Patent Description:
When the haemostatic powder is applied to a bleeding site, the particle agglomerates rapidly form a gel while at the same time binding to proteins present in the blood and in the surrounding tissue, thereby speeding up haemostasis.

Haemostasis is a tightly regulated process that maintains the blood flow through the vasculature simultaneously as a thrombotic response to tissue damage occurs. Maintaining haemostasis requires a complex interaction of the vessel wall, platelets, and the coagulation and fibrinolytic systems. There are two main phases of haemostasis: primary (ie, the cellular phase) and secondary (ie, the humoral phase).

Primary haemostasis begins immediately after endothelial disruption and is characterized by vasoconstriction, platelet adhesion, and formation of a soft aggregate plug. After the injury occurs, there is a temporary local contraction of vascular smooth muscle and the blood flow slows, promoting platelet adhesion and activation. Within <NUM> seconds of the injury, circulating von Willebrand factor attaches to the subendothelium at the site of injury and adheres to the glycoproteins on the surface of platelets. As platelets adhere to the injured surface, they are activated by contact with collagen-exposing receptors that bind circulating fibrinogen. A soft plug of aggregated platelets and fibrinogen is formed. This phase of haemostasis is short lived, and the soft plug can easily be sheared from the injured surface.

The soft platelet plug is stabilized during secondary haemostasis to form a clot. Vasoconstriction and the resultant reduction in blood flow are maintained by platelet secretion of serotonin, prostaglandin, and thromboxane while the coagulation cascade is initiated. The coagulation cascade is a series of dependent reactions involving several plasma proteins, calcium ions, and blood platelets that lead to the conversion of fibrinogen to fibrin. Coagulation factors are produced by the liver and circulate in an inactive form until the coagulation cascade is initiated. Then each step of the cascade is initiated and completed via a series of sequential and dependent coagulation factor activation reactions. In the final steps, thrombin converts the soluble plasma protein fibrinogen to the insoluble protein fibrin, while simultaneously converting factor XIII to factor Xllla. This factor conversion stabilizes the fibrin and results in cross-linking of the fibrin monomers, producing a stable clot.

During surgery, it is important to maintain a fine balance between bleeding and clotting, such that blood continues to flow to the tissues at the surgical site without excessive loss of blood, to optimize surgical success and patient outcome. Continuous bleeding from diffuse minor capillaries or small venules during surgery can obscure the surgical field, prolong operating time, increase the risk of physiologic complications, and expose the patient to risks associated with blood transfusion.

Surgeons have an array of options to control bleeding, including mechanical and thermal techniques and devices as well as pharmacotherapies and topical agents.

One of the earliest topical haemostatic agents was cotton, in the form of gauze sponges. Although such materials concentrate blood and coagulation products via physical adsorption, they are not absorbed by the body, and upon removal, the clot may be dislodged, leading to further bleeding. Absorbable topical haemostatic agents have since been developed and provide useful adjunctive therapy when conventional methods of haemostasis are ineffective or impractical. Topical haemostatic agents can be applied directly to the bleeding site and may prevent continuous unrelenting bleeding. Haemostasis using topical agents also can avoid the adverse effects of systemic haemostatic medications, such as "unwanted" blood clots. Furthermore, in surgical procedures where the amount of blood loss is unpredictable, topical haemostats can be used sparingly when blood loss is minimal and more liberally during severe bleeding.

A number of topical haemostatic agents are currently available for use in surgery. They can be divided into two categories: those that provide their mechanism of action on the clotting cascade in a biologically active manner and those that act passively through contact activation and promotion of platelet aggregation. Passive topical haemostatic agents include collagens, cellulose, and gelatins, while active agents include thrombin and products in which thrombin is combined with a passive agent to provide an active overall product.

<CIT> describes a dry haemostatic powder that is prepared by a method comprising: providing an aqueous solution comprising gelatin combined with at least one re-hydration aid; drying the solution to produce solids; grinding the solids to produce a powder; cross-linking the powder; removing at least <NUM>% (w/w) of the re-hydration aid; and drying the cross-linked gelatin to produce a powder. The re-hydration aid may comprise at least one material selected from the group consisting of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and dextran.

<CIT> describes an anhydrous fibrous sheet comprising a first component of fibrous polymer, said polymer containing electrophilic groups or nucleophilic groups, and a second component capable of crosslinking the first component when said sheet is exposed to an aqueous medium in contact with biological tissue to form a crosslinked hydrogel that is adhesive to the biological tissue; wherein:.

<CIT> describes a process for making a haemostatic composition, said process comprising: a) providing a dry granular preparation of a biocompatible polymer; and b) coating the granules in said dry granular preparation with a preparation of a coagulation inducing agent, such as a thrombin solution. The biocompatible polymer may be selected from of gelatin, soluble collagen, albumin, hemoglobin, fibrinogen, fibrin, casein, fibronectin, elastin, keratin, laminin, and derivatives or combinations thereof.

<CIT> describes a haemostatic material comprising a compacted ORC powder comprising particles having average aspect ratio from about <NUM> to about <NUM>. The haemostatic material may further comprises an additive selected from polysaccharides, calcium salt, anti-infective agent, haemostasis promoting agent, gelatin, collagen,.

<CIT> describes a haemostatic composition comprising:.

wherein the haemostatic composition is in paste form.

<CIT> describes a kit for producing a biocompatible, cross-linked polymer, said kit comprising an electrophilically activated polyoxazoline (EL-POx), said EL-POX comprising m electrophilic groups; and said nucleophilic cross-linking agent comprising n nucleophilic groups, wherein the m electrophilic groups are capable of reaction with the n nucleophilic groups to form covalent bonds; wherein m><NUM>, n><NUM> and m+n ><NUM>; wherein at least one of the m electrophilic groups is a pendant electrophilic group.

<CIT> describes adhesive haemostatic product selected from a coated mesh, a coated foam or a coated powder, said haemostatic product comprising:.

SUMMARY OF THE INVENTION The scope of this invention is defined by the claims. Any references in the description to methods of treatment refer to the products of the present invention for use in a method for treatment.

The inventors have developed a haemostatic sheet that can conveniently be used to control bleeding during surgery, even for anti-coagulated blood.

The biocompatible, flexible, haemostatic sheet of the invention comprises - a cohesive fibrous carrier structure comprising a three-dimensional interconnected interstitial space; and -distributed within the interstitial space, a haemostatic powder comprising at least <NUM> wt. % of particle agglomerates, said particle agglomerates having a diameter in the range of <NUM>-<NUM> and comprising: (a) electrophilic polyoxazoline particles containing electrophilic polyoxazoline carrying at least <NUM> reactive electrophilic groups that are capable of reacting with amine groups in blood under the formation of a covalent bond; and (b) nucleophilic polymer particles containing a water-soluble nucleophilic polymer carrying at least <NUM> reactive nucleophilic groups that, in the presence of water, are capable of reacting with the reactive electrophilic groups of the electrophilic polyoxazoline under the formation of a covalent bond between the electrophilic polyoxazoline and the nucleophilic polymer.

When applied to a bleeding site, the haemostatic powder turns into a gel while at the same time binding to proteins present in the blood and on the surrounding tissue. The haemostatic powder's outstanding ability to stop bleeding is due to highly active induced blood clotting and to the formation of a strong gelled blood clot that adheres to tissue. The haemostatic powder can easily be distributed over a bleeding site. Additional powder can be added if necessary as this will form a new layer of gelled blood clot that will stick to the underlying layer of gelled blood clots.

Although the inventors do not wish to be bound by theory, it is believed that when the haemostatic powder comes into contact with blood, the electrophilic polyoxazoline particles containing electrophilic polyoxazoline rapidly dissolve and simultaneously react with proteins in the blood and reactive nucleophilic groups of the water-soluble nucleophilic polymer in the nucleophilic polymer particles. As a result, a layer of gelled blood clot is formed. The dissolved electrophilic polyoxazoline will also react with proteins in the surrounding tissue at the bleeding site, thereby fixating the layer of gelled blood clot to the tissue and sealing off the bleeding area.

In comparison to particles comprising a molecular mixture of the electrophilic polyoxazoline and the water-soluble nucleophilic polymer, the particle agglomerates offer the advantage that they provide better sealing properties. It is believed that this is due to the fact that, when the particle agglomerates come into contact with blood, the electrophilic polyoxazoline reacts with the water-soluble nucleophilic polymer at a relatively slow rate, thereby allowing the electrophilic polyoxazoline to not only react with the water-soluble nucleophilic polymer, but also with proteins in blood and tissue at the bleeding site. In comparison to a powder mixture consisting of particles of electrophilic polyoxazoline and particles of water-soluble nucleophilic polymer, the particle agglomerates offer the advantage that a more homogeneous, strong gel is formed. It is believed that a very homogeneous dispersion of electrophilic polyoxazoline and water-soluble nucleophilic polymer is formed when the particle agglomerates come into contact in blood, and that a homogeneous strong gel is formed when the polymer components dissolve therein and start reacting.

Also disclosed (not part of the claimed invention) is a method of preparing haemostatic particle agglomerates of:.

said method comprising the step of combining <NUM> parts by weight of the electrophilic polyoxazoline particles with <NUM> to <NUM> parts by weight of the nucleophilic polymer particles in the presence of non-aqueous granulation liquid.

The inventors have unexpectedly discovered that it is possible to combine the electrophilic polyoxazoline and the water-soluble nucleophilic polymer into a single particle with minimum cross-linking reactions between, and minimum degradation of the electrophilic polyoxazoline, by using a non-aqueous granulation liquid in which the electrophilic polyoxazoline is insoluble and in which the nucleophilic polymer is somewhat soluble. Although the inventors do not wish to be bound by theory, it is believed that the use of a non-aqueous granulation liquid in which electrophilic polyoxazoline is insoluble ensures that during granulation no crosslinking reactions will occur between the electrophilic polyoxazoline and the nucleophilic polymer. Also degradation (hydrolysis) of the electrophilic polyoxazoline is minimized in this way. Contrary to the electrophilic polyoxazoline, some of the nucleophilic polymer will dissolve in the non-aqueous granulation liquid, thereby forming a sticky mass that is capable of gluing together the electrophilic polyoxazoline particles and the nucleophilic polymer particles.

