Collagen hemostats have been successfully used in the management of a variety of wounds. Collagen is an adherence protein to which blood platelets attach, when they escape from the vascular system caused by an injury of blood vessels. The vessels comprising the vascular system are surrounded by networks of collagen fibrils which are also present throughout the tissues of the body. Upon platelet attachment to collagen the binding of further platelets is initiated resulting in platelet aggregation and activation, which is the main principle of primary hemostasis to stop bleeding at the site of injury.

Microfibrillar collagen is used extensively for wide-area parenchyma bleeding and for laparoscopic procedures in which a specific amount of hemostatic agent can be delivered. On the other hand hemostatic agents in the form of collagen sponges have been shown to be particularly attractive in dermatological applications where adherence to the wound site and ease of removal are important considerations.

Despite these differences in delivery and handling, the primary function of these hemostatic agents is the rapid initiation of hemostatic plug formation through platelet adhesion, platelet activation, and gross blood coagulation.

The choice of the collagen hemostatic formulation, such as sponges, particles or fluids, mainly depends on the indication to treat either large surfaces or punctual sites of injuries. It is well known in the art to use the collagen in its fibrilla form. Collagen fibrils

are solid and easily dispersed in an aqueous medium. It is dissolved at an acid pH and solid fibrils are precipitated when neutralized, to obtain a highly viscous gel and the fibrils immersed therein.

Collagen in its fibrilla form is produced according to WO 9806444. First fetal and adult collagen is dissolved to get a solution of pH 4.0 to 6.0. This composition is suitable for packaging in an aerosol container for dispensing as a blood coagulation foam. Upon neutralization the collagen is precipitated as fibrils and thus gelling and clotted either by the coincident spraying of a neutralizing solution, or by the production of pellets resulting from centrifugation of a neutralized solution of collagen.

A dispersion of solid collagen that is mixed with thrombin is produced according to WO 9831403 by first drying the mixture and reconstituting the solid to a biomaterial that contains the solid as small particles which are flowable through a cannula of less than 1 mm. Thrombin is the activated form of the blood coagulation factor prothrombin and has enzymatic action on fibrinogen to form fibrin and thus promotes the formation of a blood clot.

Another way of producing hemostatic pellets is described in WO 0024436. The components of fibrin sealant, that are fibrinogen, factor XI II and thrombin are admixed as dry preparations to form a solid granulate. Care has to be taken to keep the water content very low or to have barrier coatings between the components, to prevent immediate reaction and fibrin clot formation before the use of the granulate.

It is further state of the art to produce particulate biological material that is coated with a protection material to minimize the reactivity with the surroundings. For instance oral preparations may be encapsulated according to FR 2524312 using collagen as protecting agent. This increases the resistance to enzymes and specifically to those contained in the gut. Another coating technique is described by WO 9908056 to form a protecting layer over microbial preparations, using water soluble polymers such as gelatin.

A collagen gel is further described in the WO 9952946, that is obtained as a neutralized solution by specific temperature control. Thus, fibrils are not precipitated in the neutral state and the collagen can be sterilized and presented as a liquid.

A similar gel is the basis for the production of hemostatic sponges according to WO 9737694. Upon lyophilization of the gel a sponge-like fleece is produced.

The above mentioned materials based on solid collagen and fibrils might not be useful for the surgical application where a quick hemostasis is necessary, because first the solid has to get intimate contact with blood to initiate hemostasis. Likewise it is difficult to apply a collagen liquid to a wound surface because of its high viscosity. There is still a difficult management of the particulate hemostats according to the prior art, because they have to be kept very dry or have barrier layers: This again impairs the immediate reaction of the hemostatic agents.

It is the object of the invention to provide a hemostatic material in the form of flowable pellets that have the specific feature of immediate contact with blood or wound fluid.

