RU2795399C2 - Fluid hemostatic agent - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to a kit for preparing a fluid hemostatic agent, including a hemostatic powder, having a composition comprising uncrosslinked fibrillar collagen, having a content of fibrous collagen and/or fibrillar collagen of at least 70 wt.% relative to the total masses of collagen; at least one monosaccharide and at least one glycosaminoglycan; a saline solution to be mixed with the hemostatic powder to form a fluid hemostatic agent, where the fluid hemostatic agent has a viscosity of 20 to 10,000 Pa·s when the hemostatic powder is mixed with the saline solution, and also refers to a method for preparing a fluid hemostatic agent using a kit.

EFFECT: group of inventions provides a hemostatic product with high hemostatic efficiency and improved efficiency, which is easy to use and that does not require a complex preparation process, and which can then be easily applied to a specific area, covering the entire required bleeding area.

29 cl, 6 dwg, 10 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to the field of hemostatic compositions, to the use of specific compounds or compositions as a hemostatic (hemostatic) agent, to a method for preparing a hemostatic composition, and to a method of hemostasis.

PRIOR ART

Wounds, regardless of whether they are external or internal, traumatic or surgical, often lead to bleeding. Such bleeding may be more or less significant. Bleeding is prevented and stopped by a series of physiological phenomena called "hemostasis (stop bleeding)". Hemostasis helps repair vascular damage and generally maintains vascular and tissue integrity.

When a blood vessel is damaged, a natural mechanism is triggered to stop bleeding, including various stages. Initially, vasoconstriction occurs, slowing down bleeding, lasting from 15 to 60 seconds and inducing a complex cascade of reactions. A fibrous network consisting of fibrin is formed around the platelet plug: the final thrombus is formed, which is protected from premature dissolution by fibrin-stabilizing factor XIII. And, finally, the fibrin mesh is pulled together (retraction), and the edges of the wound converge: the wound is reduced. Within the stable cross-linked fibrin, fibroblasts can then grow and form a connective matrix within the clot and eventually close the wound.

There is no solid fibrin in the circulating blood; if he were there, he would immediately clog vital vessels. However, a fibrin precursor, fibrinogen, is present. Under the action of thrombin, the synthesis of which is activated by coagulating factors, fibrinogen is converted into insoluble fibrin.

Finally, a few days or weeks after successful wound healing, the fibrin cluster is destroyed by fibrinolysis.

Regardless of this biochemical phenomenon, it is often necessary, especially in the case of large wounds or diffuse bleeding, to achieve "artificial" hemostasis.

There are "mechanical" solutions to help achieve hemostasis, such as pressure, ligation, and electrocoagulation, which are used as first-line therapy. However, such solutions have limited efficacy or are ineffective in a number of cases, such as weeping capillary bleeding, bleeding of hypervascularized organs such as the spleen or liver, hemorrhages leading to diffuse bleeding, for example, in bones and/or during neurosurgery.

There are also "chemical" solutions, in particular, implemented in some modern hemostatic (hemostatic) products. The components of these chemical solutions are usually either "absorbent" or "active" type.

Absorbent hemostatic products, in particular those comprising polysaccharides such as regenerated oxidized cellulose or alginates, work primarily by mechanical action and simple absorption. They often create difficulties due to excessive swelling. If said swelling results in rapid absorption of liquid, in particular blood, it can also lead to undesirable pressure when used in a "closed" environment, such as in contact with the dura or in urology.

In addition, certain products, especially those containing plant polysaccharides such as cellulose or alginates, may also cause inflammatory reactions during their resorption and/or may lead to the formation of cleavage products not recognized by the host. The consequence of this is the need to remove such products so that they do not remain in the body and therefore do not have their adverse effects.

Active hemostatic products, such as products containing thrombin or fibrin, are often blood products. The use of such products is associated with the risk of allergy and disease transmission, in particular if the disease vector is not inactivated by the conventional treatment. In addition, the treatment of these consequences is usually difficult and/or expensive. Finally, in most cases, active hemostatic products may require preparation before use, which can be limiting, moreover, inconvenient, in emergency situations.

In addition, the mechanism of action of products containing fibrin and thrombin is based on the interaction between the two blood products that make up the product. Sometimes a reaction can occur without interaction with blood, in which case the products are said to float. In other words, the product is repelled by the blood, which continues to flow, possibly causing the product to dilute or coagulate and gel over the blood without blocking the blood flow. Therefore, hemostasis cannot be performed.

The hemostatic powder, method of its preparation and method of use are disclosed in the international application published under the number WO 2012/146655 November 1, 2012, the content of which is fully incorporated into this application by reference.

Such a hemostatic powder has a satisfactory absorption capacity, high hemostatic capacity, practically no side effects, good adhesion to the wound edge, and satisfactory penetration into the bloodstream where it is used and/or limited swelling.

In addition to good hemostatic properties, the advantage of such a hemostatic powder is that it has a sufficiently high flowability that it can be sprayed onto the bleeding area. It can be used in most surgical procedures such as laparotomy, laparoscopy, celioscopy and robotic surgical techniques.

The hemostatic powder can be directly applied by the surgeon to the bleeding area without special preparation, which is another advantage.

Sometimes the use of special dispensers may be required to facilitate the application of hemostatic powder to a highly specific area of bleeding.

The object of the present invention is to provide a hemostatic product that is easy to use and in particular does not require a complex preparation process, which can then be easily applied to a specific area, covering the entire desired area of bleeding.

Another object of the present invention is to provide a hemostatic product having high hemostatic efficiency and improved efficiency compared to existing hemostatic products.

Another object of the present invention is to provide a kit for the preparation of a hemostatic product that can be used immediately without lengthy preparation with minimal manipulation of the hemostatic product, and which can be used, for example, in various surgical procedures.

SUMMARY OF THE INVENTION

To this end, a kit and method for preparing a fluid hemostatic agent is provided, such as those defined in the appended claims.

In particular, a kit for the preparation of a fluid hemostatic agent is proposed, including:

- a hemostatic powder having a composition comprising:

about collagen fibrillar type, having a content of fibrous collagen and/or fibrillar collagen of at least 70 wt. % relative to the total mass of collagen;

o at least one monosaccharide; And

o at least one glycosaminoglycan;

a saline solution to be mixed with the hemostatic powder to form a flowable hemostatic agent.

Preferably, the collagen used for the composition of the hemostatic powder is uncrosslinked. The use of uncrosslinked collagen in the composition aims in particular at simplifying the manufacturing process.

According to another preferred aspect, the saline solution is pure, consisting only of a mixture of distilled water and sodium chloride, which means that there are no additional components in the solution.

Preferred but non-limiting aspects of such a kit, taken alone or in combination, are the following:

In the composition of the hemostatic powder:

коллаген содержится в количестве от 80% до 90 мас. % относительно общей массы композиции гемостатического порошка;

collagen is contained in an amount of from 80% to 90 wt. % relative to the total weight of the hemostatic powder composition;

по меньшей мере один моносахарид содержится в количестве от 1% до 12,5 мас. % относительно общей массы композиции гемостатического порошка; и

at least one monosaccharide is contained in an amount of from 1% to 12.5 wt. % relative to the total weight of the hemostatic powder composition; And

по меньшей мере один гликозаминогликан содержится в количестве от 2% до 25 мас. % относительно общей массы композиции гемостатического порошка.

at least one glycosaminoglycan is contained in an amount of from 2% to 25 wt. % relative to the total weight of the hemostatic powder composition.

- In the composition of the hemostatic powder:

коллаген содержится в количестве от 80% до 90 мас. % относительно общей массы композиции гемостатического порошка;

collagen is contained in an amount of from 80% to 90 wt. % relative to the total weight of the hemostatic powder composition;

по меньшей мере один моносахарид содержится в количестве от 2,5% до 7,5 мас. % относительно общей массы композиции гемостатического порошка; и

at least one monosaccharide is contained in an amount of from 2.5% to 7.5 wt. % relative to the total weight of the hemostatic powder composition; And

по меньшей мере один гликозаминогликан содержится в количестве от 5% до 12,5 мас. % относительно общей массы композиции гемостатического порошка.

at least one glycosaminoglycan is contained in an amount of from 5% to 12.5 wt. % relative to the total weight of the hemostatic powder composition.

