelling tissues and hematoma), and compensatory reactions are well developed, it may stop spontaneously.

Considering the mechanism of hemostasis, local hemostatic compositions can be classified as follows:

1 . The group comprising epinephrine (adrenaline) and other vasoconstrictive drugs acting on the vessel link of the hemostasis and potentiating constrictory effects of the vessel wall.

2. The group comprising collagen, gelatin, the products of oxidation of cellulose, alginates, pectins, platelet adhesion and aggregation stimulating drugs (adhesion and aggregation inductors comprising substances such as serotonin, ADP, phosphodiesterase activators, etc.), and those functioning as primary thrombus due to their ability to absorb large amounts of blood.

3. Thromboplastin and plasma coagulation factors activating blood coagulation system.

4. Fibrinolysis inhibitors - tissue and synthetic ones (epsilon-aminocaproic acid, paraaminobenzoic acid - amben and other drugs).

5. Combined drugs.

Adrenaline (epinephrine) drug belonging to the 1 ^st group finds limited use as hemostatic due to its possible undesirable resorptive action, fast degradation, and is used predominantly in dental practice, surgery and proctology. Hemostatic properties of adrenaline are associated with its action on a1 -adrenoceptors located within the vessel walls; their activation causes vasoconstriction with consequent reduction of blood loss. Shortcomings of all drugs belonging to this group are short time of action and low specific activity, which cause a large number of undesirable effects.

The second group includes: hemostatic collagen sponge, gelatin sponge, antiseptic sponge with kanamycin, surgicel, spongostan, gelfoam, avitene, and collagen plate. Exogenic collagen and its degradation products (peptides) enhance body's own collagen synthesis, and stop the bleeding. Collagen stimulates spontaneous aggregation of platelets and is an effective hemostatic. At the same time, it readily forms complexes with many drugs and biologically active substances thus prolonging their action. Unfortunately this entire group has a number of shortcomings:

A. Due to their lightness, particles are washed out from the wound, specificity weakens significantly in contact with moist objects, and thrombus formed is poorly attached to edges of the wound, and eventually appears an electrostatic charge that causes the powder adhesion to dry objects.

B. Besides, collagen causes hyperscarring, possesses antigenic activity, and may be a carrier of hepatitis viruses and HIV (Camenzind E., Grossholz M., Urban P. et al. Collagen application versus manual compression: a prospective randomized trial for arterial puncture site closure after coronary angioplasty // J. Am. Coll. Cardiol. - 1994. - No.3. - p.655-662.)

Drugs on base of the products of oxidation of cellulose belonging to the second group are widely used as hemostatics due to their biocompatibility and low toxicity. Hemostatic effect is due to the ability of the products of oxidation of cellulose to interact with blood hemoglobin forming complexes with iron ions of hemoglobin, resulting in nonspecific platelet aggregation. Cellulose-based preparations are used in moderate bleedings.

Preparations containing thromboplastin are obtained from lung, liver, and brain tissues. Preparations from blood and its fractions possess pronounced hemostatic properties, are well sterilizable, maintain hemostatic activity for a long time, and are fully resorbable in body tissues. However, thromboplastin-containing hemostatic compositions have complex manufacturing and sterilization technology, insufficient storage stability, and are easily washed out of the wound.

A large group of hemostatics is represented with combined preparations. Combined drugs may contain hemostatic compositions from the first 4 groups, or use combination of a hemostatic and substance belonging to another group, most often antibiotic. Thus, for example, impregnation of collagen sponge with adrenaline improves hemostatic properties of the preparation without substantial increase of side effects.

Natural gel-forming ability of chitosan has been used in manufacture of hemostatic medicinal item "Celox" (Medtrade Products Ltd., Great Britain). Celox is a recognized effective drug applied in military surgery to stop arterial hemorrhages of the wounded directly on the battlefield. Blood coagulation occurs much faster when using this drug in comparison to other preparations due to its gelling ability and special treatment to enhance it. Chitosan is one of natural, low toxic, biodegradable polysaccharides commonly used as antimicrobial components. Chitosan belongs to the group of glucosamine copolymers with its n-acetylated analog n-acetyl-d-glucosamine (Sukhodub L.B. Chitosan: antibacterial activity and perspectives of the biomedical application // Annals of Mechnikov Institute, 2014, No. 3, p.8-12).