The object is solved by the subject matter of the claims. The collagen hemostatic pellets according to the invention are characterized by the collagen gel on the surface of the pellets, which gel is mediating the contact with the surrounding milieu at the wound site. Thus the collagen gel is contained in the contacting surface of the pellets. It is important that the collagen is in the gel state and provided as a liquid, essentially with no collagen fibrils immersed therein. This provides for the easy adherence to other surfaces, specifically at the wound site or prostheses, and the immediate hemostatic reaction of collagen.

The liquid collagen gel in the contacting surface of the pellets according to the invention further facilitates the resorption of the hemostat by the body. Thereby not only a quick hemostasis is achieved but also complications due to undesired adherences in the body may be avoided. The resorption time of the hemostat according to the invention usually is lower than 4 weeks, mostly less than 3 weeks. In some cases the hemostat may even be no more visible after 1 week after its application.

The pellets according to the invention may have a preferred size in the range of 10 to 1000 um, preferably 50-500 um. Even smaller pellets can be provided as long as the flowability of the product is proven. Large sized pellets or granules are specifically feasible in the case of the incorporation of carrier materials. In this case the size may be up to 5 mm.

Preferred preparations according to the invention have a transparent or translucent look resulting from the collagen that is in a gel state and do not contain particulate solids dispersed therein. Collagen preparations based on native or atelopeptide collagen specifically makes up a glass-like appearance. The transparent look may be transiently changed due to drying of the product to obtain a creamy like surface, however it is again glassy or translucent upon reconstitution.

The collagen gel is preferably presented as a solidified liquid at the surface of the pellets according to the invention. This glass-like liquid may have a decreased water content in the range of 1-25%, preferably 10-25%. Therefore a careful drying technique is necessary not to impair the natural water absorption capacity of the collagen gel.

Since the solidified gel in the contacting surface of the pellets, which surface is the outer surface, according to the invention is still a glassy liquid it maintains its elastic properties and fluid mixing and absorption capacities. The advantage of the solidified gel is the improved storage stability of the pellets. It is thus possible to provide a storage-stable pellet preparation that contains the solidified collagen gel and can be stored at room temperatures up to 35°C. If thermolabile drugs are incorporated in the preparation, it may however be necessary to keep the storage temperature in the range of 2-8°C.

The solidified gel is easily reconstituted to the original liquid state either before the application of the pellets by addition of physiological solutions, optionally containing drugs, or directly at the wound site.

The further preferred pellets contain the collagen gel at a neutral pH, e. g. pH 6 to 8. A preferred range would be 6.5 to 8.0. This is especially prudent when the collagen gel or the pellets are combined with substances that are labile in an acid milieu.

The pellets containing the liquid collagen gel in the surface area are also storage stable as a suspension. In some instances depending on the water content, the choice of stabilizing agents, the origin and type of collagen, it can be kept at temperatures up to 35°C, preferably at room temperature (20-25°C). However, if it further comprises thermolabile drugs, it should be stored at refrigerating temperatures (2-8°C), or else a stabilizer admixed to the collagen and/or the drug. The storage of the pellets according to the invention as a frozen preparation, preferably at temperatures of-30 to-10°C may be a favorite option for providing storage stability of the product, which is at least 2 years.

It is further preferred to store the pellets according to the invention in a container with an outlet for direct application to the wound or surgical site. e. g. in a flexible tube, a syringe or in vials. This is particular suitable for a ready-to-use preparation of the pellets.

According to a specific embodiment of the invention the hemostatic pellets further contain a physiologically active substance or drug, preferably in the dissolved form or as a dispersion. Among the optional additives are antibiotics, antimycotics, antiviral substances, antiphlogistics, analgesics and factors of the blood coagulation and fibrinolysis system. Antibiotics, like gentamycine, may be preferred when the local application of the substance should challenge septic conditions. The addition of a human factor of blood coagulation, like a blood coagulation factor selected from the group consisting. of factors 1, 11, V, VII, VIII, IX, X, XI, XIII, von Willebrand Factor, Fibronectin, Vitronectin, Protein C and Protein S, or activated forms of these factors may positively influence the kinetics of hemostasis by the reaction with the intrinsic and extrinsic system of coagulation to support secondary hemostasis.