- In the composition of the hemostatic powder:

коллаген содержится в количестве от 84% до 88 мас. % относительно общей массы композиции гемостатического порошка;

collagen is contained in an amount of from 84% to 88 wt. % relative to the total weight of the hemostatic powder composition;

по меньшей мере один моносахарид содержится в количестве от 4% до 6 мас. % относительно общей массы композиции гемостатического порошка; и

at least one monosaccharide is contained in an amount of from 4% to 6 wt. % relative to the total weight of the hemostatic powder composition; And

по меньшей мере один гликозаминогликан содержится в количестве от 8% до 10 мас. % относительно общей массы композиции гемостатического порошка.

at least one glycosaminoglycan is contained in an amount of from 8% to 10 wt. % relative to the total weight of the hemostatic powder composition.

- In the hemostatic powder composition, at least one monosaccharide is glucose and at least one glycosaminoglycan is chondroitin sulfate.

- In the hemostatic powder composition, at least one glycosaminoglycan is selected from chondroitin sulfate, dermatan sulfate, hyaluronic acid, and mixtures thereof.

- The hemostatic powder composition additionally contains at least one coagulating factor in an amount of less than 0.1 wt. % relative to the total weight of the hemostatic powder composition.

- The coagulating factor is thrombin.

Salt solution includes - or consists of - distilled water and sodium chloride, where sodium chloride is contained in an amount of from 0.5% to 1.5 wt. % relative to the total mass of saline, most preferably in the amount of 0.9 wt. % relative to the total mass of saline.

- The kit includes 1 g to 2 g of hemostatic powder and 4 to 10 ml of saline solution.

- The weight of the saline solution is 2 to 10 times the weight of the hemostatic powder, and preferably 4 to 5 times the weight of the hemostatic powder.

- The hemostatic powder is enclosed in a dispenser having a container part made with a bellows and a nose part located on the container part, where the nose part has an opening for filling the container part with saline to be mixed with the hemostatic powder to form a fluid hemostatic agent.

- The container portion is shaped to promote hydration of the hemostatic powder with saline to form a flowable hemostatic agent.

The dispenser also includes a cap releasably connected to the nose to cover any opening in/out of the container part.

Also proposed is a method for preparing a fluid hemostatic agent using the above kit, including the steps:

a. providing hemostatic powder in a container;

b. adding a certain amount of saline to a container containing the hemostatic powder, closing and shaking said container to accelerate the hydration of the hemostatic powder to form a fluid hemostatic agent.

Preferred but non-limiting aspects of such a method, taken alone or in combination, are the following:

the method includes a subsequent step c) in which the container containing the fluid hemostatic agent is left to stand for a certain period of rest.

- In step b), the amount of saline added to the container is between 50% and 100% of the amount of saline in the kit.

- In step b), the amount of saline added to the container is 5 ml to 10 ml, and preferably 7 ml.

- In step b) the container is shaken for 10 seconds to 30 seconds and preferably for 20 seconds.

- In step c), the rest period is at least 30 seconds, preferably at least 60 seconds and even more preferably at least 90 seconds.

- In step c), the rest period is between 30 seconds and 120 seconds, preferably 90 seconds.

BRIEF DESCRIPTION OF GRAPHICS

Other characteristics and advantages will become apparent from the following description, which is given by way of illustration only and is in no way limiting, and which should be read with reference to the accompanying drawings, in which:

Fig. 1 is an illustration of a dispenser used to apply the fluid hemostatic agent presented in this paper;

Fig. 2 is an illustration of the dispenser of FIG. 1 with open cap;

Fig. 3 is a schematic side view of the dispenser cap of FIG. 1;

Fig. 4 is a schematic side view of the nose of the dispenser of FIG. 1;

Fig. 5 is a cross section of the container portion of the dispenser of FIG. 1;

in FIG. 6 shows an example of an electrophoresis result as described in Example 10.

DETAILED DESCRIPTION OF THE INVENTION

In the following description, unless otherwise indicated, weight percentages are given relative to the total dry matter weight of the hemostatic powder composition.

In the context of the present invention, "total dry matter weight of the hemostatic powder composition" refers to the total weight of the hemostatic powder composition free of solvent, in particular water, and thus the total weight of the anhydrous product.

In addition, the weights of the components and the percentages resulting from them may correspond to the anhydrous weight of these components, in other words, the weight of the component, not including water, which it may contain. This may also apply to interest received.

The hemostatic powder composition may have a collagen content greater than or equal to 70 wt. % relative to the total weight of the composition of the hemostatic powder, in particular greater than or equal to 75 wt. %, in particular greater than or equal to 77 wt. %, preferably greater than or equal to 80 wt. %.

In addition, the hemostatic powder composition may have a collagen content of less than or equal to 99 wt. % relative to the total weight of the composition of the hemostatic powder, in particular less than or equal to 96 wt. %, in particular less than or equal to 93 wt. %, preferably less than or equal to 90 wt. %.

Thus, the hemostatic powder composition may have a collagen content in the range of 70% to 99% by weight. % relative to the total weight of the composition of the hemostatic powder, in particular in the range from 75% to 96 wt. %, in particular in the range from 77% to 93 wt. %, preferably in the range from 80% to 90 wt. %. Preferably, the content of collagen is approximately 86 wt. % of the total weight of the hemostatic powder composition.

Collagen is the main structural protein in mammals. Collagen consists of tropocollagen (TC, from tropocollagen) molecules having a length of approximately 280 to 300 nm and a diameter of approximately 1.5 nm.

The term "fibrous collagen" refers to collagen in the form of fibers corresponding to the assembly of fibrils. The fibers typically have a diameter in the range of 1 µm to 10 µm. The term "fibrillar collagen" refers to collagen in the form of fibrils. More specifically, the fibrils typically have a diameter of 10 nm to 1 µm. Thus, fibrils are formed from staggered arrays of tropocollagen molecules, and these fibrils can be arranged to form collagen fibers. Fibrous and/or fibrillar collagen is generally insoluble, while non-fibrillar collagen is very soluble.

The definition of fibrous collagen and fibrillar collagen may in particular be consistent with the definition given by Markus Buehler in "Nature designs tough collagen: explaining the nanostructure of collagen fibrils," in PNAS, August 15, 2006, v. 103, no. 33, pp. 12285-12290.

More than 28 different collagens have been identified and classified into three main categories: fibrillar-type collagens, non-fibrillar-type collagens, and FACIT collagens (fibril-associated collagens with interrupted triple helixes).

Fibrillar-type collagens are collagens containing mostly fibrillar and/or fibrous collagens and substantially no non-fibrillar collagens (eg type I collagen). Similarly, collagens of the non-fibrillar type include collagens primarily containing non-fibrillar collagens. Some non-fibrillar type collagens may only be composed of non-fibrillar collagens (eg type IV or V collagen).

Industrial extraction and purification typically consists of destructuring the original fabrics in order to: 1) remove all or most of the contaminating fibers, 2) obtain the desired level of structuring depending on the end use of the product. Extraction of collagen is usually carried out under acidic or alkaline conditions to increase the solubility of monomolecular soluble collagen, which is non-fibrillar. The final collagen naturally contains a mixture of fibrillar/fibrillar collagen and non-fibrillar collagen. The ratio between fibrillar/fibrillar collagen and non-fibrillar collagen depends on the tissue chosen for extraction and the extraction method.

The final product differs from the collagen obtained by using a synthetic mixture of only fibrillar collagen and only non-fibrillar collagen. In a paper entitled "Extraction of collagen from connective tissue by neutral salt solutions" (Proceedings of the NATIONAL ACADEMY OF SCIENCES (Proceedings of the National Academy of Sciences) - v. 41, No. I , January 15, 1955, Jerome Gross, John H. Highberger, Francis O. Schmitt) shows the differences between fibrillar and non-fibrillar collagens obtained after a specific extraction process resulting - as previously described - in a mixture of the two collagens.

The proposed hemostatic powder contains fibrillar-type collagen, including fibrous and/or fibrillar collagen in an amount of at least 60 wt. %, in particular at least 70 wt. %, in particular at least 75 wt. %, preferably at least 80 wt. % relative to the total mass of collagen.