Mechanism of chitosan action is simple enough: on coming in contact with blood, chitosan particles begin actively to "imbibe" liquid. In a few seconds they get swelled and glued together into single gelatinous mass, forming single dense thrombus. This thrombus adheres well to wet areas allowing literally to "plug" the wound independently of its type.

Chitosan is able to stop any, even the heaviest bleeding (including arterial). Heavy blood loss quickly leads to hypothermia, which is a decrease in human body temperature substantially below 35°C, whereupon the blood ceases to coagulate properly thus complicating stoppage of hemorrhages. Chitosan is able to coagulate blood even in hypothermia because it acts independently of natural blood coagulation factors.

As indicated above, chitosan is a bioabsorbable agent (prone to biodegradation) due to its ability to be degraded by enzyme lysozyme forming glucosamine, which is a sugar natural to the human body. It means that chitosan is safe for patient even when he is left unattended. Particles of the substance are biodegradable: even when granules are left in the wound, this is not dangerous. Over the subsequent 24 hours they are degraded into polysaccharides and excreted from the body in a natural way.

Besides, due to the absence in the composition of degrading procoagulant minerals or nanoparticles, chitosan is absolutely safe: it doesn't generate heat and causes no burns unlike other hemostatics. The temperature of the wound rises by less than 1 (one) degree when using chitosan, that is, no thermal or chemical burns are caused.

The advantages of contact hemostatic compositions on base of chitosan may include the following:

- the effectiveness of the action is not reduced with temperature decrease;

- its effectiveness is independent of existing diseases associated with disorders of blood coagulation factors activities (hemophilia, coagulopathies);

- chitosan presence in the wound provides the effect of gluing the injured soft tissues and prevents the resumption of bleeding during transportation.

- proven antibacterial activity against 26 species of gram-positive and gram- negative bacteria. This feature of the product allows lowering the risk of wound infection and ensuring faster healing without massive use of antibiotics. The closest prototype is Celox - chitosan-based medicinal hemostatic composition which properties are specified above.

However, independently of the advantages mentioned, Celox as a chitosan- based preparation has its shortcomings:

partly dissolves in blood.

does not affect the natural process of blood coagulation - it just physically absorbs liquid from the blood resulting in its thrombus formation.

when dissolved in blood, chitosan is degraded by lysozyme into glucosamine which, in its turn, elevates blood glucose level. Therefore, Celox should be used with caution in people with diabetes mellitus, allergy to molluscs and crustacean, iodine sensitivity, kidney disease, cataract or gastric ulcer in active phase in anamnesis.

it was found that when using Celox, chitosan was sticking so tight to the wound edges due to high adhesive properties, that its subsequent removal before the wound treatment by physician caused additional tissue injuries in the wounded.

Summary of Invention

The objective technical problem to be solved is a hemostatic composition with better efficiency, that allows transportation of the wounded after forming the thrombus, minimal movements of the wounded without risk of breaking the thrombus and reduces tissue injury while removing artificial thrombus.

The disclosure is a hemostatic composition that forms the thrombus with optimal moisture and reduced adhesive properties for preventing further injury of wound edges. The objective technical problem is solved by two-stage process of thrombus formation and ability of calcium gluconate to show absorption properties to moist natural (plant) gum.

In various embodiments, the present disclosure provides a hemostatic composition comprising calcium gluconate, and locust bean gum and/or xanthan gum and/or guar gum (hereinafter absorbent gum) as a the substance that absorbs moisture from blood, at following component ratio, % wt.:

locust bean gum and/or xanthan gum

and/or guar gum 80-99

calcium gluconate 1 -20

One embodiment of the present disclosure provides a hemostatic composition comprising the locust bean gum as the substance that absorbs moisture from blood. Another embodiment of the present disclosure provides a hemostatic composition comprising the xanthan gum as substance that absorbs moisture from blood.