Further platelet factors, enzymes or inhibitors of coagulation and fibrinolysis may be included. The most immediate action may be obtained by using activated factors of coagulation to provoke a quick secondary hemostasis. It is for instance preferred to include thrombin in the hemostatic pellets according to the invention to get a synergistic action of primary and secondary hemostasis at the bleeding wound.

The thrombin can be stabilized using proteins like human serum albumin, amino acids, polyglycols or other carbohydrates, like sugars and sugar alcohols, for a storage stable liquid at refrigerating or ambient temperatures. When using a frozen thrombin preparation the stabilizer concentration may be reduced to a minimum, and even be spared.

The preferred thrombin concentration in the pellets is in the range of 1 to 70 IU/cm3, more preferably between 5 and 60 1 U/CM3.

Further possible additives are wound healing promoting agents, growth factors, glucocorticosteroids, steroids, vitamins or vitamin derivatives, tumoricidal or tumoristatic compounds, minerals, immunomodulators, immunoglobulins, dyes, radiolabels, fluorescent labels, pofysaccharides, anesthetics and nucleic acids.

When using biological additives derived from human sources care should be taken not to transmit human pathogenic agents, such as blood born viruses. Among the relevant viruses the HIV, Hepatitis Viruses and Parvoviruses are well-known. Therefore effective methods of inactivating and/or depleting potentially present viruses in the preparation of the biologicals or additives are employed. Those methods are for instance the treatment with solvent and detergent, such as according to the EP 0 131 740, the heat treatment methods of pasteurization or heating in the dry state, irradiation methods and filtration methods.

The preferred addition of one or more of the above mentioned physiologically active substances or drugs is the direct coating of the pellets with the additives and/or the mixture of the additives and the collagen gel. A coating layer containing the additive and optionally a further carrier substance, like a bioresorbable polymer, preferably collagen, is applied to the pellets, advantageously after or during drying the pellets or during the pelleting process. This may be effected by overlaying the pellets with a solution or dispersion of the additive and optionally the carrier mixture in a solute, and evaporating the solute to leave the additive and optionally the carrier substances as a coating on the surface. As a suitable solute water or an organic solute commonly used in encapsulation procedures, like ethanol, may be used.

The further option is to include the physiologically active substance and/or a carrier substance, such as a biomaterial, in the pellets according to the invention. The biomaterial is preferably bioresorbable and selected from the group consisting of collagen, gelatin, fibrin, cellulose and hydroxyapatite. The carrier of the hemostatic pellets may consist of liquids or specific solid particles or granules that are encapsulated by at least one layer of the collagen gel.

Likewise it is advantageous to provide hemostatic pellets according to the invention with a homogeneous composition throughout the pellet. This means that the collagen gel is not only in the surface area of the pellet or in a layer, but also throughout the pellet, thus forming the basis for the hemostatic preparation.

On the other hand it may be preferred to have different layers of distinct compositions, that enable the incorporation of at least two different physiologically active substances, such as coagulation factors like fibrinogen and thrombin. Provided their presence in one layer these substances would immediately react, specifically in the presence of the usual water content of the pellets according to the invention, that is more than 5% (w/w). The separate layers of the pellets however provide for the reaction only after floating the pellets to the wound site.

The pellets according to the invention preferably make use of a source of native collagen. The source material for producing the hemostatic pellets according to the invention is preferably collagen from mammalian sources, most preferred from species which bear reportedly no risk of transmissible spongiform encephalitis (TSE). The pathogens of TSE are not yet characterized. It is however known that cows, sheep and goats may contain pathogens causing transmissing spongiform encephalitis, like BSE (bovine spongiform encephalitis), scrapie (sheep TSE) or CJD (Creutzfeld Jakob Disease). According to the recommendation of the world health organization WHO the source of collagen material for use in pharmaceuticals should be carefully selected to reduce the risk of transmitting those pathogens, also known as prions. The further recommendation is a treatment with chemicals to reduce the potential infectivity of the product. A preferred method is the treatment of collagen with a high concentration of NaOH. The treatment with the chemicals further reduces the risk of transmitting other pathogens like viruses that could be harmful to humans.