More specifically, collagen comprises at least 85%, in particular at least 90%, in particular at least 95%, preferably at least 98 wt. % fibrous and/or fibrillar collagen relative to the total mass of collagen in the hemostatic powder composition.

Preferably the composition has a content of fibrous and/or fibrillar collagen in the range from 85% to 95 wt. % relative to the total mass of collagen in the composition and, most preferably, from 85% to 90 wt. %.

This means that, according to a preferred embodiment, the hemostatic powder composition has a non-fibrillar collagen content in the range of 5% to 15% by weight. % relative to the total mass of collagen in the composition and, most preferably, from 10% to 15 wt. %.

It is advantageous to have a composition with such a ratio of fibrous and/or fibrillar collagen relative to non-fibrillar collagen, in particular for use as a hemostatic powder formulation. Preferably, fibrous and/or fibrillar collagen should be present in an amount sufficient to effect hemostasis, and non-fibrillar collagen should be present in an amount sufficient to cohesive the product, but not too much to avoid excessive swelling.

The collagen may be selected from type I collagens or type I and III collagens. Collagen can be extracted from various source tissues, in particular from skin and/or veins, of all species, more specifically pigs, cows or horses.

Collagen can generally be obtained from fibrous collagen of porcine origin extracted from skin and/or veins. In the case of vein collagen, the extraction can be as described in international patent application WO 2010/125086.

The above collagen, in particular fibrous and/or fibrillar collagen, can be obtained by acid or basic extraction. According to a particular embodiment, said collagen is obtained by a basic extraction. According to a particular embodiment, the collagen may be as described in patent application FR 2944706.

Preferably, the collagen is obtained by primary extraction, which maximizes the amount of fibrous and/or fibrillar collagen in the collagen to be extracted. In addition, such a basic extraction can be optimized in terms of controlling the ratio of fibrillar/fibrillar collagen and non-fibrillar collagen in the collagen to be extracted. Unlike acid extraction, basic extraction allows the hydrolysis of proteoglycans to occur. This action leads to the destruction of tissues and the separation of fibers without changing their shape. Under acidic conditions, swelling of the intrinsic collagen molecules in the fibers leads to their partial destruction during the process, releasing more non-fibrillar soluble collagen.

After extraction, the collagen can be used as such, i.e. without further processing, or it can be cross-linked, in particular using classical cross-linking methods such as thermal dehydration, the use of bridge-forming agents, for example, formaldehyde and/or glutaraldehyde; using oxidized polysaccharides, for example according to the method described in international patent application WO 2010/125086; and/or with oxidized amylopectins or glycogen. However, collagen crosslinking is not preferred because it complicates the process without necessarily increasing hemostatic efficiency.

Thus, preferably, the collagen used in the composition does not undergo any further processing and, in particular, is uncrosslinked. The use of uncrosslinked collagen is an advantage as it simplifies the process.

The hemostatic powder composition contains at least one monosaccharide, alone or in admixture with other monosaccharides. Said monosaccharides may be selected from ribose, sucrose, fructose, glucose and mixtures thereof. The monosaccharide present in the composition according to the invention alone or in admixture with monosaccharides is, in particular, glucose.

The composition of the hemostatic powder may have a monosaccharide content in the range from 1% to 12.5 wt. % relative to the total weight of the composition, in particular from 1.5% to 10 wt. %, in particular from 2% to 8 wt. % and, in particular, from 2.5% to 7.5 wt. %. Most preferably, the monosaccharide content is about 5 wt. % relative to the total weight of the composition.

The hemostatic powder composition may have a collagen/monosaccharide weight ratio in the range of 5 to 100, in particular 7 to 65, more in particular 10 to 50 and even more in particular 11 to 40. Most preferably, the composition has a weight ratio collagen/monosaccharide approximately 19.

A monosaccharide, namely ribose, sucrose, fructose, glucose and mixtures thereof, and in particular glucose, can in particular make it possible to obtain particles containing mainly fibrous and/or fibrillar collagen and monosaccharides with the desired characteristics, namely, size and density. The inclusion of the monosaccharide in the collagen mixture allows further reduction of the electrical charges within the composition, which makes it possible to obtain a powder that can be placed inside a container such as tubes, powder blowers, sprayers or dispensers.

In particular, the presence of a monosaccharide can make it easier and/or cheaper to obtain particles of the desired density and/or size, especially in terms of improving the hemostatic properties of the composition powder.

Grinding collagen fibers without additives leads to a decrease in the size of the fibers and reduces the density of the powder. In addition, the final product contains a significant amount of electrical charges, which prevents manipulation of the final product. The addition of the monosaccharide prior to collagen milling causes the mix to solidify, allowing rapid milling (limiting denaturation), thereby allowing the preparation of a powder with reduced electrical charges (suitable for placement in containers such as dispensers) and a final density suitable for application and reconstitution compositions.

Contrary to what might be expected, this addition of the monosaccharide does not affect the final activity of the product. In particular, it does not change the biological activity of the final product. The monosaccharide does not have a hemostatic effect.

Moreover, this addition of a monosaccharide does not cause it to behave like a blowing agent, as is the case in WO 01/97873. In patent document WO 01/97873, heating a dilute solution entails the formation of gelatin. To obtain a very concentrated solution, a high concentration of gelatin can be made, but the final product contains gelatin and does not contain collagen. Gelatin is known to be less hemostatic than collagen because platelet aggregation requires the presence of collagen fibrils and native collagen structures that are absent in gelatin.

According to one embodiment, the composition contains, preferably consists of particles containing, preferably consisting of collagen and a monosaccharide, in particular selected from ribose, sucrose, fructose, glucose and mixtures thereof, in particular glucose.

The composition may contain at least one coagulating factor. Such coagulating factors are well known to those skilled in the art. Preferably, one of the coagulating factors is thrombin. Even more preferably, the hemostatic powder composition contains only thrombin as a coagulating factor.

Said coagulating factor, in particular thrombin, can be obtained from animal sources (extracted from animal tissues and fluids) or from recombinant sources (obtained using cultures of genetically modified cells). The coagulating factor may be, for example, thrombin extracted from human tissues and fluids.

If a coagulating factor, in particular thrombin, is present, its content is preferably less than 0.1 wt. % relative to the total weight of the hemostatic powder composition.

In relation to thrombin, international units (ME, English IU, international units) are usually used. Thus, the composition may have a thrombin content in the range from 0.01 IU/mg to 20 IU/mg of the composition, in particular from 0.05 IU/mg to 10 IU/mg, in particular from 0.1 IU/mg up to 5 IU/mg, preferably from 0.2 IU/mg to 2 IU/mg. Most preferably, the thrombin content - if any - is about 0.83 IU/mg of the composition.

In addition to the monosaccharide, the composition may contain at least one other carbohydrate compound, which may be a glycosaminoglycan. Such a carbohydrate compound may be part of the composition, regardless of the presence or absence of a coagulating factor such as thrombin.

Said glycosaminoglycan can be selected from chondroitin sulfate, dermatan sulfate, hyaluronic acid and mixtures thereof, in particular from chondroitin sulfate.

Glycosaminoglycans can improve the rate at which the hemostatic composition absorbs blood. More specifically, glycosaminoglycans can accelerate the contact between blood and hemostatic products, in particular collagen and thrombin.

The composition may have a glycosaminoglycan content in the range from 2% to 25 wt. % relative to the total weight of the composition, in particular in the range from 3% to 20 wt. %, in particular from 4% to 15 wt. %, in particular from 5% to 12.5 wt. %. Most preferably, the content of glycosaminoglycan - if any - is approximately 9 wt. % of the total weight of the composition.

The composition may have a collagen/glycosaminoglycan weight ratio in the range of 2.5 to 50, in particular 3.5 to 35, more in particular 5 to 25 and even more in particular 6.5 to 20.

According to one embodiment, the composition comprises at least one, in particular one, monosaccharide, and at least one, in particular one, glycosaminoglycan, in particular as defined above and in particular in the amounts previously defined. .

Carbohydrate compounds, in particular, are monosaccharides and glycosaminoglycans.

The composition may have a carbohydrate content ranging from 2% to 25% by weight. % relative to the total weight of the composition, in particular in the range from 5% to 23 wt. %, in particular from 7% to 21 wt. %, in particular from 10% to 18 wt. %.