A further embodiment of the present disclosure provides a hemostatic composition comprising the guar gum as the substance that absorbs moisture from blood.

Still another embodiment of the present disclosure provides a hemostatic composition comprising the mixture of locust bean gum, xanthan gum, and guar gum as the substance that absorbs moisture from blood.

Yet another embodiment of the present disclosure provides a hemostatic composition comprising calcium gluconate, the mixture of locust bean gum, xanthan gum, and guar gum as the substance that absorbs moisture from the blood, at following component ratio, % wt.:

locust bean gum

xanthan gum

guar gum

calcium gluconate

Yet another embodiment of the present disclosure provides a hemostatic composition comprising calcium gluconate, the mixture of locust bean gum, xanthan gum, and guar gum as the substance that absorbs moisture from the blood, at following component ratio, % wt.:

locust bean gum

xanthan gum

guar gum

calcium gluconate

Yet another embodiment of the present disclosure provides a hemostatic composition comprising calcium gluconate, the mixture of locust bean gum, xanthan gum, and guar gum as the substance that absorbs moisture from the blood, at following component ratio, % wt.:

locust bean gum

xanthan gum

guar gum

calcium gluconate

In pharmacy, locust bean gum belongs to the group of gelling agents, (hydrocolloids), which also comprises xanthan and guar gums. Hydrocolloids are widely used for gelling or stabilization of aqueous colloid systems, including use in pharmaceutical industry to prepare gels, ointments, and oral syrups, locust bean gum is a neutral hydrocolloid consisting of D-mannose and D-galactose monomers in a ratio about 1 :4. It is obtained from the fruits of carob tree Ceratonia siliqua L. (Fabaceae) by grinding and following screening.

It was decided to add calcium gluconate into the composition to improve hemostatic properties of the technical solution of the present disclosure.

Role of calcium gluconate in hemostasis is caused by involving calcium ions in all stages of thrombus formation, beginning with stimulating the release of tissue factors after violation of integrity of the vessel wall and finishing with involving in all three phases of the coagulation cascade. Use of calcium gluconate as local hemostatic, as compared to calcium chloride, is preferred since the latter exerts a strong irritating effect.

Surprisingly, it was determined that calcium gluconate together with locust bean gum or xanthan gum, or guar gum, or their mixture, demonstrate synergism with respect to the blood coagulation due to their combined effect on different mechanisms affecting the blood coagulation. The absorbent gum exerts mechanical action by plugging the wound and absorbing the effluent blood, and forms the artificial thrombus, while calcium gluconate is acting directly as one of the blood coagulation factors, that accelerates the activation of the process, and begins to show absorbing properties, although not with respect to wound liquids, but to the liquid that was absorbed by the absorbent gum, thus creating a new mechanism of thrombus formation and hemorrhage stopping.

The disclosure provided for simultaneous start of absorbent gums and calcium gluconate action when the hemostatic composition is applied to the injured area: the gum absorbs the blood, and calcium gluconate in contact with blood starts activation of the thrombus formation. However, it was found experimentally that the process of artificial thrombus formation proceeds in two stages. On the first stage blood is actively absorbed by the gum, and on the second stage calcium gluconate due to its hygroscopicity starts to absorb moisture from the gum, thus providing optimal moisture content level for the artificial thrombus. Owing to this consistency, the artificial thrombus is firmly held in the wound, allowing not only to perform minimal movements of the wounded and to transport him/her, but also to remove the artificial thrombus from the wound without inflicting tissues additional injuries, as distinct from Celox prototype which absorbs the moisture from tissues so much that it "glues" to the edges of the wound. Modes for Carrying out the Invention

Some embodiments of the present disclosure are described in Examples 1 to 60. In one embodiment composition of a batch of hemostatic includes following steps:

Method 1 .