Provided is a biocompatible, flexible, haemostatic sheet comprising:.

Accordingly, the invention relates to a biocompatible, flexible, haemostatic sheet comprising: - a cohesive fibrous carrier structure comprising a three-dimensional interconnected interstitial space; and -distributed within the interstitial space, a haemostatic powder comprising at least <NUM> wt. % of particle agglomerates, said particle agglomerates having a diameter in the range of <NUM>-<NUM> and comprising: (a) electrophilic polyoxazoline particles containing electrophilic polyoxazoline carrying at least <NUM> reactive electrophilic groups that are capable of reacting with amine groups in blood under the formation of a covalent bond; and (b) nucleophilic polymer particles containing a water-soluble nucleophilic polymer carrying at least <NUM> reactive nucleophilic groups that, in the presence of water, are capable of reacting with the reactive electrophilic groups of the electrophilic polyoxazoline under the formation of a covalent bond between the electrophilic polyoxazoline and the nucleophilic polymer.

The term "particle agglomerate" as used herein refers to a granule that comprises two or more particles that are all bound together.

The term "polyoxazoline" as used herein refers to a poly(N-acylalkylenimine) or a poly(aroylalkylenimine) and is further referred to as POx. An example of POx is poly(<NUM>-ethyl-<NUM>-oxazoline). The term "polyoxazoline" also encompasses POx copolymers.

The term "water-soluble nucleophilic polymer" as used herein refers to a nucleophilic polymer that has a water-solubility in demineralised water of <NUM>, at a pH in the range of <NUM> to <NUM>, of at least <NUM>/L. Chitosan is an example of a water-soluble nucleophilic polymer. Chitosan is water-soluble in water at pH<<NUM>. In order to determine water solubility of a nucleophilic polymer at different pH, pH of the demineralised water is adjusted using hydrochloric acid.

The term "collagen" as used herein refers the main structural protein in the extracellular space of various connective tissues in animal bodies. Collagen forms a characteristic triple helix of three polypeptide chains. Depending upon the degree of mineralization, collagen tissues may be either rigid (bone) or compliant (tendon) or have a gradient from rigid to compliant (cartilage). Unless indicated otherwise, the term "collagen" also encompasses modified collagens other than gelatin (e.g. crosslinked collagen).

The term "gelatin" as used herein refers to a mixture of peptides and proteins produced by partial hydrolysis of collagen extracted from the skin, bones, and connective tissues of animals such as domesticated cattle, chicken, pigs, and fish. During hydrolysis, the natural molecular bonds between individual collagen strands are broken down into a form that rearranges more easily. The term "gelatin" as used herein also encompasses modified gelatins, such a crosslinked gelatins and reduced crosslinked gelatins.

The term "reduced crosslinked gelatins" as used herein refers to a crosslinked gelatin that has been partly hydrolysed. Partial hydrolysis of the peptide bonds in the cross-linked gelatin can be achieved by e.g. alkaline treatment. Hydrolysis of the cross-linked gelatin results in an increased density of free carboxyl and free amine groups and in increased water solubility.

The term "haemostatic sheet" as used herein, unless indicated otherwise, refers to a sheet having the ability to stop bleeding from damaged tissue. The haemostatic sheet of the present invention may achieve haemostasis by turning blood into a gel and/or by forming a seal that closes off the wound site.

The term "water-resistant" as used herein in relation to the fibrous carrier structure means that this structure is not water soluble and does not disintegrate in water to form a colloidal dispersion, at neutral pH conditions (pH <NUM>) and a temperature of <NUM>.

The term "interstitial space" as used herein refers to the void ("empty") space within the fibrous carrier structure. The interstitial space within the fibrous carrier structure allows the introduction of haemostatic powder into the structure. Also blood and other bodily fluids can enter the interstitial space, thereby allowing the haemostatic powder to exert its haemostatic effect and/or to provide tissue-adhesiveness to the haemostatic sheet.

The term "protein" as used herein, unless indicated otherwise, also encompasses cross-linked and hydrolysed proteins. Likewise, unless indicated otherwise, whenever reference is made to a particular protein species, such as gelatin or collagen, also hydrolysed and cross-linked versions of that protein species are encompassed.

The diameter distribution of the haemostatic powder, of the particle agglomerates, of the electrophilic polyoxazoline particles and of the nucleophilic polymer particles may suitably be determined by means of laser diffraction using a Malvern Mastersizer <NUM> in combination with the Stainless Steel Sample Dispersion Unit. The sample dispersion unit is filled with approx. <NUM> of cyclohexane, which is stabilized for <NUM> to <NUM> minutes at a stirring speed of <NUM> rpm, followed by a background measurement (blanc measurement). The sample tube is shaken and turned horizontally for <NUM> times. Next, about <NUM> is dispersed in the sample dispersion unit containing the cyclohexane. After the sample is introduced in the dispersion unit, the sample is stirred for one and a half minute at <NUM> rpm to ensure that all particles are properly dispersed, before carrying out the measurement. No ultrasonic treatment is performed on the dispersed particles. Mean particle size is expressed as D [<NUM>,<NUM>], the volume weighted mean diameter (ΣniDi<NUM>)/(ΣniDi<NUM>).

Besides the particle agglomerates comprising the electrophilic polyoxazoline particles and the nucleophilic polymer particles, the haemostatic powder of the present invention may contain other particulate components, e.g. biocompatible (bio)polymers like gelfoam or starch. Preferably, the haemostatic powder contains at least <NUM> wt. %, more preferably at least <NUM> wt. % and most preferably at least <NUM> wt. % of the particle agglomerates.

The particle agglomerates in the haemostatic powder preferably contain at least <NUM> wt. %, more preferably at least <NUM> wt. % and most preferably at least <NUM> wt. % of the electrophilic polyoxazoline.

The nucleophilic polymer is preferably is contained in the particle agglomerates in a concentration of at least <NUM> wt. %, more preferably of at least <NUM> wt. % and most preferably of at least <NUM> wt.

The combination of the electrophilic polyoxazoline and the nucleophilic polymer typically constitutes at least <NUM> wt. % of the particle agglomerates. More preferably, the combination of these two polymers constitutes at least <NUM> wt. %, most preferably at least <NUM> wt. % of the particle agglomerates.

Besides the electrophilic polyoxazoline and the nucleophilic polymer the particle agglomerates may contain one or more other components, e.g. polysaccharides. Examples of polysaccharides that may be employed in include amylose, maltodextrin, amylopectin, starch, dextran, hyaluronic acid, heparin, chondroitin sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, dextran sulfate, pentosan polysulfate, alginate and combinations thereof. Typically, the amount of polysaccharide in the particle agglomerates does not exceed <NUM> wt. If polysaccharide is contained in the particle agglomerates, the combination of the electrophilic polyoxazoline, the nucleophilic polymer and the polysaccharide preferably constitutes at least <NUM> wt. %, more preferably at least <NUM> wt. % and most preferably at least <NUM> wt. % of the particle agglomerates.

In accordance with another advantageous embodiment the particle agglomerates contain a dry buffering system. Preferably, the buffering system has a buffering pH in the range of <NUM> to <NUM>, more preferably in the range of <NUM> to <NUM>. Preferably, the buffering system has a buffer capacity of at least <NUM> mmol. More preferably, the buffer capacity is a least <NUM> mmol. pH-<NUM>, most preferably the buffer capacity is at least <NUM> mmol. Examples of biocompatible buffering systems that may be employed in the particle agglomerates include sodium phosphate /sodium carbonate buffer; sodium borate decahydrate buffer; tris buffered protein; HEPES buffered saline and sodium bicarbonate/carbonate buffer.

As will be explained below, the particle agglomerates of the present invention can be prepared without the use of a granulation binder, i.e. a component that is employed during granulation to provide adhesion between the electrophilic polyoxazoline particles and the nucleophilic polymer particles. Accordingly, in a preferred embodiment, the particle agglomerates do not contain a granulation binder.

The haemostatic powder of the present invention preferably contains at least <NUM> wt. % of the particle agglomerates having a diameter in the range of <NUM>-<NUM>, more preferably at least <NUM> wt. % of the particle agglomerates having a diameter in the range of <NUM>-<NUM>, and most preferably at least <NUM> wt. % of the particle agglomerates having a diameter in the range of <NUM>-<NUM>.

The haemostatic powder preferably has a volume weighted mean diameter (D [<NUM>,<NUM>], (ΣniD <NUM>)/(ΣniD<NUM>)) in the range of <NUM>-<NUM>, more preferably in the range of <NUM>-<NUM>, most preferably in the range of <NUM>-<NUM>.

The particle agglomerates in the haemostatic powder preferably have a D [<NUM>,<NUM>] in the range of <NUM>-<NUM>, more preferably in the range of <NUM>-<NUM>, most preferably in the range of <NUM>-<NUM>.

The electrophilic polyoxazoline particles in the particle agglomerates preferably have a volume weighted mean diameter (D [<NUM>,<NUM>]) in the range of <NUM>-<NUM>, more preferably in the range of <NUM>-<NUM>, most preferably in the range of <NUM>-<NUM>.

The nucleophilic polymer particles in the particle agglomerates preferably have a volume weighted mean diameter (D [<NUM>,<NUM>]) is in the range of <NUM>-<NUM>, more preferably in the range of <NUM>-<NUM>, most preferably in the range of <NUM>-<NUM>.