The composition may have a collagen/carbohydrate compound weight ratio in the range of 2 to 40, in particular 2.5 to 30, more in particular 3 to 20 and still more in particular 3.5 to 15.

The expression "total weight of carbohydrate compounds" refers to the sum of the weight of the monosaccharides defined above and the weight of other carbohydrate compounds mentioned above.

According to one of the embodiments, the composition contains, preferably consists of:

- collagen, including mainly fibrous and/or fibrillar collagen with a content of at least 50 wt. % relative to the total mass of collagen, and

- at least one, in particular one, monosaccharide.

In particular, the composition contains, preferably consists of:

- collagen, in particular, in an amount of from 70% to 99 wt. % relative to the total weight of the composition, in particular in the range from 75% to 96 wt. %, in particular from 77% to 93 wt. %, preferably from 80% to 90 wt. %, where said collagen includes fibrous and/or fibrillar collagen with a content of at least 50 wt. % relative to the total mass of collagen, and

- at least one monosaccharide, in particular glucose, in an amount of from 1% to 12.5 wt. % relative to the total weight of the composition, namely, in the range from 1.5% to 10 wt. %, in particular from 2% to 8 wt. % and, in particular, from 2.5% to 7.5 wt. %.

According to another embodiment, the composition comprises, preferably consists of:

- collagen, including mainly fibrous and/or fibrillar collagen with a content of at least 50 wt. % relative to the total mass of collagen,

- at least one, in particular one, monosaccharide,

- at least one, in particular one, coagulating factor.

In particular, the composition contains, preferably consists of:

- collagen, in particular, in an amount of from 70% to 99 wt. % relative to the total weight of the composition, in particular from 75% to 96 wt. %, in particular from 77% to 93 wt. %, preferably from 80% to 90 wt. %, where the specified collagen content includes fibrous and/or fibrillar collagen with a content of at least 50 wt. % relative to the total mass of collagen,

- at least one monosaccharide, in particular glucose, in an amount of from 1% to 12.5 wt. % relative to the total weight of the composition, in particular in the range from 1.5% to 10 wt. %, in particular from 2% to 8 wt. % and, in particular, from 2.5% to 7.5 wt. %, And

- at least one, in particular one, coagulating factor, in particular thrombin, in an amount of from 0.01 IU/mg to 20 IU/mg of the composition, in particular from 0.05 IU/mg to 10 IU/mg , in particular from 0.1 IU/mg to 5 IU/mg, preferably from 0.2 IU/mg to 2 IU/mg.

According to another embodiment, the composition comprises, preferably consists of:

- collagen, including mainly fibrous and/or fibrillar collagen with a content of at least 50 wt. % relative to the total mass of collagen,

- at least one, in particular one, monosaccharide, and

- at least one, in particular one, glycosaminoglycan.

In particular, the composition contains, preferably consists of:

- collagen, in particular, in an amount of from 70% to 99 wt. % relative to the total weight of the composition, in particular in the range from 75% to 96 wt. %, in particular from 77% to 93 wt. %, preferably from 80% to 90 wt. %, where the specified collagen content includes fibrous and/or fibrillar collagen with a content of at least 50 wt. % relative to the total mass of collagen,

- at least one monosaccharide, in particular glucose, in an amount of from 1% to 10 wt. % relative to the total weight of the composition, in particular in the range from 1% to 12.5 wt. %, in particular from 1.5% to 10 wt. %, in particular from 2% to 8 wt. % and, in particular, from 2.5% to 7.5 wt. %, And

- at least one glycosaminoglycan, in particular chondroitin sulfate, in an amount of from 2% to 25 wt. % relative to the total weight of the composition, in particular in the range from 3% to 20 wt. %, in particular from 4% to 15 wt. %, in particular from 5% to 12.5 wt. %.

According to another embodiment, the composition comprises, preferably consists of:

- collagen, including fibrous and/or fibrillar collagen with a content of at least 50 wt. % relative to the total mass of collagen,

- at least one, in particular one, monosaccharide,

- at least one, in particular one, coagulating factor, and

- at least one, in particular one, glycosaminoglycan.

In particular, the composition contains, preferably consists of:

- collagen, in particular, in an amount of from 70% to 99 wt. %, in particular in the range from 75% to 96 wt. %, in particular from 77% to 93 wt. %, preferably from 80% to 90 wt. % relative to the total weight, in particular, relative to the dry matter weight, compositions, where the specified collagen includes fibrous and/or fibrillar collagen with a content of at least 50 wt. % relative to the total mass of collagen,

- at least one monosaccharide, in particular glucose, in an amount of from 1% to 10 wt. % relative to the total weight of the composition, namely, from 1% to 12.5 wt. %, namely, from 1.5% to 10 wt. %, in particular from 2% to 8 wt. % and, in particular, from 2.5% to 7.5 wt. %,

- at least one coagulating factor, in particular thrombin, in an amount of from 0.01 IU/mg to 20 IU/mg of the composition, in particular from 0.05 IU/mg to 10 IU/mg, in particular from 0 1 IU/mg to 5 IU/mg, preferably 0.2 IU/mg to 2 IU/mg, and

- at least one glycosaminoglycan, in particular chondroitin sulfate, in an amount of from 2% to 25 wt. % relative to the total weight of the composition, namely, from 3% to 20 wt. %, in particular from 4% to 15 wt. %, in particular from 5% to 12.5 wt. %.

According to another specific embodiment, the composition comprises, preferably consists of:

- fibrillar-type collagen, mainly including fibrous and/or fibrillar collagen, said fibrillar-type collagen obtained, for example, by extraction in a basic medium and contained in an amount of approximately 85 wt. % relative to the total weight of the composition,

- glucose, in the amount of approximately 4.9 wt. % relative to the total weight of the composition,

- thrombin, in the amount of 0.2 IU/mg to 2 IU/mg of the composition, and

- chondroitin sulfate, in an amount of approximately 10 wt. % relative to the total weight of the composition.

According to another particular embodiment, the composition contains, preferably consists of:

- fibrillar-type collagen, mainly including fibrous and/or fibrillar collagen, said fibrillar-type collagen obtained, for example, by extraction in a basic medium and contained in an amount of approximately 85 wt. % relative to the total weight of the composition,

- glucose, in the amount of 5 wt. % relative to the total weight of the composition,

- and chondroitin sulfate, in the amount of 10 wt. % relative to the total weight of the composition.

In the context of the present invention, the expression "amount of approximately X%" refers to a deviation of plus or minus 20%, that is, an amount of approximately 10% means from 8% to 12%, in particular, a deviation of plus or minus 10%, preferably, plus or minus 5%.

When adding a coagulating factor in the form of a powder, in particular thrombin, such a powdered coagulating factor is preferably mixed with the powdered homogeneous molecular mixture of collagen/monosaccharide prepared previously.

In case both glycosaminoglycan (e.g. chondroitin sulfate) and a coagulating factor (e.g. thrombin) are added, they are preferably first mixed with each other, and then this mixture is added to the previously prepared collagen/monosaccharide mixture (already ground to powder form) .

Thrombin is not stabilized by either carbohydrate or collagen. Thrombin is never in contact with the monosaccharide solution (contrary to WO 98/57678), which prevents any denaturation of the protein and rehydration of the powder, resulting in the inability to dry it properly again.

The composition in powder form may, in particular, contain or consist of:

- particles comprising or consisting of fibrillar-type collagen and at least one monosaccharide, in particular glucose, where, in particular, said particles have a size, particle size distribution and/or density such as defined in the present description, and

- optionally, particles comprising or consisting of at least one glycosaminoglycan, in particular chondroitin sulfates, and / or at least one coagulating factor, in particular thrombin, where, in particular, these particles have a size, particle size distribution and/or density, such as defined herein.

The hemostatic powder composition preferably contains at least 50 wt. % particles whose size is from 200 µm to 400 µm.

The particles that make up the hemostatic powder preferably have an average particle size distribution in the range from 10 µm to 500 µm, in particular from 50 µm to 400 µm.

Preferably, at least 90 wt. %, in particular 100 wt. %, the particles that make up the specified hemostatic powder can pass through a sieve with a mesh size of 500 μm, in particular 400 μm.