1 . Calcium gluconate is grinded in a special device;

2. Absorbent gum of one kind locust bean gum or xanthan gum or guar gum) is grinded in a special device.

3. Grinded calcium gluconate and absorbent gum of one kind chosen in step 2 are mixed in 1 :1 ratio.

4. The mixture obtained in step 3 is mixed in 1 :1 ratio with absorbent gum of one kind chosen in step 2.

5. Thereafter, step 4 is repeated until the desired ratio of components listed in Table 1 , g:

Method 2.

1 . Calcium gluconate is grinded in a special device;

2. Grinded calcium gluconate is mixed with absorbent gum of one kind without preliminary grinding of the absorbent gum, in 1 :1 ratio.

3. The mixture obtained in step 2 is mixed in 1 :1 ratio with absorbent gum of one kind chosen in step 2, without preliminary grinding of the absorbent gum.

4. Thereafter, step 3 is repeated until the desired ratio of components listed in Table 1 , g:

Method 3.

1 . Calcium gluconate without preliminary grinding is mixed with absorbent gum of one kind, without preliminary grinding of the absorbent gum, in 1 :1 ratio.

2. The mixture obtained in step 1 is mixed in 1 :1 ratio with absorbent gum of one kind chosen in step 2, without preliminary grinding of the absorbent gum.

3. Thereafter, step 2 is repeated until the desired ratio of components listed in Table 1 , g:

Table 1 locust bean gum q.s. to 100

Example 2

calcium gluconate 2 locust bean gum q.s. to 100

Example 3

calcium gluconate 3 locust bean gum q.s. to 100

Example 4

calcium gluconate 4 locust bean gum q.s. to 100

Example 5

calcium gluconate 5 locust bean gum q.s. to 100

Example 6

calcium gluconate 6 locust bean gum q.s. to 100

Example 7

calcium gluconate 7 locust bean gum q.s. to 100

Example 8

calcium gluconate 8 locust bean gum q.s. to 100

Example 9

calcium gluconate 9 locust bean gum q.s. to 100

Example 10

calcium gluconate 10 locust bean gum q.s. to 100

Example 1 1

calcium gluconate 1 1 locust bean gum q.s. to 100

Example 12

calcium gluconate 12 locust bean gum q.s. to 100

Example 13

calcium gluconate 13 locust bean gum q.s. to 100

Example 14

calcium gluconate 14 locust bean gum q.s. to 100

Example 15

calcium gluconate 15 locust bean gum q.s. to 100

Example 16

calcium gluconate 16 locust bean gum q.s. to 100

Example 17

calcium gluconate 17

Example 18 locust bean gum q.s. to 100 calcium gluconate 18 locust bean gum q.s. to 100