Collagen from horses, pigs or birds, even from humans are preferred sources for the hemostat according to the invention. Provided the security recommendations of the WHO are considered also bovine, or other ruminant sources may be used. The material usually is derived from tendons, skins or placenta.

It is preferred to use native collagen that is extracted using a procedure to maintain the integrity of the polypeptide chains and the helical structure of the molecule. It may also be treated with enzymes such as pepsin to obtain atelopeptide collagen, which specifically results in the collagen gel of the translucent or transparent look.

Native, including atelopeptide collagen specifically comprises all fractions of collagen including the acid soluble and acid insoluble fraction. Such a native material has significantly improved hemostatic activity.

Yet it is not preferred to use collagen modified by chemical reactions, like cross-linking.

Different collagen types, like type 1, 11, 111 or IV can be treated to produce the hemostat according to the invention, the preferred collagen is however of type I or a preparation containing more than 80% type I collagen.

It is the further subject of the invention to provide a method for producing the hemostatic pellets by dissolving the collagen source to a gel and pelleting by centrifugation, spray-drying, fluid-bed drying of the gel and/or encapsulating with the gel.

The collagen is contained in the source material mostly in the fibrilla form. The fibrils are then dissolved to form a collagen liquid in an acid milieu, preferably at a pH between 3 and 5. The collagen is advantageously acidified by addition of acetic acid, citric acid, lactic acid or monochlor acetic acid to obtain the collagen in the gel state.

The further optional neutralization of the gel is carefully carried out under temperature control, specifically not to exceed the denaturation temperature of about 35 to 50°C.

Neutralization usually is effected by the admixture of alkali solutions to achieve a pH of about 6 to 8.

The method according to the invention may further comprise the process steps of washing, cutting the collagen source material, delipidation and optionally elimination of aminoglycans by treatment with salts.

Various pelleting processes known in the art may be used for the production of the hemostatic pellets according to the invention. Care should however be taken not to exceed the denaturation temperature of the collagen gel, which usually is in the range of 35 to 50°C. A process that enables the temperature control to lower than 40°C is preferred.

Among the useful pelleting processes is the centrifugation of a suspension or a gel.

Semisolid, viscous pellets are resulting from centrifugation of a neutralized solution of collagen. For example, the collagen is spinned in a centrifuge to yield a collagen pellet mass. The water content of the pellet mass can be controlled by the centrifugal force employed. The stronger the centrifugal force, the less water and the higher concentration of collagen up to 150 mg/ml can be obtained.

Ball milling of a solid or the solidified collagen gel for a time sufficient to form the particulate structure is a further option for producing the pellets according to the invention.

The encapsulation of liquids or solid materials by the collagen gel is preferably effected by suspending or dissolving the collagen in a solute, covering the liquids or solid materials with the collagen, and evaporating the solute to leave a reduced collagen gel layer on the surface of the pellets. Suitable solutes are for instance aliphatic monoalcohols, like methanol or ethanol that rapidly evaporate at temperatures lower than the denaturation temperature of collagen.

The direct polymerization of the collagen on the carrier materials, which is effected by elevated temperatures up to 40° to 60°C is a preferred encapsulation method. A collagen gel of high concentration, such as more than 2%, preferably 3-6%, even up to 10%, can be used for coating and directly polymerizing the collagen for building the hemostatic contacting surface. Thereby no organic solutes are necessary. A suitable collagen gel for this encapsulation method is particularly at acid pH.