At least 90 wt. % and, in particular, at least 95 wt. %, the particles that make up the specified hemostatic powder can be retained by a sieve with a mesh size of 10 μm, namely, 20 μm, preferably 30 μm, preferably 50 μm.

This separation was chosen to ensure the hydration of the powder. If the particle size is too small, the powder does not form a hydrated matrix that meets the required characteristics and aspects.

The hemostatic composition in powder form, in particular, includes:

- particles containing collagen and monosaccharide, and

optionally at least a glycosaminoglycan and/or a coagulating factor such as thrombin.

The hemostatic powder composition may include:

- particles containing collagen, monosaccharide and optionally at least one glycosaminoglycan and/or coagulating factor,

particles containing collagen, a monosaccharide and optionally a coagulating factor and optionally glycosaminoglycan particles,

- particles containing collagen and monosaccharide and particles containing at least one glycosaminoglycan and/or coagulating factor.

In the context of the present invention, the expression "dry powder" means that the composition contains a limited amount of solvent, in particular water. The specified limited amount may be less than 5 wt. %, in particular less than 3 wt. % and, in particular, less than 1 wt. % relative to the total weight of the composition.

Said dry form can be obtained by simple evaporation of the solvent used, by dehydration with organic solvents.

As mentioned above, the described hemostatic powder is formed from non-crosslinked collagen, because it is much simpler in terms of manufacturing technology, and it has been proven to have high efficiency in hemostasis even though the collagen of the powder is non-crosslinked.

The inventors unexpectedly found that, despite the fact that the collagen is uncrosslinked, mixing the specific hemostatic powder described above with saline made it possible to obtain a hemostatic product with a viscosity that allows it to be directly applied to the bleeding area to accelerate hemostasis.

This was really unexpected, because, on the contrary, it was known that in order to prepare a hemostatic collagen paste from a mixture of collagen-based powder and saline, in order for such a paste to work and be stable, cross-linked collagen must be used. This was specifically disclosed in US Pat. No. 4,891,359, published January 2, 1990. Cross-linking is known to impart stability to molecules by adding chemical bonds to the corresponding molecular structure, such additional chemical bonds are usually required to keep the molecules in aqueous form.

Therefore, according to a preferred embodiment, a dry hemostatic powder such as described above, in which the collagen is uncrosslinked, must be hydrated with saline to form a flowable hemostatic agent to be applied to the bleeding area.

The term "fluid" as used herein applies to compositions whose consistency allows the composition to retain a certain shape without the application of any stress, while being able to deform if stress, such as pressure, is applied to the composition.

A fluid is not a liquid, sponge, or powder, but a kind of paste, gel, or matrix that has a certain viscosity. Preferably, the fluid has a viscosity of 20 Pa.s to 10,000.Pa.s (corresponding to a fluidity range of 0.0001 (Pa.s) ^-1 to 0.05 (Pa.s) ^-1 ).

Fluid refers to a composition capable of, for example, passing through a syringe and/or cannula.

In the present description, the terms fluid hemostatic agent, fluid hemostat and hemostatic matrix are independently used to refer to the same specific composition.

Thus, in order to obtain a flowable hemostatic agent as defined above, the hemostatic powder and the saline solution are mixed.

The saline solution is preferably the standard sterile saline solution used in the operating room.

It preferably consists of distilled water containing sodium chloride in an amount of 0.5% to 1.5% and preferably about 0.9%.

The saline solution is preferably pure, which means that it consists of a mixture of sodium chloride and distilled water without the addition of any other components.

Saline solution can be stored in various forms, such as bulk in a large volume container or in a special container of a certain volume, such as pre-filled syringes.

Preferably, the saline solution is part of a kit for the preparation of a flowable hemostatic agent, such as a kit that further includes a certain amount of hemostatic powder in a container.

Preferably, the hemostatic powder from the kit is stored in a special dispenser such as shown in FIG. 1 and 2.

Thus, prior to preparation of a flowable hemostatic agent, all active ingredients are held together, in powder form, within the dispenser. This is very convenient in several respects. Firstly, it facilitates the storage of the product, since in this case, special care must be taken in the container containing the hemostatic powder, and not in the saline solution, which is a widely available product. It is also convenient from a preparation point of view, since only the hemostatic powder needs to be sterilized before storage, unlike the situation where some components are first mixed with saline (for example, thrombin) and then combined with the hemostatic powder.

Preferably, the dispenser 1 has a container portion 10 and a nose portion 20 configured to dispense a fluid hemostatic agent from the container portion 10.

The container part 10, shown more clearly in FIG. 5 is preferably shaped to promote mixing of the saline solution with the hemostatic powder and to increase the rate of hydration of the hemostatic powder.

In addition, the container portion 10 may have a bellows 11 allowing the user to apply the fluid hemostatic by simply squeezing the bellows 11 by hand, pushing the fluid hemostatic out of the dispenser 1 through the nose 20.

Thus, the nose portion 20 is designed to allow flowable hemostatic agent to flow out of the dispenser when the bellows 11 of the container portion 12 is compressed.

To facilitate manual compression of the bellows 11 by the user, the nose 20 may have a finger rest 21 so that at least one, but preferably two fingers can be placed on the rest 21 holding the dispenser 1 against the compression force acting on the bellows 11.

Preferably, as shown in FIG. 4, the finger support 21 includes two elongated elements extending radially from the longitudinal axis of the dispenser 1, said longitudinal axis corresponding to the compression axis of the bellows 11.

The nose portion 20 is also preferably designed to allow the user to fill the container portion 10 with saline through the nose portion 20, thereby eliminating the need to separate the nose portion 20 from the container portion 10 and avoiding the risk of contamination of the hydrated hemostatic powder.

To this end, the nose portion 20 may, for example, include a tube 22 through which a saline solution may be injected into the inside of the container portion 10. In addition, the tube 22 is designed in such a way that a fluid hemostatic agent can easily flow out of the dispenser 1 through the tube 22 when compressed. bellows 11.

Preferably, the dispenser further comprises a cap 30 that can be fitted onto the nose 20 in a removable manner.

This cap 30 preferably provides a hermetic seal for the dispenser 1, which is especially important during the storage and mixing steps.

When the dispenser 1 is not in use, the cap 30 is preferably closed, as shown in FIG. 1. This can help limit the danger of contamination of the inside of the device.

The cap 30 used in the dispenser and shown in FIG. 3 is preferably a screw cap which facilitates its removal, opening and closing.

The amount of hemostatic powder for one set is preferably 1 g to 2 g.

The amount of saline solution for one set is preferably 4 ml to 10 ml, more preferably 5 ml to 10 ml.

According to a preferred example, the kit contains 1.65 g of hemostatic powder to be mixed with 7 ml of pure saline.

Preferably, in the kit, the weight of the saline solution used to hydrate the hemostatic composition is 2 to 10 times the weight of the hemostatic powder, preferably 4 to 5 times the weight of the hemostatic powder.

When a user, such as a surgeon, needs to use the inventive fluid hemostatic agent, he can prepare it using the kit described above.

To this end, the user opens the application dispenser (also referred to as the applicator) containing the hemostatic powder by removing the corresponding cap 30.

Next, the user should pour a certain amount of sterile saline solution into dispenser 1.

Depending on the amount of hemostatic powder in dispenser 1, the amount of saline used is between 5 ml and 10 ml, preferably 7 ml.

To pour the saline solution into the container part 10 of the dispenser 1, the user can use, for example, a syringe. Such a syringe is preferably provided in the kit, whereby it has a volume corresponding to the exact amount of saline required to form a flowable hemostatic agent by hydration of the hemostatic powder.

During the pouring of the saline solution into the dispenser 1, the container part 10 is preferably rotated, for example around its own axis, in order to facilitate the incorporation of the saline solution into the hemostatic powder. If the saline solution is administered manually by the user, the rotation of the container part 10 can also be performed using the hand. However, if necessary, the process can be automated.

Tapping and/or gentle shaking of the container portion 10 during saline infusion may also be beneficial in accelerating the incorporation of saline into the hemostatic powder.

After the saline solution has been poured into the dispenser 1, the opening in the nose 20 is closed, preferably with the cap 30 of the dispenser 1, and the container is shaken to mix the hemostatic powder with the saline solution.

Shaking is preferably performed for at least 15 seconds, even more preferably at least 30 seconds. A shaking time of 10 seconds to 30 seconds, such as 20 seconds, is already effective in terms of hydration of the hemostatic powder.