Example 19

calcium gluconate 19 xanthan gum q.s. to 100

Example 20

calcium gluconate 20 xanthan gum q.s. to 100

Example 21

calcium gluconate 1 xanthan gum q.s. to 100

Example 22

calcium gluconate 2 xanthan gum q.s. to 100

Example 23

calcium gluconate 3 xanthan gum q.s. to 100

Example 24

calcium gluconate 4 xanthan gum q.s. to 100

Example 25

calcium gluconate 5 xanthan gum q.s. to 100

Example 26

calcium gluconate 6 xanthan gum q.s. to 100

Example 27

calcium gluconate 7 xanthan gum q.s. to 100

Example 28

calcium gluconate 8 xanthan gum q.s. to 100

Example 29

calcium gluconate 9 xanthan gum q.s. to 100

Example 30

calcium gluconate 10 xanthan gum q.s. to 100

Example 31

calcium gluconate 1 1 xanthan gum q.s. to 100

Example 32

calcium gluconate 12 xanthan gum q.s. to 100

Example 33

calcium gluconate 13 xanthan gum q.s. to 100

Example 34

calcium gluconate 14 xanthan gum q.s. to 100

Example 35

calcium gluconate 15 xanthan gum q.s. to 100

Example 36

calcium gluconate 16 xanthan gum q.s. to 100

Example 37

calcium gluconate 17 xanthan gum q.s. to 100

Example 38

calcium gluconate 18 xanthan gum q.s. to 100

Example 39

calcium gluconate 19 xanthan gum q.s. to 100

Example 40

calcium gluconate 20 guar gum q.s. to 100

Example 41

calcium gluconate 1 guar gum q.s. to 100

Example 42

calcium gluconate 2 guar gum q.s. to 100

Example 43

calcium gluconate 3 guar gum q.s. to 100

Example 44

calcium gluconate 4 guar gum q.s. to 100

Example 45

calcium gluconate 5 guar gum q.s. to 100

Example 46

calcium gluconate 6 guar gum q.s. to 100

Example 47

calcium gluconate 7 guar gum q.s. to 100

Example 48

calcium gluconate 8 guar gum q.s. to 100

Example 49

calcium gluconate 9 guar gum q.s. to 100

Example 50

calcium gluconate 10

Example 51 guar gum q.s. to 100 calcium gluconate 1 1

guar gum q.s. to 100

Example 52

calcium gluconate 12

guar gum q.s. to 100

Example 53

calcium gluconate 13

guar gum q.s. to 100

Example 54

calcium gluconate 14

guar gum q.s. to 100

Example 55

calcium gluconate 15

guar gum q.s. to 100

Example 56

calcium gluconate 16

guar gum q.s. to 100

Example 57

calcium gluconate 17

guar gum q.s. to 100

Example 58

calcium gluconate 18

guar gum q.s. to 100

Example 59

calcium gluconate 19

guar gum q.s. to 100

Example 60

calcium gluconate 20

Method 4.

1 . Calcium gluconate is grinded in a special device;

2. The required absorbent gums are mixed in a certain ratio to obtain the absorbent gum (mixture).

3. The absorbent gum (mixture) is grinded.

4. Grinded calcium gluconate and absorbent gum (mixture) are mixed in 1 :1 ratio.

5. The mixture obtained in step 4 is mixed in 1 :1 ratio with absorbent gum (mixture).

6. Thereafter, step 5 is repeated until the desired ratio of components listed in Table 2, g:

Method 5.

1 . Calcium gluconate is grinded in a special device; 2. The required absorbent gums are mixed without preliminary grinding in a certain ratio to obtain absorbent gum (mixture) in a certain ratio.

3. Ground calcium gluconate and absorbent gum (mixture), without preliminary grinding, are mixed in 1 :1 ratio.

4. The mixture obtained in step 3 is mixed in 1 :1 ratio with absorbent gum (mixture), without preliminary grinding of the absorbent gums.

5. Thereafter, step 4 is repeated until the desired ratio of components listed in Table 2, g:

Table 2

Method 6. 1 . The required absorbent gums are mixed without preliminary grinding in a certain ratio to obtain absorbent gum (mixture).

2. Calcium gluconate, without preliminary grinding, and absorbent gum (mixture) from step 1 are mixed in 1 :1 ratio.

3. The mixture obtained in step 2 is mixed in 1 :1 ratio with absorbent gum (mixture) from step 1 .

4. Thereafter, step 3 is repeated until the desired ratio of components listed in Table 2, g:

The six examples of the mixtures selected from the wide range of values of substance contents are not intended to limit the scope of the present disclosure.

Following ratios were found to be preferred:

Data in Tables 3 to 5

Embodiment 1

Table 3

For these component ratios, preferred level of thrombus moisture content was achieved. However, for other components ratios, the purpose of the disclosure was also achieved. To determine advantages of the disclosure, a number of studies were conducted:

1 . Natural (plant) gum biodegradation studies have been conducted in order to ascertain the possibility of safe use of the natural (plant) gums for stopping hemorrhages.

In vitro experiment had been conducted to compare biodegradation of natural (plant) gums and chitosan.