The electrophilic polyoxazoline particles preferably contain at least <NUM> wt. %, more preferably at least <NUM> wt. % and most preferably at least <NUM> wt. % of the electrophilic polyoxazoline.

The electrophilic polyoxazoline preferably has a solubility in distilled water of <NUM> of at least <NUM>/L, more preferably of at least <NUM>/L.

The electrophilic polyoxazoline preferably has a solubility in isopropyl alcohol at <NUM> of less than <NUM>/L, more preferably of less than <NUM>/L and most preferably of less than <NUM>/L.

The electrophilic polyoxazoline preferably has a molecular weight of at least <NUM> kDa. More preferably, the electrophilic polyoxazoline has a molecular weight of <NUM> to <NUM> kDa, most preferably of <NUM> to <NUM> kDa.

As explained herein before, the inventors have found a way to combine the electrophilic polyoxazoline and the water-soluble nucleophilic polymer into a single particle with minimum cross-linking reactions between, and minimum degradation of the electrophilic polyoxazoline. Accordingly, in a very preferred embodiment of the invention, the electrophilic polyoxazoline the particle agglomerates has a polydispersity index (PDI) of less than <NUM>, more preferably of less than <NUM> and most preferably of less than <NUM>.

The electrophilic polyoxazoline preferably contains at least <NUM> reactive electrophilic groups, more preferably at least <NUM> reactive electrophilic groups, even more preferably at least <NUM> reactive electrophilic groups and most preferably at least <NUM> reactive electrophilic groups.

The electrophilic polyoxazoline typically carries on average at least <NUM>, more preferably at least <NUM> reactive electrophilic groups.

The electrophilic polyoxazoline is preferably derived from a polyoxazoline whose repeating units are represented by the following formula (I):.

wherein R<NUM>, and each of R<NUM> are independently selected from H, optionally substituted C<NUM>-<NUM> alkyl, optionally substituted cycloalkyl, optionally substituted aralkyl, optionally substituted aryl; and m being <NUM> or <NUM>.

Preferably, R<NUM> and R<NUM> in formula (I) are selected from H and C<NUM>-<NUM> alkyl, even more preferably from H and C<NUM>-<NUM> alkyl. R<NUM> most preferably is H. The integer m in formula (I) is preferably equal to <NUM>.

According to a preferred embodiment, the polyoxazoline is a polymer, even more preferably a homopolymer of <NUM>-alkyl-<NUM>-oxazoline, said <NUM>-alkyl-<NUM>-oxazoline being selected from <NUM>-methyl-<NUM>-oxazoline, <NUM>-ethyl-<NUM>-oxazoline, <NUM>-propyl-<NUM>-oxazoline, <NUM>-butyl-<NUM>-oxazoline and combinations thereof. Preferably, the polyoxazoline is a homopolymer of <NUM>-propyl-<NUM>-oxazoline or <NUM>-ethyl-oxazoline. Most preferably, the polyoxazoline is a homopolymer of <NUM>-ethyl-oxazoline.

According to a particularly preferred embodiment, the electrophilic polyoxazoline comprises at least <NUM> oxazoline units, more preferably at least <NUM> oxazoline units and most preferably at least <NUM> oxazoline units. The electrophilic polyoxazoline preferably comprises on average at least <NUM> reactive electrophilic groups per oxazoline residue. Even more preferably, the electrophilic polyoxazoline comprises on average at least <NUM> reactive electrophilic groups per oxazoline residue. Most preferably, the electrophilic polyoxazoline comprises on average <NUM>-<NUM> reactive electrophilic groups per oxazoline residue.

Polyoxazoline can carry reactive electrophilic groups in its side chains (pendant reactive electrophilic groups), at its termini, or both. The electrophilic polyoxazoline that is employed in accordance with the present invention advantageously contains one or more pendant reactive electrophilic groups. Typically, the electrophilic polyoxazoline contains <NUM>-<NUM> pendant reactive electrophilic groups per monomer, more preferably <NUM>-<NUM> pendant reactive electrophilic groups per monomer, even more preferably <NUM>-<NUM> pendant reactive electrophilic groups per monomer.

In accordance with a preferred embodiment, the reactive electrophilic groups of the electrophilic polyoxazoline are selected from carboxylic acid esters, sulfonate esters, phosphonate esters, pentafluorophenyl esters, p-nitrophenyl esters, p-nitrothiophenyl esters, acid halide groups, anhydrides, ketones, aldehydes, isocyanato, thioisocyanato, isocyano, epoxides, activated hydroxyl groups, olefins, glycidyl ethers, carboxyl, succinimidyl esters, sulfo succinimidyl esters, maleimido (maleimidyl), ethenesulfonyl, imido esters, aceto acetate, halo acetal, orthopyridyl disulfide, dihydroxy-phenyl derivatives, vinyl, acrylate, acrylamide, iodoacetamide and combinations thereof. More preferably, the reactive electrophilic groups are selected from carboxylic acid esters, sulfonate esters, phosphonate esters, pentafluorophenyl esters, p-nitrophenyl esters, p-nitrothiophenyl esters, acid halide groups, anhyinidrides, ketones, aldehydes, isocyanato, thioisocyanato, isocyano, epoxides, activated hydroxyl groups, glycidyl ethers, carboxyl, succinimidyl esters, sulfo succinimidyl esters, imido esters, dihydroxy-phenyl derivatives, and combinations thereof. Even more preferably, the reactive electrophilic groups are selected from halo acetals, orthopyridyl disulfide, maleimides, vinyl sulfone, dihydroxyphenyl derivatives, vinyl, acrylate, acrylamide, iodoacetamide, succinimidyl esters and combinations thereof. Most preferably, the reactive electrophilic groups are selected from maleimides, vinyl, acrylate, acrylamide, succinimidyl esters, sulfo succinimidyl esters and combinations thereof.

Examples of succinimidyl esters that may be employed include succinimidyl glutarate, succinimidyl propionate, succinimidyl succinamide, succinimidyl carbonate, disuccinimidyl suberate, bis(sulfosuccinimidyl) suberate, dithiobis(succinimidylpropionate), bis(<NUM>-succinimidooxycarbonyloxy) ethyl sulfone, <NUM>,<NUM>'-dithiobis(sulfosuccinimidyl-propionate), succinimidyl carbamate, sulfosuccinimidyl(<NUM>-iodoacetyl)aminobenzoate, bis(sulfosuccinimidyl) suberate, sulfosuccinimidyl-<NUM>-(N-maleimidomethyl)-cyclohexane-I-carboxylate, dithiobis-sulfosuccinimidyl propionate, disulfo-succinimidyl tartarate; bis[<NUM>-(sulfo-succinimidyloxycarbonyloxyethylsulfone)], ethylene glycol bis(sulfosuccinimiclylsuccinate), dithiobis-(succinimidyl propionate).

Examples of dihydroxyphenyl derivatives that may be employed include dihydroxyphenylalanine, <NUM>,<NUM>-dihydroxyphenylalanine (DOPA), dopamine, <NUM>,<NUM>-dihydroxyhydroccinamic acid (DOHA) , norepinephrine, epinephrine and catechol.

The water-soluble nucleophilic polymer that is contained in the nucleophilic polymer particles preferably has a solubility in demineralised water of <NUM>, at a pH in the range of <NUM> to <NUM>, of at least <NUM>/L, more preferably of at least <NUM>/L and most preferably of at least <NUM>/L. The present invention may suitably employ a nucleophilic polymer that is soluble at acidic pH if the electrophilic polyoxazoline releases acidic substances when it reacts with blood components, tissue and/or the nucleophilic polymer. This is the case, for instance, when the electrophilic polyoxazoline contains N-hydroxysuccinimide groups.

The nucleophilic polymer particles preferably contain at least <NUM> wt. %, more preferably at least <NUM> wt. % and most preferably at least <NUM> wt. % of the nucleophilic polymer.

According to a particularly preferred embodiment, the water-soluble nucleophilic polymer that is employed in the agglomerate particles dissolves relatively slowly in water. As explained herein before, it is believed that when the electrophilic polyoxazoline reacts with the water-soluble nucleophilic polymer at a relatively slow rate, the electrophilic polyoxazoline can also react with proteins in blood and tissue at the bleeding site. By employing a water-soluble nucleophilic polymer that dissolves relatively slowly, dissolved electrophilic polyoxazoline has the opportunity to react with proteins in blood and tissue as well as with the (slow) water-soluble nucleophilic polymer, thereby creating a strong homogeneous sealing gel.

Water-soluble nucleophilic polymers having a high molecular weight tend to dissolve relatively slowly in water. Accordingly, in a very preferred embodiment, the water-soluble nucleophilic polymer that is contained in the nucleophilic polymer particles has a molecular weight of at least <NUM> kDa, more preferably of at least <NUM> kDa and most preferably of <NUM> to <NUM>,<NUM> kDa.

The nucleophilic polymer preferably contains at least <NUM> reactive nucleophilic groups, more preferably at least <NUM> reactive nucleophilic groups and most preferably at least <NUM> reactive nucleophilic groups. Most preferably, these reactive nucleophilic groups are amine groups, most preferably primary amine groups.

Examples of water-soluble nucleophilic polymers that can suitably be used in the nucleophilic polymer particles include protein, chitosan, nucleophilic polyoxazoline, nucleophilic polyethylene glycol, polyethyleneimine and combinations thereof. More preferably, the nucleophilic polymer is selected from gelatin, collagen, chitosan, nucleophilic polyoxazoline and combinations thereof.

According to a particularly advantageous embodiment, the nucleophilic polymer particles employed in accordance with the invention provide haemostasis by accelerating the coagulation process. Collagen is capable of activating the intrinsic pathway of the coagulation cascade. Collagen has a large surface area, which acts as a matrix for platelet activation, aggregation, and thrombus formation. Gelatin particles have the ability to restrict blood flow and to provide a matrix for clot formation. Accordingly, in a very preferred embodiment, the nucleophilic polymer is selected from gelatin, collagen and combinations thereof. Most preferably, the nucleophilic polymer is gelatin, even more preferably crosslinked gelatin.