Shaking is preferably carried out manually, but it can also be automated.

Mixing, performed manually, may consist in moving the dispenser 1 up and down a certain number of times. For example, the dispenser 1 may move up and down at least 10 to 30 times, preferably 20 times. To improve mixing efficiency, the dispenser 1 can also be turned upside down and then moved up and down a certain number of times. In this second manual mixing step, the dispenser 1 can also be moved up and down at least 10 to 30 times, preferably 20 times.

After agitation, the dispenser 1 containing the resulting hemostatic fluid is preferably allowed to stand for at least 30 seconds, preferably at least 60 seconds and even more preferably at least 90 seconds.

The residence time can be from 30 seconds to 120 seconds, preferably about 90 seconds.

This dormant period allows for hydration of the hemostatic powder and initial swelling to form a hydrated flowable hemostatic agent.

The advantage of the flowable hemostatic agent thus obtained is its homogeneity. In particular, the fluid hemostatic agent has a substantially uniform fluidity within the dispenser. This is particularly advantageous since the application of the fluid hemostatic agent will be the same whether it is the initial portion of the product from the dispenser or the remainder of the product.

After a fluid hemostatic agent is formed by hydration of the hemostatic powder with saline, it can be used for several hours, for example, for at least 8 hours, without loss of properties or activity.

The finished fluid hemostatic agent can be used as follows:

- Step 1: Excess blood should be blotted from the target site of bleeding with gauze/pad or aspiration so that the fluid hemostatic agent can be applied directly to the source of bleeding. The surface of the wound should be as dry as possible before application.

- Step 2: while squeezing the bellows, apply a fluid hemostatic agent to the source of bleeding. Apply enough product to completely cover the source of bleeding.

- Step 3: Immediately use a gauze/pad, preferably moistened with saline rather than blood, to hold the hemostatic material at the target site of bleeding, fitting it to the damaged site.

- Step 4: the hemostatic material should be maintained at the bleeding target site for a certain time, for example, for at least two to three minutes, in order to form a complex of hemostatic clots. Then you should carefully remove the gauze and inspect the area.

- Step 5: in case hemostasis has not been achieved, repeat steps 1 to 4 or use an alternative method of hemostatic treatment.

- Step 6: Throw away any unused opened food.

Depending on the viscosity (or fluidity) of the fluid hemostatic, skip step 3, particularly if the fluid hemostatic is sufficiently viscous to be held in place without the use of a gauze pad.

For best results, it is recommended not to disturb the clot complex with physical force.

In addition, after bleeding has been controlled, any excess fluid hemostatic agent not included in the hemostatic clot should be removed by gentle rinsing.

One of the advantages of the proposed flowable hemostatic agent is that it can be used at a time, i. using the entire product contained in the dispenser at once or multiple times, for example if there are multiple areas of bleeding that need to be treated at the same time, or if several consecutive bleedings occur during surgery.

A flowable hemostatic agent, such as described above, has the advantage of improving the surface of contact with the wound, in particular, contact with the bleeding area is deeper. This is of particular interest when the area of bleeding is soft tissue or parenchymal organs.

In addition, the flowable hemostatic agent can be easily applied to a wound or bleeding area, for example directly by the surgeon by hand using a special applicator. This makes it possible, for example, to cover the entire bleeding area without leaving areas uncovered by the flowable hemostatic agent.

Another advantage of the proposed flowable hemostatic agent is that it is familiar to surgeons who use a hemostatic product in the form of a paste to treat bleeding.

In addition, it gives the surgeon the choice of the form of hemostatic product he prefers to use, depending on his skills, the specific conditions of the operation, and so on. Therefore, he can use either a hemostatic powder such as described in WO 2012/146655 or a flowable hemostatic agent as described in this paper.

A hemostatic powder such as described above may be prepared, for example, according to a method comprising at least the following steps:

a) forming an aqueous suspension comprising, preferably, consisting of fibrillar-type collagen - mainly containing fibrous and/or fibrillar collagen - and a monosaccharide such as glucose,

b) recovering the product in the form of a precipitate, paste or gel, in particular by centrifugation or decantation,

c) drying the product, for example by evaporation.

d) grinding the product to the desired particle size, in particular by means of a hammer mill, and

e) optionally adding thrombin and/or chondroitin sulfates, in particular in solid form, in particular in powder form.

The formation in step a) of an aqueous suspension containing fibrous/fibrillar collagen and a monosaccharide results in a homogeneous redistribution of the monosaccharide around the collagen molecules. In addition, close contact between the molecular particles of collagen and monosaccharide leads, after dehydration, to the formation of a solid layer of sediment, suitable for obtaining - by grinding - a powder with the desired high density. In contrast, mixing powdered collagen with powdered glucose does not result in a homogeneous and sprayable powder, in particular due to density and electrical charges.

In step a), collagen may be present at a concentration of 30 g/l to 150 g/l.

The monosaccharide may be added to the suspension or to the homogeneous collagen paste in an amount as defined in the description, and more specifically, from about 2% to 5 wt. % relative to the mass of collagen.

In step a), the monosaccharide may be present at a concentration of 0.3 g/l to 10 g/l.

The aqueous suspension of collagen in step a) may be acidic and in particular contain an acid such as hydrochloric acid. Said acid may be present in a concentration of 0.01M to 0.5M, and in particular 0.02M to 0.1M, preferably about 0.05M. Said suspension may be in the form of a homogeneous paste.

Step b) may include pouring the suspension into a mould.

Step c) is carried out in such a way as to obtain a cake layer as thick as possible (exceeding the final desired particle size), with a very high density and as few air bubbles as possible (less than 5%) inside the sediment layer.

Step d) may be followed by a step of screening the powder, in particular to obtain the desired particle size.

According to a preferred embodiment, stage a) is the formation of a mixture containing 95 wt. % collagen fibrillar type and 5 wt. % glucose. After drying (stage b) and grinding (stage c), the mixture is added with chondroitin sulfate in an amount of 10 wt. % of the total weight of the mixture, so that the final composition contains:

- collagen: 86.36 wt. % relative to the total weight of the composition;

- glucose: 4.54 wt. % relative to the total weight of the composition;

- chondroitin sulfate: 9.09 wt. % relative to the total weight of the composition.

If thrombin is also added, its final content is less than 0.01 wt. % relative to the total weight of the composition. In the above mixture, thrombin may be present in an amount of 0.083 IU/mg of composition.

It is quite obvious that more or less fine grinding can be applied to all the above powdered products to obtain a powder with different particle sizes, depending on the type of grinding and its duration.

A flowable hemostatic agent made from a hemostatic powder mixed with a certain amount of saline can be used as a hemostatic agent.

Such a fluid hemostatic agent can also be used as a pharmaceutical composition, in particular a hemostatic preparation.

As described above, a method of hemostasis is also provided, comprising applying a fluid hemostatic agent, such as defined above, to a bleeding body part of an animal, including a human. In particular, the fluid hemostatic agent can be used in surgical procedures such as laparotomy, laparoscopic, celioscopy and robotic procedures.

The fluid hemostatic agent described above can also be used as a healing agent for internal and external wounds. The term "healing agent" refers to a product that produces clinically satisfactory scarring of the tissues with which it comes into contact.

DESCRIPTION OF EXAMPLES OF CARRYING OUT THE INVENTION

Example 1

In Vitro Hemostatic Capacity Measurement Protocol

Citrated (approximately 0.1 M) human blood is kept in a water bath at 37° C. throughout the measurement. The test product (10 mg) is placed in a 5 ml polypropylene tube with a snap cap and fresh citrated blood (2 ml) is added. Then add CaCl ₂ so that the final concentration of CaCl ₂ in the blood was 15 mm, after which the tube is closed. Then the contents are mixed by vigorous inversion (10 times), after which the tube is immersed in a water bath; the tube is returned to the vertical position every 10 seconds. The time required for the formation of a clot is noted, which corresponds to the hemostatic capacity.

Example 2

Particle Sizing Protocol

A known amount of product, in particular powder, is sifted through 50 µm, 100 µm, 200 µm, 300 µm and 400 µm sieves for 2 minutes (per sieve). The fractions from each sieve are weighed. The proportion of each particle size range is determined.