The following enzymes were used in the experiment:

1 ) Lysozyme (lysozyme from chicken egg white, Sigma-Aldrich)

2) Alpha-amylase (alpha-amylase from porcine pancreas, Sigma-Aldrich)

3) Amyloglucosidase (amyloglucosidase from Aspergillus niger, Sigma-Aldrich)

The aim of this study was to establish the possibility of hydrolysis of locust bean gum, xanthan gum, and guar gum, and "Celox" preparation (chitosan) by natural human enzymes.

Table 6

Comparison of biodegradation substances studied

Study of the influence of hemostatic composition on metabolism of rats.

Table 7 presents data relating to the effect of hemostatic composition on basic biochemical blood indicators of white rats.

As can be seen from the results in Table 7, no substantial effect on metabolism indicators has been observed in male and female rats for the hemostatic composition tested in intragastric administration in dose of 170 mg/kg for 7 days. Total protein concentration, AST, ALT, creatinine, urea, alkaline phosphatase, glucose, and potassium in the blood serum were within the normal range. However, quite substantial, although non-significant decrease in sodium ion concentration in the blood serum had been observed both in female and male rats as compared to intact control. It can be assumed that this is due to adsorptive properties of gel-like suspension. Thus, local hemostatic composition studied has no toxic effect on metabolism of rats in intragastric administration in powder form.

Table 7

Data relating to the effect of hemostatic composition on basic biochemical blood indicators of white rats

It was found that natural (plant) gums are not absorbed in the body and excreted unchanged, which proves that they do not affect the body metabolism and have no harmful effects, do not increase blood sugar level, which allows their use for the people with diabetes mellitus, allergy to molluscs and crustacean, iodine sensitivity, kidney disease, cataract, or peptic ulcer disease in active phase in medical history, providing advantages over the prototype.

2. Studies have been conducted in order to determine hemostatic effects of the present disclosure.

Results of the studies of blood-stopping action for hemostatic composition and prototype in a model of incised wound of the liver are presented in Table 9. In the experiment with capillary parenchymatous hemorrhage, the blood loss in the groups administered hemostatic agents also decreased significantly by 49% in comparison with the PC (positive control) group when using local hemostatic composition in powder form, and by 44% for prototype, which testifies to their pronounced hemostatic effect. Incised wound of the liver and profuse blood loss cause violation of individual indicators of the blood coagulation system in all animals. Thus, PT level increases significantly by 157% and APTT by 16% in the PC group over the NC (negative control) group, with simultaneous significant increase in platelets count by 137%, which probably balances the hemostasis system. As a result, blood coagulation time remains on the level with NC group. In the group administered the local hemostatic composition in powder form, PT increase by 46% over NC group had been observed without affecting the integral state of the blood coagulation system. In the group receiving prototype, fibrinogen level increases by 33% on the background of APTT increase by 37%, which probably also equilibrated the hemostasis system and did not affect post-hemorrhagic state of the blood coagulation system. At the same time, hyperfibrinogenemia may be a predictor of a further hypercoagulation shift.

Considering the possibility of infection with pathogenic microorganisms in wounding and injury with further disturbance of wound healing, the important role belongs to antimicrobial and immunostimulatory action of hemostatic compositions. It is known from the literature that chitosan-based prototype possesses pronounced antimicrobial properties.

Therefore, phagocytic activity of neutrophils had been investigated as an important cellular link of non-specific immunity in mammals, as well as state of oxygen- containing phagocytic systems of neutrophils.

The results obtained (Table 9) demonstrate that single administration of tested local hemostatic composition in powder form and "Celox" prototype in a model of incised wound of the liver does not provide a stimulating action on the phagocytic activity of neutrophils. So, number of phagocytic cells (Phi) and number of yeast cells which they engulf (PhN) do not change significantly by the drugs studied. Significant increase in the number of formazan-positive cells is accompanied by activation of the oxygen-containing bactericidal systems of phagocytes on the background of hemostatic administration.