One of the best modes of pelleting is however the drying in a fluid bed or under eddy gas conditions, e. g. by spray drying. These techniques enable the combination of layers by the sequential processing of components, the collagen gel layer being the last component to be dried on the surface. It also can produce homogeneous pellets of collagen gel and optional additives in the specified size range. Sometimes core substances like sugars or one of the carrier substances mentioned above are used when producing the pellets.

During the drying process in the fluidized bed it is preferred to carefully monitor and control the granulation, agglomeration and coating process. The spray rate, the drying gas temperature and the volume flow are exemplary parameters well-know in the art to be regulated to obtain the suitable pellet form. i) Usually the collagen gel or the liquid source material is sprayed in a fluid bed chamber and contacted with a drying medium or gas. The preferred gas is inert, such as nitrogen or carbon dioxide. The agitation by the gas stream in the chamber is a welcome tool for increasing the contacting surface of the material to facilitate the drying of the material. A coating material which is solid at room temperature may further be melted, sprayed in the interior of the particle bed and onto the particles and pellets, respectively, and solidified by cooling. It is advantageous to obtain a continuous and smooth coating with the collagen gel by the specific process steps. Rotatable means for shaping the pellets or agglomerated materials may be used.

Although well-know techniques are suitable to produce the inventive product, it was still surprising to experience that the gel character of the collagen surface of the pellets still remains intact and provides for the excellent hemostastic properties, even after pelleting. For most surgical procedures it is required to achieve hemostasis within 3 to 5 minutes. The hemostatic pellets according to the invention fulfil this requirement, and occasionally stop bleeding within 1 or 2 minutes.

The pharmaceutical preparation based on the hemostatic pellets may also be presented in a matrix to form an ointment, a gel, gel cream, cream, aerosol and occlusive film.

The collagen gel further may be characterized by a relatively high viscosity between 20 and 10 000 cp, preferably between 50 and 5 000 cps. Drying the pellets increases the viscosity even more. Nevertheless the product is then easily applied to the wound site because of the flowability of the pellets.

The hemostatic pellets according to the invention are further provided as a sterile preparation. The process of manufacturing the pellets or intermediate materials thus comprises the process step of sterilization. It is hereby advantageous to either sterilize the collagen by chemical treatment, filtration through a bacteria filter or by irradiation using beta or gamma rays, for example with an intensity of 15-30 kGy, usually about 25 kGy. The collagen may be chemically treated using ethylene oxide or peroxides.

Since the hemostatic pellets according to the invention may contain a relatively high concentration of collagen and however is a flowable presentation form of a hemostat, it is especially suitable for punctual application during surgical interventions. It may however also be applied to large hemorrhaging wounds, and most suitably in cases of diffuse and oozing bleeding.

It is also very useful for being placed in contact with any hemorrhagic site of uneven surface and more particularly in thoracic, orthopedic, plastic (skin grafts), ENT or neurosurgery. It may further be applied to a puncture site of organ tissue.

A preferred mode of application is by laparoscopic procedures. The indication of microinvasive or minimal invasive surgery is most interesting for such a flowable and easily applicable material like the hemostatic pellets according to the invention, especially in the abdomen, the thoracic and cardiovascular indications. Generally, hemostasis at all wound sites may be achieved. This includes surgical wounds, burns, ulcers and lacerations.

The following example is illustrating the present invention.

Example 1: Preparation of the collagen hemostatic pellets according to the invention (microcapsules)

150 g dry collagen powder obtained by extraction from horse skin and sterilized by irradiation are dissolved in 10 liters of acetic acid solution to get a pH of 4. Undissolved components are separated by centrifugation, the supernatant diluted collagen gel is treated with acetone at a ratio of 1: 6. Thereby the collagen gel precipitates, which precipitate is separated by centrifugation. The precipitate is washed and solubilized in methanol at a concentration of 2 g/I.

This solution is transferred to the reservoir of a pistol pulverizator and extruded by pressure of 1 bar. Granules of the exemplary carrier material fibrinogen are agitated at about 50 to 100 tours per minute and coated with the extruded collagen to obtain the capsules after evaporation of the methanol.