Example 3

Composition Swelling Protocol

Weigh a 15 ml flask (m ₀ in mg) and add X mg of dry composition powder (m _0+X in mg) to it. Add 0.15 M aqueous NaCl solution (2 ml), and the composition is left to swell for 20 minutes; then the flask is centrifuged at a rotation speed of 1000 rpm (eng. rpm, revolution per minute - revolutions per minute).

Excess NaCl is removed with a Pasteur pipette, and drops are removed by inverting the flask on filter paper; then the flask with the wet powder is weighed (m ₁ in mg).

The swelling ratio is calculated as follows: ((m ₁ -m ₀ )/(m _0+X -m ₀ )).

Example 4

Preparation of fibrillar-type collagen by basic extraction

Pieces of porcine dermis (30 kg), defatted with acetone, are left to swell for 3 hours in 100 kg of 0.05 M NaOH solution. The pieces of dermis are thinly cut with a cutting mill and the resulting paste is diluted with 50 liters of 0.05 M NaOH solution. The mixture is then pressure filtered through a 1 mm sieve. The pH value of the resulting paste is adjusted to pH 6-7.5 with HCl, the precipitate formed is collected by centrifugation or filtration through a 1 mm sieve.

The retentate is dehydrated with acetone according to methods known to those skilled in the art. This dehydrated retentate consists of fibrillar-type collagen with a high content of fibrillar/fibrillar collagen compared to non-fibrillar collagen. As a rule, such extracted collagen includes from 85% to 95 wt. % fibrillar/fibrous collagen relative to the total mass of collagen and from 5% to 15 wt. % non-fibrillar collagen relative to the total mass of collagen.

Example 5

Preparation of hemostatic powder No.1

30 g of the fibrillar-type collagen obtained in Example 4 was added to 1 L of 0.02 M HCl aqueous solution, and the mixture was stirred for 5 hours. Then powdered fructose is added to the obtained homogeneous paste in the amount of 2 wt. % (0.6 g) relative to the mass of collagen.

The mixture is homogenized for 1 hour, then poured and dehydrated. After drying, the dry product was milled at 25 g/min using a Fitzpatrick hammer mill at 7000 rpm under controlled heat conditions. The product is then screened by mechanical screening to remove particles larger than 400 µm.

After that, dermatan sulfate is added to the powder in an amount of 2 wt. % relative to the dry weight of the powder (0.612 g).

Next, the mixture was homogenized with a ball mill, lyophilized thrombin was added to the mixture in an amount of 15 IU/mg of powder, and finally the mixture was homogenized with a ball mill.

Example 6

Preparation of hemostatic powder No.2

7.5 kg of the fibrillar-type collagen obtained in Example 4 was added to 50 L of 0.05 M HCL aqueous solution, and the mixture was stirred for 16 hours. Then powdered fructose is added to the obtained homogeneous paste in the amount of 5 wt. % (375 g) relative to the mass of collagen.

The mixture is homogenized for 3 hours, then distributed on trays and dehydrated. After drying, the dry product is milled into fractions at a rate of 5 g/min using a hammer mill at 12,000 rpm under controlled heat conditions. The product is then screened by mechanical screening to remove particles larger than 400 µm and smaller than 50 µm.

Determine the particle size distribution to ensure that the particle size distribution of 60 wt. % of the sample has a particle size distribution greater than 200 µm.

Then purified chondroitin sulfates are added to the powder in an amount of 20 wt. % relative to the dry weight of the powder (1.575 kg). The mixture is homogenized using a ball mill.

Finally, lyophilized thrombin is added to the mixture in an amount of 10 IU/mg of powder. As before, the mixture is homogenized using a ball mill.

Example 7

Preparation of hemostatic powder No.3

1000 g of the fibrillar-type collagen obtained in Example 4 was added to 60 ml of 0.02 M HCl aqueous solution, and the mixture was stirred for 5 hours. Then crushed glucose is added to the obtained homogeneous paste in the amount of 5 wt. % (50 g) relative to the mass of collagen.

The mixture is homogenized for 1 hour, then poured and dehydrated. After drying, the dry product was milled at 25 g/min using a Fitzpatrick hammer mill at 7000 rpm under controlled heat conditions. The product is then screened by mechanical screening to remove particles larger than 400 µm and smaller than 50 µm.

Then chondroitin sulfate is added to the powder in an amount of 10 wt. % relative to the dry weight of the powder (105 g). The mixture is then homogenized using a ball mill.

Such a powder composition has a bulk density of approximately 0.408 g/ml.

Example 8

Preparation of hemostatic powder No.4

500 g of fibrillar-type collagen obtained in Example 4 was added to 30 ml of 0.02 M HCl aqueous solution, and the mixture was stirred for 5 hours. Then crushed glucose is added to the obtained homogeneous paste in the amount of 5 wt. % (25 g) relative to the mass of collagen.

The mixture is homogenized for 1 hour, then poured and dehydrated. After drying, the dry product was milled at 25 g/min using a Fitzpatrick hammer mill at 7000 rpm under controlled heat conditions. The product is then screened by mechanical screening to remove particles larger than 400 µm and smaller than 50 µm.

Then, chondroitin sulfate mixed with powdered thrombin is added to the powder in an amount of 10 wt. % relative to the dry weight of the powder (52.5 g). Thrombin is added to the mixture in a final amount of 0.85 U/mg. The mixture is then homogenized using a ball mill.

Such a powder composition has a bulk density of approximately 0.425 g/ml.

Example 9

Preparation of hemostatic powder No.5

750 g of the fibrillar type collagen obtained in Example 4 is mixed with 6675 ml of highly purified water. The mixture was stirred at a first stirring speed of 20 rpm for 10 minutes and then at a second stirring speed of 40 rpm for 15 minutes.

The above mixture is then stirred again at a first stirring speed of 20 rpm with added glucose solution (37.5 g glucose in 300 ml water). The amount of added glucose corresponds to 5 wt. % relative to the mass of collagen used in the mixture. Next, this new mixture is stirred at a second stirring speed of 40 rpm for 10 minutes. The resulting preparation is then stored for 16 hours.

Next, 87.5 ml of 1 M aqueous HCl solution was added to the preparation with stirring at a speed of 30 rpm. Then this new mixture is mixed at the first stirring speed of 35 rpm for 1 minute and at the second stirring speed of 40 rpm for 1 minute, after which several mixing sessions are performed for 5 minutes at the same speed of 40 rpm, making a quick pause in mixing between two sessions.

After that, the thick paste obtained in the previous step is divided into several pieces having the same shape and weight. Then these pieces of paste are placed for 24 hours in a sealed chamber containing an atmosphere saturated with ammonia. After the neutralization step, the paste pieces are dried at 20° C. for 96 hours, and then the dry products are ground at a rate of 1 kg/h using a Forplex cryogenic mill at 8500 rpm. The resulting powdered product is sieved by mechanical sieving to remove particles larger than 200 µm and smaller than 50 µm, thereby obtaining a collagen-glucose powder.

Then, powdered chondroitin sulfate (CS), consisting of particles ranging in size from 50 μm to 200 μm, is added to the collagen-glucose powder in an amount of 10 wt. % relative to the dry weight of the collagen-glucose powder. For example, 30 g of powdered chondroitin sulfate is mixed with 300 g of collagen-glucose powder. Freeze-dried thrombin is also added to hemostatic powder No.5 in the amount of 1000 U/g. Next, the mixture is homogenized using a V-shaped mixer. The final hemostatic powder has a bulk density of approximately 0.4 g/ml.

Example 10

Characterization of collagen→presence of soluble collagen in collagen, determination of the ratio between fibrillar/fibrillar collagen and non-fibrillar collagen

The purpose of the experiment is to determine the content of collagen (extracted collagen or powdered collagen) of fibrillar/fibrillar collagen and non-fibrillar collagen. This content can be determined by assessing the collagen content of insoluble collagen (corresponding to fibrillar/fibrous collagen) and soluble collagen (corresponding to non-fibrillar collagen).