Thus, the hemostatic composition studied in a model of incised wound of the liver in capillary parenchymatous hemorrhage has pronounced blood stopping effect. Injury of internal organ and profuse bleeding is reflected in some indicators of rats' blood coagulation system, but it does not lead to significant changes of the blood coagulation time in posthemorrhagic period in all animal groups, which indicates to the local effect of the local hemostatic composition in powder form, similar to the prototype. Drugs studied do not affect the phagocytic activity after single administration, but the hemostatic composition of the present disclosure activates oxygen-dependent bactericidal systems of phagocytes according to the results of NBT test.

Table 8

Effects of local hemostatic composition in powder form and prototype on indicators of blood coagulation system and immunity after single administration in a model of incised wound of the liver in rats (n

Notes: * - significant difference from negative control indicator, p<0.05;

** - significant difference from positive control indicator, p<0.05;

*** - significant difference with indicator values for the group of animals administered local hemostatic composition in powder form, p<0.05;

Phi - phagocytic index; PhN - phagocytic number.

3. Blood stopping effect of the hemostatic composition on base of absorbent gum and calcium gluconate was studied in a model of hemorrhage from mechanically injured femoral artery, as well as in a model of capillary parenchymatous hemorrhage from incised wound of the liver in rats. In each experiment, rats were divided into 4 groups of 6 animals. The first group comprised pseudo-operated rats in which surgical access to femoral artery or liver had been provided under anesthesia in order to equalize the conditions of the experiment - negative control (NC). The second group consisted of animals with model pathology without treatment, or positive control (PC); the third group consisted of animals with model pathology administered hemostatic composition on base of absorbent gum and calcium gluconate; and fourth group comprised animals with model pathology which were administered prototype.

In 3 ^rd and 4 ^th groups, animals were treated with hemostatic compositions studied after cleaning the wounds by applying them to the vessel injured in dose of 170 mg/kg, after which bleeding time and blood loss were determined.

Table 9

Effects of single administration of hemostatic compositions on indicators of blood coagulation system in hemorrhages from femoral artery in rats (n = 6), M ± m

Notes: ^* - significant difference with indicator value for negative control group (NC), p<0.05; ^** - significant difference with indicator value for positive control group (PC), p<0.05.

Prothrombin time (PT), thrombin time (TT), and activated partial thromboplastin time (APTT) were determined in blood plasma. Also the following indicators were determined: phagocytic index (Phi) - number of phagocytic cells per 100 cells counted; and phagocytic number (PhN) - mean number of yeast cells engulfed per single neutrophil. State of the oxygen-dependent bactericidal systems of neutrophils was determined using nitro blue tetrazolium reduction test (NBT test).

The results obtained lead to the conclusion that the hemostatic composition tested, which is based on absorbent gum and calcium gluconate, shows blood-stopping effect in hemorrhages from femoral artery, without affecting systemic hemostasis. In effect, the tested hemostatic composition is more efficient than the comparison drug - prototype.

4. Clinical application.

Efficiency and convenience in clinical application was estimated assessed through a survey of surgeons providing qualified medical aid to those injured in medical institutions. The purpose of the survey was to reveal the degree of satisfaction of physicians with hemostatic compositions on base of absorbent gum and calcium gluconate, and chitosan.

42 surgeons participated in the survey.

Site of the survey - Medical dispensary institutions: stationary: Vinnitsa Regional Center of Emergency Medicine, Kramatorsk municipal healthcare facility City Hospital No. 1 ; Odessa municipal facility Regional Center for Emergency Medical Care and Disaster Medicine, Slavutich Specialized Medico-Sanitary Unit No. 5 of Ministry of Health of Ukraine; Kherson Emergency Medical Care Center, Kharkiv municipal health agency Center for Emergency Medical Care and Disaster Medicine; Uman' District Center for Primary Medico-Sanitary Care.

The survey was conducted on 4 issues. In order to ensure objective assessment, for each issue, rating scale from 1 to 3 points was developed for each issue:

1 ^st issue - bleeding during transportation:

3 points - bandage is clean or slightly soaked with serous hemorrhagic discharge;

2 points - bandage is soaked with hemorrhagic discharge;

1 point - bandage is soaked with hemorrhagic discharge, signs of ongoing bleeding.