The crosslinked gelatin, preferably has a molecular weight in the range of <NUM>-<NUM>,<NUM> kDa, more preferably in the range of <NUM> to <NUM>,<NUM> kDa, most preferably in the range of <NUM> to <NUM>,<NUM> kDa.

The average primary amine content of the crosslinked gelatin is in the range of <NUM> × <NUM>-<NUM> to <NUM> × <NUM>-<NUM> µmol, more preferably <NUM> × <NUM>-<NUM> to <NUM> × <NUM>-<NUM> µmol of primary amine per µg of reduced crosslinked gelatin.

According to another advantageous embodiment, the nucleophilic polymer is nucleophilic polyethylene glycol (PEG). Preferably, the nucleophilic PEG contains at least <NUM> more preferably at least <NUM> and most preferably <NUM> reactive nucleophilic groups.

In accordance with a further preferred embodiment, the nucleophilic polymer is chitosan. Chitosan is a biodegradable, nontoxic, complex carbohydrate derivative of chitin (poly-N-acetyl-D-glucosamine), a naturally occurring substance. Chitosan is the deacetylated form of chitin. The chitosan applied in accordance with the present invention preferably has a degree of deacetylation of more than <NUM>%. Chitosan employed in accordance with the present invention preferably has a molecular weight of at least <NUM> kDa, more preferably of <NUM>-<NUM>,<NUM> kDa.

According to another very advantageous embodiment, the nucleophilic polymer is nucleophilic polyoxazoline. Preferably, the nucleophilic polyoxazoline contains at least <NUM> more preferably at least <NUM> and most preferably <NUM> to <NUM> reactive nucleophilic groups.

In comparison to naturally occurring nucleophilic biopolymers such as gelatin, collagen and chitosan, the use of synthetic nucleophilic polymers, such as nucleophilic polyoxazoline and nucleophilic PEG, offers the advantage that the particle agglomerates containing these synthetic nucleophilic polymers exhibit more predictable (reproducible) haemostatic properties.

The reactive nucleophilic groups of the water-soluble nucleophilic polymer are preferably selected from amine groups, thiol groups and combinations thereof.

According to a preferred embodiment, the water-soluble nucleophilic polymer contains two or more amine groups and the reactive electrophilic groups in the electrophilic polyoxazoline are selected from carboxylic acid esters, sulfonate esters, phosphonate esters, pentafluorophenyl esters, p-nitrophenyl esters, p-nitrothiophenyl esters, acid halide groups, anhydrides, ketones, aldehydes, isocyanato, thioisocyanato, isocyano, epoxides, activated hydroxyl groups, glycidyl ethers, carboxyl, succinimidyl esters, sulfosuccinimidyl esters, imido esters, dihydroxy-phenyl derivatives, and combinations thereof.

According to another preferred embodiment, the water-soluble nucleophilic polymer contains two or more thiol groups and the reactive electrophilic groups of the electrophilic polyoxazoline are selected from halo acetals, orthopyridyl disulfide, maleimides, vinyl sulfone, dihydroxyphenyl derivatives, vinyl, acrylate, acrylamide, iodoacetamide, succinimidyl esters, sulfosuccinmidyl esters and combinations thereof. More preferably, the reactive electrophilic groups are selected from succinimidyl esters, sulfosuccinimidyl esters, halo acetals, maleimides, or dihydroxyphenyl derivatives and combinations thereof. Most preferably, the reactive electrophilic groups are selected from maleimides or dihydroxyphenyl derivatives and combinations thereof.

According to a particularly preferred embodiment, the ratio between the total number of reactive electrophilic groups provided by the electrophilic polyoxazoline and the total number of reactive nucleophilic groups provided by the nucleophilic polymer lies in the range of <NUM>:<NUM> to <NUM>:<NUM>, more preferably in the range of <NUM>:<NUM> to <NUM>:<NUM> and most preferably in the range of <NUM>:<NUM> to <NUM>:<NUM>.

The inventors have unexpectedly discovered that particle agglomerates showing excellent adhesion to organs such as spleen and kidney can be obtained if the particle agglomerates contain <NUM>-<NUM> wt. %, more preferably <NUM>-<NUM> wt. % and most preferably <NUM>-<NUM> wt. % of a non-reactive non-ionic polymer. This non-reactive non-ionic polymer does not contain reactive electrophilic groups or reactive nucleophilic groups.

In a very preferred embodiment, the agglomerated particles are coated with the non-reactive non-ionic polymer.

The non-reactive non-ionic polymer preferably has a melting point in the range of <NUM>-<NUM>, more preferably in the range of <NUM>-<NUM> and most preferably in the range of <NUM>-<NUM>. Here the melting point refers to the temperature at which the polymer is completely melted.

Examples of non-reactive non-ionic polymers that may suitably be applied in the agglomerate particles of the present invention include poloxamers, polyethylene glycols and combinations thereof. Poloxamer is a non-ionic triblock copolymer composed of a central hydrophobic chain of polyoxypropylene (polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)) and is represented by formula (I)
<CHM>
wherein a is an integer of from <NUM> to <NUM> and b is an integer of from <NUM> to <NUM>. When a is <NUM> and b is <NUM>, this polymer is known as poloxamer <NUM>. Other known poloxamers useful in the present invention are poloxamer <NUM> (a = <NUM>; and b = <NUM>), poloxamer <NUM> (a = <NUM>; and b = <NUM>) and poloxamer <NUM> (a = <NUM>; and b = <NUM>). Further poloxamers that are known and can be useful in the present invention include poloxamer <NUM>, poloxamer <NUM>, poloxamer <NUM>, poloxamer <NUM>, poloxamer <NUM>, poloxamer <NUM>, poloxamer <NUM>, poloxamer <NUM>, poloxamer <NUM> and poloxamer <NUM>.

According to a particularly preferred embodiment, the non-reactive non-ionic polymer is a poloxamer, even more preferably a poloxamer having an average molecular mass of <NUM>,<NUM>-<NUM>,<NUM>, most preferably a poloxamer having an average molecular mass of <NUM>,<NUM>-<NUM>,<NUM>. The poloxamer applied in the particle agglomerates preferably is a solid at room temperature.

In another advantageous embodiment, the haemostatic powder is bioresorbable, allowing the powder to be used in abdominal surgery.

Another aspect of the invention relates to a method of preparing haemostatic particle agglomerates of:.

said method comprising combining <NUM> parts by weight of the electrophilic polyoxazoline particles with <NUM> to <NUM> parts by weight of the nucleophilic polymer particles in the presence of non-aqueous granulation liquid.

The electrophilic polyoxazoline particles that are employed in the present method are preferably identical to the electrophilic polymer particles described herein before. Likewise, the nucleophilic polymer particles that are employed are preferably identical to the nucleophilic polymer particles described herein before.

According to a preferred embodiment, both the electrophilic polyoxazoline particles and the nucleophilic polymer particles that are employed in the present method have a water content of less than <NUM> wt. %, more preferably of less than <NUM> wt. To achieve such a low water content, it may be necessary to dry the polymer particles before the granulation.

The electrophilic polyoxazoline preferably has a solubility in the non-aqueous granulation liquid at <NUM> of less than <NUM>/L, more preferably of less than <NUM>/L, even more preferably of less than <NUM>/L and most preferably of less than <NUM>/L.

The nucleophilic polymer preferably has a solubility in the non-aqueous granulation liquid at <NUM> of at least <NUM>/L, more preferably of at least <NUM>/L, even more preferably of <NUM>-<NUM>/L and most preferably of <NUM>-<NUM>/L.

In a preferred embodiment, the method comprises combining <NUM> parts by weight of the electrophilic polyoxazoline particles with <NUM> to <NUM> parts by weight, more preferably <NUM> to <NUM> parts by weight of the nucleophilic polymer particles, in the presence of the non-aqueous granulation liquid.

The amount of non-aqueous granulation liquid employed in the present method preferably is in the range of <NUM>-<NUM>% by weight of the combined amount of electrophilic polyoxazoline particles and nucleophilic polymer particles that is used in the method. More preferably, the amount of non-aqueous granulation liquid employed is in the range of <NUM>-<NUM> %, most preferably <NUM>-<NUM> % by weight of the combined amount of electrophilic polyoxazoline particles and nucleophilic polymer particles.

According to a particularly preferred embodiment, the method comprises the step of wetting the electrophilic polyoxazoline particles with the non-aqueous granulation liquid, followed by the step of combining the wetted polyoxazoline particles with the nucleophilic polymer particles. This particular embodiment offers the advantage that it yields a granulate that is very homogeneous in terms of particle size and composition.

The electrophilic polyoxazoline particles used in the preparation of the powder blend preferably have an volume weighted mean diameter (D [<NUM>,<NUM>]) in the range of <NUM>-<NUM>, more preferably in the range of <NUM>-<NUM>, most preferably in the range of <NUM>-<NUM>.

The volume weighted mean diameter (D [<NUM>,<NUM>]) of the nucleophilic polymer particles used in the preparation of the powder blend preferably is in the range of <NUM>-<NUM>, more preferably in the range of <NUM>-<NUM>, most preferably in the range of <NUM>-<NUM>.

The agglomerated powder that is obtained by the present method preferably has a volume weighted mean diameter (D [<NUM>,<NUM>]) in the range of <NUM>-<NUM>, more preferably in the range of <NUM>-<NUM>, most preferably in the range of <NUM>-<NUM>.