The experiment consists of dissolving approximately 2.5 g of the collagen to be tested in 166 ml of water at pH 13 for 16 hours. The solution is then centrifuged (10,000 rpm for 10 minutes). The supernatant (supernatant) (corresponding to non-fibrillar collagen) and the residue (corresponding to fibrous/fibrillar collagen) are separated. The residue is immediately dried by successive washes with acetone under controlled air flow. The pH of the supernatant was adjusted to pH 3 with acetic acid and hydrochloric acid at 6 M. Solid collagen from the supernatant was obtained by adding 0.6 M NaCl followed by centrifugation. It is then dried by successive washes with acetone under controlled air flow.

Calculate the masses of collagen from the residue (M _residue ) and from the supernatant (M _supernatant ), and using the formula M _residue / (M _residue +M _supernatant ) × 100, get the percentage of fibrous collagen from the total amount of collagen.

In the invention, the ratio M _residue /(M _residue + M _supernatant ) must be above 80% in both the collagen used to prepare the powder and in the final collagen powder. Preferably, the ratio is greater than 85%.

For example, the experiment described above with three batches of collagen prepared as described in Example 4 gives very similar ratios of 92.67%, 94.60% and 91.51%, respectively. After milling the collagen from these three batches, the ratio remains very close at 91.63%, 88.02% and 88.69%, respectively.

Another way to show the presence of fibrous / fibrillar collagen and soluble collagen is to perform SDS page electrophoresis (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - polyacrylamide gel electrophoresis with sodium dodecyl sulfate).

On FIG. 6 shows such an electrophoresis where sample S1 corresponds to the supernatant of the first batch (obtained from collagen extracted as in Example 4), sample S2 corresponds to the remainder of this first batch, sample S3 corresponds to the supernatant of the second batch (also obtained from collagen extracted as in Example 4) , sample S4 corresponds to the remainder of this second batch.

The results show that in the case of residue-derived collagen, more fibers cannot pass through the acrylamide gel and stain when the gel is stopped. The sample preparation does not allow the separation of each chain from the collagen. Thus, alpha chains are present in very low amounts. The collagen from the supernatant is able to move completely in the gel, there are no fibers blocked at the top, the collagen chains are properly separated during the electrophoresis process.

BIBLIOGRAPHICAL DATA

WO 2012/146655

"Nature designs tough collagen: explaining the nanostructure of collagen fibrils," Markus Buehler (PNAS, August 15, 2006, v. 103, No. 33, pp.12285-12290)

"Extraction of collagen from connective tissue by neutral salt solutions" by Jerome Gross, John H. Highberger and Francis O. Schmitt (Proceedings of the NATIONAL ACADEMY OF SCIENCES, v. 41, No. I, January 15, 1955)

WO 2010/125086

FR 2944706

WO 01/97873

US 4891359

Claims (48)

1. A set for the preparation of a fluid hemostatic agent, including: - a hemostatic powder having a composition comprising: non-crosslinked fibrillar-type collagen having a content of fibrous collagen and/or fibrillar collagen of at least 70 wt.% relative to the total mass of collagen; at least one monosaccharide; And at least one glycosaminoglycan; a saline solution to be mixed with the hemostatic powder to form a fluid hemostatic agent, where the fluid hemostatic agent has a viscosity of 20 to 10,000 Pa·s when the hemostatic powder is mixed with the saline solution. 2. The kit according to claim 1, where in the composition of the hemostatic powder: - collagen is contained in an amount of from 80 to 90 wt.% relative to the total weight of the hemostatic powder composition; - at least one monosaccharide is contained in an amount of from 1 to 12.5 wt.% relative to the total weight of the hemostatic powder composition; And - at least one glycosaminoglycan is contained in an amount of 2 to 25 wt.% relative to the total weight of the hemostatic powder composition. 3. The kit according to claim 1, where in the composition of the hemostatic powder: - collagen is contained in an amount of from 80 to 90 wt.% relative to the total weight of the hemostatic powder composition; - at least one monosaccharide is contained in an amount of from 2.5 to 7.5 wt.% relative to the total weight of the hemostatic powder composition; And - at least one glycosaminoglycan is contained in an amount of 5 to 12.5 wt.% relative to the total weight of the hemostatic powder composition. 4. The kit according to claim 1, where in the hemostatic powder composition: - collagen is contained in an amount of from 84 to 88 wt.% relative to the total weight of the hemostatic powder composition; - at least one monosaccharide is contained in an amount of from 4 to 6 wt.% relative to the total weight of the hemostatic powder composition; And - at least one glycosaminoglycan is contained in an amount of 8 to 10 wt.% relative to the total weight of the hemostatic powder composition. 5. Set according to any one of paragraphs. 1-4, where in the hemostatic powder composition at least one monosaccharide is glucose and at least one glycosaminoglycan is chondroitin sulfate. 6. Set according to any one of paragraphs. 1-5, where in the hemostatic powder composition at least one glycosaminoglycan is selected from chondroitin sulfate, dermatan sulfate, hyaluronic acid and mixtures thereof. 7. Set according to any one of paragraphs. 1-6, where the hemostatic powder composition further comprises at least one coagulating factor in an amount of less than 0.1 wt.% relative to the total weight of the hemostatic powder composition. 8. The kit of claim 7 wherein the coagulating factor is thrombin. 9. Set according to any one of paragraphs. 1-8, where the saline solution includes distilled water and sodium chloride, and sodium chloride is contained in an amount of from 0.5 to 1.5 wt.% relative to the total weight of the salt solution. 10. Set according to any one of paragraphs. 1-8, where the saline solution includes distilled water and sodium chloride, and sodium chloride is contained in an amount constituting 0.9 wt.% relative to the total weight of the salt solution. 11. Set according to any one of paragraphs. 1-8 where the brine is pure. 12. Set according to any one of paragraphs. 1-11, including from 1 to 2 g of hemostatic powder and from 4 to 10 ml of saline. 13. Set according to any one of paragraphs. 1-11, where the mass of the saline solution is 2-10 times the mass of the hemostatic powder. 14. Set according to any one of paragraphs. 1-11, where the mass of the saline solution is 4-5 times the mass of the hemostatic powder. 15. Set according to any one of paragraphs. 1-14, where the hemostatic powder is enclosed in a dispenser having a container part made with a bellows, and a nose part located on the container part, where the nose part has an opening for filling the container part with saline to be mixed with the hemostatic powder to form a fluid hemostatic agent . 16. The kit of claim 15 wherein the container portion is shaped to promote hydration of the hemostatic powder with saline to form a flowable hemostatic agent. 17. Set according to any one of paragraphs. 15, 16, where the dispenser additionally has a cap releasably connected to the nose to cover any opening in/out of the container part. 18. Method for preparing a fluid hemostatic agent using a kit according to any one of paragraphs. 1-17, including stages, where: a. placing the hemostatic powder in a container; b. adding a saline solution to the container containing the hemostatic powder, closing and shaking said container to accelerate the hydration of the hemostatic powder to form a fluid hemostatic agent. 19. The method of claim 18, comprising a subsequent step c) wherein the container containing the fluid hemostatic agent is left to stand. 20. The method according to any one of paragraphs. 18, 19, where in step b) the amount of brine added to the container is 50 to 100% of the amount of brine in the kit. 21. The method according to any one of paragraphs. 18-20, where in step b) the amount of saline added to the container is 5 to 10 ml. 22. The method according to any one of paragraphs. 18-20, where in step b) the amount of saline added to the container is 7 ml. 23. The method according to any one of paragraphs. 18-22, where in step b) the container is shaken for 10 to 30 seconds. 24. The method according to any one of paragraphs. 18-22, where in step b) the container is shaken for 20 seconds. 25. The method according to any one of paragraphs. 19-24, where in step c) the fluid hemostatic agent is allowed to stand for a rest period of at least 30 seconds. 26. The method according to any one of paragraphs. 19-24, where in step c) the fluid hemostatic agent is allowed to stand for a rest period of at least 60 seconds. 27. The method according to any one of paragraphs. 19-24, where in step c) the fluid hemostatic agent is allowed to stand for a rest period of at least 90 seconds. 28. The method according to any one of paragraphs. 19-24, where in step c) the fluid hemostatic agent is allowed to stand for a rest period of 30 seconds to 120 seconds. 29. The method according to p. 18, where - in step a) 1.65 mg of hemostatic powder is placed in the container; - in step b), 7 ml of saline is added to the container and the container is shaken for 20 seconds.

Images