2 ^nd issue - integrity of artificial thrombus:

3 points - artificial thrombus without signs of injury;

2 points -partly fragmented artificial thrombus;

1 point - destroyed artificial thrombus, bleeding.

3 ^rd issue - intensity of hemorrhage after removal of artificial thrombus: 3 points - minor capillary bleeding;

2 points - moderate bleeding of wound surface;

1 point - active spurting bleeding or bleeding from pulsating vessel.

4 ^th issue - complexity of artificial thrombus removal procedure:

3 points - artificial thrombus is easily removed from the wound surface;

2 points - removal of artificial thrombus causes moderate traumatization of wound surface;

1 point - removal of artificial thrombus is complex and results in resumption of hemorrhage.

Table 10

Results of the survey "Studies of satisfaction of physicians with hemostatic composition on base of absorbent gum and calcium gluconate, and chitosan"

Analysis of satisfaction of physicians with hemostatic compositions on base of absorbent gum and calcium gluconate revealed that the hemostatic meets the requirements of most physicians by all criteria, and 83% of physicians consider the hemostatic composition to be less traumatic and more efficient than prototype, by certain parameters.

In the course of the survey conducted among surgeons, the responses have also been collected and summarized concerning the differences noticed by surgeons between prototype - hemostatic agent Celox, and the hemostatic composition claimed herein, in their practical application during the fighting.

According to responses of surgeons, hemostatic agent Celox displays too high adhesion of the thrombus forming in the wound to body tissues and tendency of the thrombus formed to hardening, resulting in formation on the wound of a thick crust very firmly adherent to the tissues. This leads to the following problems. If the wounded with a wound treated with Celox is admitted to hospital an hour or more after getting wounded, it is often impossible to separate the crust formed from the wound by simple mechanical force, or removing of the crust leads to avulsion of tissues near the wound, in which case physicians have to cut out the crust from the wound resulting (in) new injures to body tissues and new bleeding.

Besides, the surgeons have also noted instances when Celox does not close wound properly due to instability of fresh thrombus forming in the wound in the presence of water - if water gets on a fresh thrombus, it can disintegrate into small ones forming a slush in the wound with water penetrating to the tissues, which can lead to infection.

Problems were also noted to occur with military men, who were forced to open the package with Celox powder holding the edge of the package with teeth. In case of arm wound, it is impossible to open the package containing the hemostatic composition powder with two hands; therefore man opens a package by holding the edge of the package with teeth and pulling the package away. In this case, some small quantity of hemostatic composition powder may be getting into mouth, nose and eyes of a man. Celox irritates mucous membranes of a man by getting on them, with eye surface being irritated particularly strong (something like a burn), and can strongly adhere to the tongue if getting there.

According to responses of physicians, the studied forms a thick gel-like thrombus in the wound which remains unhardened for a long time and is easily enough removed from the wound - withdrawal of this thrombus in most cases did not lead to new tissue injuries. In case of water getting on such a thrombus in the wound it does not change the thrombus, even the fresh one, and the latter continues to close the tissue in the wound and water does not penetrate to the tissues. For the present disclosure, no effects were noted after getting in contact with mucous membranes of a man, with gel formed separating in a natural way.

On the basis of the above, one can see that the present disclosure has the following advantages over the prototype: 1 . Fastens the process of stopping hemorrhages due to synergistic action of absorbent gum and calcium gluconate.

2. Is not biodegradable substance, which extends the possibilities of its application in people with different diseases.

3. Has no allergenic effects, because it is not absorbed by the body, resists fermentation, is excreted unchanged, contains no allergen substances (seafood traces, iodine and other).

4. Is firmly held in the wound, thus providing for the possibility of transporting the wounded and allowing them to make certain moves.

5. Is removed from the wound without inflicting additional injuries to the wounded.