The non-aqueous granulation liquid employed in the wet granulation preferably contains at least <NUM> wt. % of an organic solvent selected from isopropyl alcohol, ethanol, methanol, diethyl ether, heptane, hexane, pentane, cyclohexane, dichloromethane, acetone and mixtures thereof. More preferably, the non-aqueous granulation liquid contains at least <NUM> wt. %, most preferably at least <NUM> wt. % of an organic solvent selected from isopropyl alcohol, ethanol and mixtures thereof. Even more preferably, the non-aqueous granulation liquid contains at least <NUM> wt. %, most preferably at least <NUM> wt. % of isopropyl alcohol.

The non-aqueous granulation liquid preferably contains not more than <NUM> wt. % water, more preferably not more than <NUM> wt.

The present method offers the advantage that it does not require the use of granulation binder. Accordingly, in a preferred embodiment of the method, no granulation binder is used. According to a particularly preferred embodiment, the only materials used in the preparation method are the electrophilic polyoxazoline particles, the nucleophilic polymer particles and the non-aqueous granulation liquid.

The particle agglomerates of the present invention are applied in haemostatic sheets to improve the haemostatic properties thereof. Accordingly, the invention relates to a biocompatible, flexible, haemostatic sheet comprising:.

In accordance with a particularly preferred embodiment, the cohesive fibrous carrier structure is water resistant.

The electrophilic polyoxazoline in the haemostatic powder that is distributed throughout the fibrous carrier structure dissolve rapidly when the sheet comes into contact with blood or other aqueous bodily fluids that can penetrate the interstitial space. Thus, upon application of the sheet onto a wound site, rapid covalent cross-linking occurs between on the one hand the electrophilic polyoxazoline, and on the other hand the nucleophilic polymer in the nucleophilic polymer particles, blood proteins and tissue, leading to the formation of a gel which seals off the wound surface and stops the bleeding and further leading to strong adhesion of the haemostatic sheet to the tissue The chitosan applied in accordance with the present invention preferably has a degree of deacetylation of more than <NUM>%. The water-resistant fibrous carrier structure provides mechanical strength during and after application, and prevents excessive swelling.

Since the electrophilic polyoxazoline and the nucleophilic polymer are contained in a single particle, it is ensured that these two reactive components can be homogeneously distributed throughout the haemostatic sheet, that no segregation occurs during transport and handling, and that these components can react immediately with each other when the particle agglomerates come into contact with blood.

According to a particularly preferred embodiment, the haemostatic sheet of the present invention is bioabsorbable, meaning that the carrier structure, the particle agglomerates and any other components of the haemostatic sheet are eventually absorbed in the body. Absorption of the carrier structure and the particle agglomerates typically requires chemical decomposition (e.g. hydrolysis) of the polymers contained therein. Complete bioabsorption of the haemostatic sheet by the human body is typically achieved in <NUM> to <NUM> weeks, preferably in <NUM> to <NUM> weeks.

The haemostatic sheet of the present invention typically has a non-compressed mean thickness of <NUM>-<NUM>. More preferably, the non-compressed mean thickness is in the range of <NUM>-<NUM>, most preferably in the range of <NUM>-<NUM>.

The dimensions of the haemostatic sheet preferably are such that the top and bottom of the sheet each have a surface area of at least <NUM><NUM>, more preferably of at least <NUM><NUM> and most preferably of <NUM>-<NUM><NUM>. Typically, the sheet is rectangular in shape and has a length of <NUM>-<NUM>, an a width of <NUM>-<NUM>.

The haemostatic sheet preferably has a non-compressed density of less than <NUM>/cm<NUM>, more preferably of less than <NUM>/cm<NUM> and most preferably of <NUM>-<NUM>/cm<NUM>.

The haemostatic sheet of the present invention preferably is essentially anhydrous. Typically, the haemostatic sheet has a water content of not more than <NUM> wt. %, more preferably of not more than <NUM> wt. % and most preferably of not more than <NUM> wt.

The water absorption capacity of the haemostatic sheet preferably is at least <NUM>%, more preferably lies in the range of <NUM>% to <NUM>%, most preferably in the range of <NUM>% to <NUM>%.

The haemostatic sheet of the present invention is preferably sterile.

The use of a fibrous carrier structure in the haemostatic sheet of the present invention offers the advantage that the haemostatic powder can be homogeneously distributed throughout this carrier structure without difficulty. Such a homogeneous distribution is much more difficult to achieve in, for instance, foamed carrier structures.

The fibres in the fibrous carrier structure preferably have a mean diameter of <NUM>-<NUM>, more preferably of <NUM>-<NUM> and most preferably of <NUM>-<NUM>. The mean diameter of the fibres can suitably be determined using a microscope.

Typically, at least <NUM> wt. %, more preferably at least <NUM> wt. % of the fibres in the fibrous carrier structure have a diameter of <NUM>-<NUM> and a length of at least <NUM>.

Preferably, at least <NUM> wt. %, more preferably at least <NUM> wt. % of the fibres in the fibrous carrier structure have an aspect ratio (ratio of length to diameter) of at least <NUM>.

The fibrous carrier structure that is employed in accordance with the present invention preferably is a felt structure, a woven structure or a knitted structure. Most preferably, the fibrous carrier structure is a felt structure. Here the term "felt structure" refers to a structure that is produced by matting and pressing fibres together to form a cohesive material.

The fibrous carrier structure preferably comprises at least <NUM> wt. %, more preferably at least <NUM> wt. % and most preferably at least <NUM> wt. % fibres containing gelatin, collagen, cellulose, modified cellulose, carboxymethyldextran, PLGA, sodium hyaluronate/carboxy methylcellulose, polyvinyl alcohol, chitosan or a combination thereof.

In an embodiment of the invention, the fibrous carrier structure does not comprise oxidised regenerated cellulose.

Preferred fibrous carrier structures have an open pore structure with a permeability to air of at least <NUM>/min × cm<NUM>, more preferably of at least <NUM>/min × cm<NUM>. The air permeability is determined in accordance with EN ISO <NUM>:<NUM> (Textiles - Determination of the permeability of fabrics to air).

The fibres in the fibrous carrier structure can be produced by means of methods known in the art, such as electrospinning, electro-blown spinning and high speed rotary sprayer spinning. Production of fibrous carrier structure by means of high speed rotary sprayer spinning is described in <CIT>. It is also possible to use commercially available haemostatic fibrous sheets as the fibrous carrier structure.

The haemostatic powder is preferably present in the haemostatic sheet of the present invention in an amount of <NUM>-<NUM>%, more preferably <NUM>-<NUM>%, even more preferably <NUM>-<NUM>% and most preferably <NUM>-<NUM>%, by weight of the fibrous carrier structure.

In general: wherever residual moisture (i.e., residual water in dried powder, granulate and/or cohesive fibrous carrier structures) after drying is not explicitly mentioned, levels are below <NUM>% w/w.

Haemostatic powders without fibrous carrier structure are not according to the claimed invention.

NHS-side chain activated poly[<NUM>-(ethyl/hydroxy-ethyl-amide-ethyl/NHS-ester-ethyl-ester-ethyl-amide-ethyl)-<NUM>-oxazoline] terpolymer containing <NUM>% NHS-ester groups (= EL-POx, <NUM>% NHS) was synthesized as follows:.

<NUM> of NHS-POx powder were dissolved in water and mixed with <NUM> Brilliant Blue FCF (Sigma Aldrich) using a high-performance dispersing instrument (Ultra-Turrax, IKA). Directly after mixing (<NUM> minutes) the solution was frozen at -<NUM> and subsequently freeze dried overnight. The freeze dried powder so obtained was dried in a Rotavap at <NUM> until the residual water content was below <NUM>% w/w as determined via Karl Fischer titration. Next, the dried (blue) powder was grinded using a ball mill (Retch MM400) until a blue dyed NHS-POx powder having an average particle size of not more than <NUM> (D [<NUM>,<NUM>]) and vacuum sealed in alu-alu bags.

A <NUM>µg/mL solution of <NUM>,<NUM>'-(ethylenedioxy)-bis-(ethylamine) (EDEA, Aldrich, Mw <NUM>) was prepared in a mixture of dichloromethane (DCM) and isopropanol (IPA) by dissolving <NUM> of EDEA in <NUM> of DCM/IPA <NUM>:<NUM> (v/v). This solution was diluted <NUM> times with DCM/IPA <NUM>:<NUM> (v/v) and <NUM>µL of the EDEA solution were added to <NUM>µL of a <NUM>/mL solution of NHS-POx in DCM/IPA <NUM>:<NUM> (v/v) corresponding with <NUM> mole% amine groups with respect to NHS groups. Directly after addition of the EDEA solution, the reaction mixture was thoroughly mixed using a vortex mixer. The reaction mixture was agitated at <NUM> for <NUM> hours and all volatiles were removed by rotary evaporation under reduced pressure.

The dried sample was reconstituted in the size exclusion chromatographic (SEC) mobile phase N,N-dimethylacetamide containing <NUM> lithium chloride. SEC was measured against poly(methyl methacrylate) standards. From the obtained size exclusion chromatogram, the Mn, Mw and PDI were determined. The PDI was more than <NUM>.

A <NUM>/mL solution of NHS-POx in DCM/IPA <NUM>:<NUM> (v/v), without the addition of EDEA, following the same SEC procedure resulted in a PDI of <NUM>, indicating that only <NUM> mole% crosslinking of NHS groups already results in a significant increase in PDI of the polymer.

Polyoxazoline containing ethyl and amine groups in the alkyl side chain was synthesized by CROP of EtOx and MestOx and subsequent amidation of the methyl ester side chains with ethylene diamine to yield a poly(<NUM>-ethyl/aminoethylamidoethyl-<NUM>-oxazoline) copolymer (NU-POx). The NU-POx contained <NUM>% NH<NUM> according to <NUM>H-NMR. NU-POx was dissolved between <NUM>-<NUM> in water (<NUM> in <NUM>), cooled at minus <NUM> for half an hour an freeze dried. The freeze dried powder so obtained was dried in a Rotavap at <NUM> until the water content was below <NUM>% w/w as determined via Karl Fischer titration. This dry powder was grinded in a table top grinding machine until the average particle size was not more than <NUM> (D [<NUM>,<NUM>]) and vacuum sealed in alu-alu bags.

Blue or white (non-dyed) NHS-POx powder was wetted with isopropyl alcohol (IPA) in a high shear mixer until a homogeneous snow-like powder was obtained containing about <NUM>-<NUM>% w/w IPA. After this, NU-POx powder was added and mixed. The wetted blue NHS-POx powder was mixed with NU-POx powder in a molar ratio of <NUM>:<NUM>, said molar ratio referring to the ratio of the number of NHS groups provided by NHS-POx to the number of amine groups provided by the NU-POx. The wetted non-dyed NHS-POx powder was also mixed with NU-POx powder in other molar ratios (<NUM>:<NUM>; <NUM>:<NUM> and <NUM>:<NUM>).

After mixing, the wet granulates were dried under reduced pressure until the IPA content was less than <NUM>% w/w as determined via <NUM>H-NMR. The dried granulates were grinded using a ball mill (Retch MM400) until the average particle size was not more than <NUM> (D [<NUM>,<NUM>]) and vacuum sealed in alu-alu bags.

The particle size distribution of the granulates so obtained was approximately: <NUM> vol. % < <NUM>, <NUM> vol. % < <NUM> and <NUM> vol.

The NHS-POX/NU-POx granulate (<NUM>:<NUM>) was analysed using <NUM>H-NMR spectroscopy. <NUM> of granulate were dissolved in trifluoroacetic acid (<NUM>) by sonicating for <NUM> minutes. After complete dissolution of the granulate, the sample was diluted with deuterated dimethylsulfoxide (DMSO-d<NUM>) containing maleic acid (<NUM>/mL) as an internal standard (<NUM>), transferred to an NMR tube and a <NUM>H-NMR spectrum was recorded. From the acquired spectrum, the amount of NHS bound to NHS-POx can be calculated, along with the molar ratio of NHS and amine groups present in the granulate. The amount of NHS bound to NHS-POx in the granulate was equal to the amount of NHS bound to NHS-POx starting material indicating no decay or cross linking during granulation.

The total polymer recovery, i.e. the combination of NHS-POx and NU-POx, in the NMR sample was determined using a known amount of internal standard (maleic acid) and a calibration curve constructed from <NUM>H-NMR spectra recorded of NHS-POx and NU-POx in different concentrations. The total polymer recovery was measured to be <NUM> percent, indicating that no insoluble crosslinked material was formed.

The NHS-POX/NU-POx granulate (<NUM>:<NUM>) was further analysed by means of size exclusion chromatography. <NUM> of the granulate was treated with acetic anhydride (<NUM>) for <NUM> hour at <NUM>. Subsequently, methanol (<NUM>) was added and the mixture was stirred for an additional hour at <NUM>. An aliquot (<NUM>) was taken and all volatiles were removed under reduced pressure. The sample was taken up in N,N-dimethylacetamide containing <NUM> lithium chloride (<NUM>), which was the eluent for SEC analysis. SEC was measured against poly(methyl methacrylate) standards and from the obtained size exclusion chromatogram, the Mn, Mw and PDI were determined. The PDI was not more than <NUM>, indicating no cross linking had occurred during granulation. Analytical validation of this size exclusion chromatographic method indicated that intentional cross linking of NHS-POx with NU-POx at a level of <NUM> mol% increased the PDI to more than <NUM>.

Co-freeze dried NHS-POx/NU-POx powders were prepared as follows: <NUM> of NU-POx were dissolved in <NUM> of glacial acetic acid. After complete dissolution of the polymer, <NUM> of NHS-POx were added and the sample was sonicated for <NUM> minutes. The solution was flash frozen in liquid nitrogen and freeze dried. The resulting sticky solid was further dried under reduced pressure (< <NUM> kPa) and vacuum sealed in an alu-alu bag.

Co-freeze dried NHS-POx / NU-POx samples could not be analyzed by means of <NUM>H-NMR spectroscopy and size exclusion chromatography because the samples were neither soluble in trifluoroacetic acid nor acetic anhydride due to a high degree of crosslinking between NHS-POx and NU-POx.

NHS-POx/NU-POx mixtures were prepared as follows: <NUM> of NHS-POx and <NUM> of NU-POx were dry mixed by means of tumble mixing for <NUM> minutes. The resulting powder was dried under reduced pressure (< 1kPa) and vacuum sealed in an alu-alu bag.

The powder was analysed using <NUM>H-NMR spectroscopy. <NUM> of powder were dissolved in trifluoroacetic acid (<NUM>) by sonicating for <NUM> minutes. After complete dissolution of the powder, the sample was diluted with deuterated dimethylsulfoxide (DMSO-d<NUM>) containing maleic acid (<NUM>/mL) as an internal standard (<NUM>), transferred to an NMR tube and a <NUM>H-NMR spectrum was recorded. From the obtained spectrum, the amount of non-reacted NHS was calculated to be <NUM> percent compared to NHS-POx.

The powder was further analysed by means of size exclusion chromatography. <NUM> of the powder was treated with acetic anhydride (<NUM>) for <NUM> hour at <NUM>. Subsequently, methanol (<NUM>) was added and the mixture was stirred for an additional hour at <NUM>. An aliquot (<NUM>) was taken and all volatiles were removed under reduced pressure. The sample was taken up in N,N-dimethylacetamide containing <NUM> lithium chloride (<NUM>), which was the eluent for SEC analysis. SEC was measured against poly(methyl methacrylate) standards and from the obtained size exclusion chromatogram, the Mn, Mw and PDI were determined. The PDI was not more than <NUM>, indicating that no cross linking had occurred during dry mixing.

The following powders were produced by freeze drying:.

<NUM> of sodium phosphate dibasic dihydrate and <NUM> of sodium phosphate monobasic monohydrate were dissolved in <NUM> ultrapure water. After complete dissolution, the pH was adjusted to <NUM> by addition of <NUM> of a <NUM> molar aqueous sodium hydroxide solution. The solution was frozen in liquid nitrogen and freeze dried. The resulting powder was dried under reduced pressure and vacuum sealed in an alu-alu bag.

A <NUM>:<NUM> mole/mole mixture of sodium carbonate and sodium hydrogen carbonate was prepared by dissolving <NUM> of sodium carbonate and <NUM> of sodium hydrogen carbonate in <NUM> ultrapure water. The solution was flash frozen in liquid nitrogen and freeze dried. The resulting powder was dried under reduced pressure and vacuum sealed in an alu-alu bag.

<NUM> of citric acid were dissolved in <NUM> ultrapure water. The solution was cooled between <NUM>-<NUM>. Subsequently, <NUM> of NHS-POx were added and dissolved with the aid of a high-performance dispersing instrument (Ultra-Turrax, IKA). Directly after mixing (<NUM> minutes), the solution was flash frozen using liquid nitrogen and freeze dried overnight. The resulting powder was dried under reduced pressure and vacuum sealed in an alu-alu bag.

Next, a carbonate-containing NU-POx powder was produced as follows:<NUM> of NU-POx and <NUM> of the carbonate powder were dissolved in <NUM> ultrapure water. The solution was flash frozen using liquid nitrogen and freeze dried. The resulting powder was dried under reduced pressure and vacuum sealed in an alu-alu bag.

NHS-POx / NU-POx (sequestered) granulate was prepared as follows: <NUM> of NHS-POx/Citric acid powder and <NUM> of the phosphate powder were mixed in a high shear mixer. After obtaining a homogeneous mixture, <NUM> IPA were added slowly, while the mixing was continued, until a homogeneous snow-like powder was formed. Next, <NUM> of the NU-POx/Carbonate powder were added and the mixing was stopped once a homogeneous granulate was formed. The granulate was dried under reduced pressure pressure until the IPA content was less than <NUM> % w/w. The dried granulate was milled in a coffee grinder until the average particle size was not more than <NUM> (D [<NUM>,<NUM>]) and vacuum sealed in an alu-alu bag.

Reactive NHS-POx / NU-POx granules as described previously were coated with Pluronic P188. <NUM>% w/w P188 coated reactive NHS-POx / NU-POx granulate was prepared by heating the NHS-POx / NU-POx granulate together with P188 powder in a high shear mixer at <NUM> for <NUM> minutes followed by cooling down to ambient conditions. The coated granulate was grinded using a ball mill (Retch MM400) until the average particle size was not more than <NUM> (D [<NUM>,<NUM>]) and vacuum sealed in alu-alu bags.

The NHS-POx/NU-POx/P188 granulate was analysed using <NUM>H-NMR spectroscopy. <NUM> of powder were dissolved in trifluoroacetic acid (<NUM>) by sonicating for <NUM> minutes. After complete dissolution of the granulate, the sample was diluted with deuterated dimethylsulfoxide (DMSO-d<NUM>) (<NUM>), transferred to an NMR tube and a <NUM>H-NMR spectrum was recorded. From the obtained spectrum, the amount of non-reacted NHS was calculated to be <NUM> percent compared to NHS-POx.

The NHS-POx/NU-POx/P188 granulate was further analysed by means of size exclusion chromatography. <NUM> of the granulate was treated with acetic anhydride (<NUM>) for <NUM> hour at <NUM>. Subsequently, methanol (<NUM>) was added and the mixture was stirred for an additional hour at <NUM>. An aliquot (<NUM>) was taken and all volatiles were removed under reduced pressure. The sample was taken up in N,N-dimethylacetamide containing <NUM> lithium chloride (<NUM>), which was the eluent for SEC analysis. SEC was measured against poly(methyl methacrylate) standards and from the obtained size exclusion chromatogram, the Mn, Mw and PDI were determined. The PDI was not more than <NUM>, indicating that no cross linking had occurred during granulation.

Reduced crosslinked gelatin (RXL) was prepared according to three procedures:.

NHS-POx/RXL reactive granules were prepared as follows: <NUM> of blue NHS-POx powder were wetted with IPA in a high shear mixer until a homogeneous snow like powder was obtained containing about <NUM>-<NUM>% w/w IPA. After this, <NUM> of RXL, RXL-LS or RXL-HS powder were added and mixed. After mixing, the wet granulates were dried under reduced pressure until the IPA content was less than <NUM>% w/w as determined via <NUM>H-NMR. The dried granulates were milled in a coffee grinder until the average particle size was not more than <NUM> (D [<NUM>,<NUM>]) and vacuum sealed in alu-alu bags.

The granulate containing RXL was analysed by means of <NUM>H-NMR spectroscopy analysis. To this end deuterated chloroform (CDCl3) containing <NUM> %(v/v) acetic acid (<NUM>) was added to <NUM> of the granulate. NHS-POx was selectively extracted by sonicating the sample for <NUM> minutes. The dispersion was passed through a <NUM> filter, transferred to an NMR tube and a <NUM>H-NMR spectrum was recorded. From the obtained spectrum, the amount of non-reacted NHS was calculated to be <NUM> percent compared to NHS-POx.

The recovery of NHS-POx in the NMR sample was determined using trimethylsilane as an internal standard and a calibration curve constructed from <NUM>H-NMR spectra of NHS-POx in different concentrations. The total NHS-POx recovery was measured to be <NUM> percent, indicating that no insoluble crosslinked material was formed.

The NHS-POx/RXL granulate was further analysed by means of size exclusion chromatography. Therefore, an aliquot (<NUM>) was taken from the solution used for <NUM>H-NMR spectroscopy analysis. This solution was diluted with N,N-dimethylacetamide containing <NUM> lithium chloride (<NUM>), which was the eluent for SEC analysis. SEC was measured against poly(methyl methacrylate) standards and from the obtained size exclusion chromatogram, the Mn, Mw and PDI were determined. The PDI was not more than <NUM>, again indicating that no cross linking had occurred during granulation.

Co-freeze dried NHS-POx/RXL powders were prepared as follows: <NUM> of RXL powder were dissolved in <NUM> of ultrapure water. The pH was adjusted to <NUM> by the addition of acetic acid and the solution was cooled to <NUM>. Subsequently, <NUM> of NHS-POx was added and dissolved with the aid of high shear stirring. Directly after complete dissolution of the NHS-POx, the solution was flash frozen in liquid nitrogen and freeze dried. The resulting powder was dried under reduced pressure and vacuum sealed in an alu-alu bag.

The co-freeze dried NHS-POx/RXL powder was analysed by means of <NUM>H-NMR spectroscopy analysis. To this end deuterated chloroform (CDCl<NUM>) containing <NUM> %(v/v) acetic acid (<NUM>) was added to <NUM> of the granulate. NHS-POx was selectively extracted by sonicating the sample for <NUM> minutes. The dispersion was passed through a <NUM> filter, transferred to an NMR tube and a <NUM>H-NMR spectrum was recorded. From the obtained spectrum, the amount of non-reacted NHS was calculated to be <NUM> percent compared to NHS-POx.

The recovery of NHS-POx in the NMR sample was determined using trimethylsilane as an internal standard and a calibration curve constructed from <NUM>H-NMR spectra of NHS-POx in different concentrations. The total NHS-POx recovery was measured to be <NUM> percent, indicating that to some extent insoluble crosslinked material was formed.

The NHS-POx/RXL powder was further analysed by means of size exclusion chromatography. An aliquot (<NUM>) was taken from the solution used for <NUM>H-NMR spectroscopy analysis. This solution was diluted with N,N-dimethylacetamide containing <NUM> lithium chloride (<NUM>), which was the eluent for SEC analysis. SEC was measured against poly(methyl methacrylate) standards and from the obtained size exclusion chromatogram, the Mn, Mw and PDI were determined. The PDI was <NUM> indicating that cross linking had occurred during co-freeze drying of both components.

NHS-POx/RXL mixtures were prepared as follows: <NUM> of RXL powder and <NUM> of NHS-POx were dry mixed by means of tumble mixing for <NUM> minutes. The resulting powder was dried under reduced pressure and vacuum sealed in an alu-alu bag.

The powder was analysed by means of <NUM>H-NMR spectroscopy analysis. To this end deuterated chloroform (CDCl<NUM>) containing <NUM> %(v/v) acetic acid (<NUM>) was added to <NUM> of the granulate. NHS-POx was selectively extracted by sonicating the sample for <NUM> minutes. The dispersion was passed through a <NUM> filter, transferred to an NMR tube and a <NUM>H-NMR spectrum was recorded. From the obtained spectrum, the amount of non-reacted NHS was calculated to be <NUM> percent compared to NHS-POx.

The NHS-POX/RXL powder was further analysed by means of size exclusion chromatography. Therefore, an aliquot (<NUM>) was taken from the solution used for <NUM>H-NMR spectroscopy analysis. This solution was diluted with N,N-dimethylacetamide containing <NUM> lithium chloride (<NUM>), which was the eluent for SEC analysis. SEC was measured against poly(methyl methacrylate) standards and from the obtained size exclusion chromatogram, the Mn, Mw and PDI were determined. The PDI was not more than <NUM>, indicating that no cross linking had occurred during dry mixing.

First, a <NUM>:<NUM> mole/mole mixture of sodium carbonate and sodium hydrogen carbonate was prepared by dissolving <NUM> of sodium carbonate and <NUM> of sodium hydrogen carbonate in <NUM> ultrapure water. The solution was flash frozen in liquid nitrogen and freeze dried. The resulting powder was dried under reduced pressure and vacuum sealed in an alu-alu bag.

The NHS-POx/RXL/carbonate granulates were prepared as follows: <NUM> of RXL-LS or RXL-HS and <NUM> of the sodium carbonate / sodium hydrogen carbonate were mixed using a high shear mixer. Next, <NUM> of blue NHS-POx were added containing about <NUM>-<NUM>% w/w IPA and mixed until a homogeneous powder was obtained. After mixing, the wet granulates were dried under reduced pressure until the IPA content was less than <NUM>% w/w as determined via <NUM>H-NMR. The dried granulates were milled in a coffee grinder until the average particle size was not more than <NUM> (D [<NUM>,<NUM>]) and vacuum sealed in alu-alu bags.

NHS-POx (<NUM>,<NUM>) was wetted with IPA in a high shear mixer until a homogeneous snow-like powder was obtained containing about <NUM>-<NUM>% w/w IPA. Subsequently, <NUM> of amine-PEG-amine, <NUM>-arm, MW <NUM> (ex Creative PEGWorks) were added (molar ratio of <NUM>:<NUM>, said molar ratio referring to the ratio of the number of NHS groups provided by NHS-POx to the number of amine groups provided by the PEG-amine). The formed granulate was dried under reduced pressure until the IPA content was less than <NUM> % w/w as determined via <NUM>H-NMR. The dried granulate was milled in a coffee grinder until the average particle size was not more than <NUM> (D [<NUM>,<NUM>]) and vacuum sealed in alu-alu bags.

The NHS-POX/NH2-PEG granulate was analysed using <NUM>H-NMR spectroscopy. <NUM> of granulate were dissolved in trifluoroacetic acid (<NUM>) by sonicating for <NUM> minutes. After complete dissolution of the granulate, the sample was diluted with deuterated dimethylsulfoxide (DMSO-d<NUM>) (<NUM>), transferred to an NMR tube and a <NUM>H-NMR spectrum was recorded. From the obtained spectrum, the amount of non-reacted NHS was calculated to be <NUM> percent compared to NHS-POx.

The NHS-POX/NH2-PEG granulate was further analysed by means of size exclusion chromatography. <NUM> of the granulate was treated with acetic anhydride (<NUM>) for <NUM> hour at <NUM>. Subsequently, methanol (<NUM>) was added and the mixture was stirred for an additional hour at <NUM>. An aliquot (<NUM>) was taken and all volatiles were removed under reduced pressure. The sample was taken up in N,N-dimethylacetamide containing <NUM> lithium chloride (<NUM>), which was the eluent for SEC analysis. SEC was measured against poly(methyl methacrylate) standards and from the obtained size exclusion chromatogram, the Mn, Mw and PDI were determined. The PDI was not more than <NUM>, indicating that no cross linking occurred during granulation.

The following starch powders were produced by freeze drying:.

<NUM> of starch (Arista™ AH, BARD) was dispersed in <NUM> of ultrapure water using a high shear mixer. The dispersion was cooled between <NUM>-<NUM> and <NUM> of NHS-POx were added and dissolved with the aid of high shear mixing.

Directly after dissolution of the NHS-POx, the solution was flash frozen using liquid nitrogen and freeze dried. The resulting powder was dried under reduced pressure and vacuum sealed in an alu-alu bag.

<NUM> of starch (Arista™ AH, BARD) were dispersed in <NUM> of ultrapure water using a high shear mixer. Subsequently, <NUM> of NU-POx were added and allowed to dissolve. After complete dissolution of the NU-POx, the dispersion was flash frozen using liquid nitrogen and freeze dried. The resulting powder was dried under reduced pressure and vacuum sealed in an alu-alu bag.

<NUM> of starch (Arista™ AH, BARD) were dispersed in <NUM> of ultrapure water using a high shear mixer. Subsequently, <NUM> of NU-POx and <NUM> of sodium carbonate were added and allowed to dissolve. After complete dissolution of the NU-POx, the dispersion was flash frozen using liquid nitrogen and freeze dried. The resulting powder was dried under reduced pressure and vacuum sealed in an alu-alu bag.

Two granulates were prepared from these three powders. Starch / NHS-POx / NU-POx granulates were prepared as follows:.

<NUM> of the NHS-POx / Starch powder, <NUM> of the NU-POx / Starch powder and <NUM> of IPA were mixed using a pestle and mortar. After obtaining a homogeneous granulate, residual IPA was removed under reduced pressure until the IPA content was less than <NUM> % w/w. The dried granulate was milled in a coffee grinder until the average particle size was not more than <NUM> (D [<NUM>,<NUM>]) and vacuum sealed in an alu-alu bag.

<NUM> of NHS-POx / Starch powder, <NUM> of the NU-POx / Starch / Sodium Carbonate powder <NUM> and <NUM> of IPA were mixed using a pestle and mortar. After a homogeneous granulate was formed, residual IPA was removed under reduced pressure until the IPA content was less than <NUM> % w/w. The dried granulate was milled in a coffee grinder until the average particle size was not more than <NUM> (D [<NUM>,<NUM>]) and vacuum sealed in an alu-alu bag.

<NUM> of pre-dried gelatin powder (Gelita-SPONO, ex Gelita Medical GmbH), having a water content of less than <NUM>% w/w, was dispersed in dichloromethane (<NUM>) using a high shear mixer operating at <NUM>,<NUM> rpm for <NUM> minutes. Subsequently, NHS-POx (<NUM>) was added and the stirring was continued for <NUM> minutes. NHS-POx did not dissolve. All volatiles were removed from the suspension under reduced pressure. The obtained powder was milled using a coffee grinder until the average particle size was not more than <NUM> (D [<NUM>,<NUM>]) and vacuum sealed in alu-alu bags, further dried under reduced pressure and vacuum sealed in an alu-alu bag.

The granulate was analysed by means of <NUM>H-NMR spectroscopy analysis. To this end deuterated chloroform (CDCl<NUM>) containing <NUM> %(v/v) acetic acid (<NUM>) was added to <NUM> of the granulate. NHS-POx was selectively extracted by sonicating the sample for <NUM> minutes. The dispersion was passed through a <NUM> filter, transferred to an NMR tube and a <NUM>H-NMR spectrum was recorded. From the obtained spectrum, the amount of non-reacted NHS was calculated to be <NUM> percent compared to NHS-POx.

The granulate was further analysed by means of size exclusion chromatography (SEC) analysis. An aliquot (<NUM>) of the filtered NHS-POx extract described above was diluted with N,N-dimethylacetamide containing <NUM> lithium chloride (<NUM>), which was the eluent for SEC analysis. The sample was analysed by SEC against poly(methyl methacrylate) standards and the PDI was <NUM> indicating that no cross linking had occurred.

NHS-POx/Chitosan reactive granules were prepared as follows: <NUM> of NHS-POx powder were wetted with IPA in a high shear mixer until a homogeneous snow like powder was obtained containing about <NUM>-<NUM>% w/w IPA. After this, <NUM> of Chitosan powder (Shanghai Waseta International, <NUM>% DAC degree) were added and mixed. After mixing, the wet granulates were dried under reduced pressure until the IPA content was less than <NUM>% w/w as determined via <NUM>H-NMR. The dried granulates were milled in a coffee grinder until the average particle size was not more than <NUM> (D [<NUM>,<NUM>]) and vacuum sealed in alu-alu bags. The particle size distribution of the granulates so obtained was approximately: <NUM> vol. % < <NUM>, <NUM> vol. % < <NUM> and <NUM> vol.

The NHS-POX/Chitosan granulate was further analysed by means of size exclusion chromatography. Therefore, an aliquot (<NUM>) was taken from the solution used for <NUM>H-NMR spectroscopy analysis. This solution was diluted with N,N-dimethylacetamide containing <NUM> lithium chloride (<NUM>), which was the eluent for SEC analysis. SEC was measured against poly(methyl methacrylate) standards and from the obtained size exclusion chromatogram, the Mn, Mw and PDI were determined. The PDI was not more than <NUM>, indicating that no cross linking had occurred during granulation.

Co-freeze dried NHS-POx/Chitosan powders were prepared as follows: <NUM> of Chitosan powder (Shanghai Waseta International, <NUM>% DAC degree) were dissolved in <NUM> of a <NUM>% v/v acetic acid solution in ultrapure water. The pH was adjusted to <NUM> by the addition of acetic acid and the solution was cooled to <NUM>. Subsequently, <NUM> of NHS-POx was added and dissolved with the aid of high shear stirring. Directly after complete dissolution of the NHS-POx, the solution was flash frozen in liquid nitrogen and freeze dried. The resulting powder was dried under reduced pressure and vacuum sealed in an alu-alu bag.

The granulate was analysed by means of <NUM>H-NMR spectroscopy analysis. To this end deuterated chloroform (CDCl<NUM>) containing <NUM> %(v/v) acetic acid (<NUM>) was added to <NUM> of the granulate. NHS-POx was selectively extracted by sonicating the sample for <NUM> minutes. The dispersion was passed through a <NUM> filter, transferred to an NMR tube and a <NUM>H-NMR spectrum was recorded. From the obtained spectrum, the amount of non-reacted NHS was calculated to be <NUM> percent compared to NHS-POx, which implies that crosslinking and/or hydrolysis occurred.

The recovery of NHS-POx in the NMR sample was determined using trimethylsilane as an internal standard and a calibration curve constructed from <NUM>H-NMR spectra of NHS-POx in different concentrations. The total NHS-POx recovery was measured to be <NUM> percent (in line with the NMR results), indicating that insoluble crosslinked material was formed.

The NHS-POX/Chitosan granulate was further analysed by means of size exclusion chromatography. Therefore, an aliquot (<NUM>) was taken from the solution used for <NUM>H-NMR spectroscopy analysis. This solution was diluted with N,N-dimethylacetamide containing <NUM> lithium chloride (<NUM>), which was the eluent for SEC analysis. SEC was measured against poly(methyl methacrylate) standards and from the obtained size exclusion chromatogram, the Mn, Mw and PDI were determined. The PDI was determined to be <NUM>, indicating that crosslinking took place.

NHS-POx/Chitosan mixtures were prepared as follows: <NUM> of Chitosan powder (Shanghai Waseta International, <NUM>% DAC degree) and <NUM> of NHS-POx were dry mixed by means of tumble mixing for <NUM> minutes. The resulting powder was dried under reduced pressure and vacuum sealed in an alu-alu bag.

The NHS-POX/Chitosan powder was further analysed by means of size exclusion chromatography. Therefore, an aliquot (<NUM>) was taken from the solution used for <NUM>H-NMR spectroscopy analysis. This solution was diluted with N,N-dimethylacetamide containing <NUM> lithium chloride (<NUM>), which was the eluent for SEC analysis. SEC was measured against poly(methyl methacrylate) standards and from the obtained size exclusion chromatogram, the Mn, Mw and PDI were determined. The PDI was not more than <NUM>, indicating that no cross linking had occurred during dry mixing.

The following commercially available haemostatic product was selected to be used as fibrous carrier structures in the preparation of tissue-adhesive sheets according to the present invention:.

Standardized ex-vivo and in-vivo porcine bleeding models were used to assess haemostatic efficacy. All models use heparin to increase clotting time of blood to about <NUM> to <NUM> times activated coagulation time (ACT).

Ex-vivo model: live ex-vivo pig model with a fresh liver, perfused with heparinized fresh blood from the slaughterhouse to mimic real in-vivo situations a closely as possible. Livers are mounted onto a perfusion machine by which oxygenation, pH of blood, temperature and blood pressure are kept within vivo boundaries. Two livers and <NUM> litres of heparinized blood (<NUM> units/L) are collected at the slaughterhouse. Livers are transported on ice; blood at ambient temperature. Within two hours after collection, livers are inspected for lesions which are closed with gloves and cyanoacrylate glue.

In-vivo model: standardized combined penetrating spleen rupture is inflicted in anesthetized swine (Domestic Pig, Male, Body Weight Range: <NUM>, Adult). A midline laparotomy is performed to access the spleen and other organs. Using a scalpel, n=<NUM> (S1. S3) subcapsular standardized lesions (<NUM> × <NUM>) are made. The haemostatic products are applied with gentle pressure by a pre-wetted gauze (saline) and held for <NUM>. After application of the product the time to haemostasis (TTH) is assessed. If TTH equals zero, this means that after <NUM> minute pressure haemostasis had already been achieved.

The haemostatic properties of the different reactive granulates was evaluated in the ex vivo and in vivo bleeding tests described above. The results are summarised in Table <NUM> and table <NUM>.

Using mechanical shaking, Gelita Tuft-It® patches (50x75 mm, appr. <NUM>) were impregnated with blue dyed NHS-POx/NU-POx (<NUM>:<NUM>) granulate. A paint shaking machine was used (VIBA PRO V of Collomix GmbH) to introduce the powder (appr. <NUM>) into the patch. The array with the carrier structures holder was clamped in the machine. The array was vibrated vertically.

The impregnated samples were put on a PMMA plate and placed in an oven in which samples were subjected to different heat treatments. To evaluate powder fixation, samples were ticked twice on the white PMMA plate. If no blue powder was released, the outcome was regarded as fixated. The results are shown in Table <NUM>.

The NHS-POx/NU-POx granulate is hygroscopic. At ambient temperature and relative humidity (RH) lower than <NUM>%, fibrous carrier structures can be impregnated within half an hour of exposure, reproducibly. However, if impregnation is performed at, for instance, RH <NUM>% and <NUM> the granulate gets sticky within minutes, leading to non-reproducible, inhomogeneous impregnation characteristics.

Hemostatic patches (Gelita Tuft-It®; <NUM> × <NUM>, appr. <NUM>) were impregnated with the reactive NHS-POx/NU-POx (<NUM>:<NUM>) granulate previously described. One gram of the granulate was distributed throughout the patches as described in Example <NUM>. Next, the hemostatic patches were packed in alu-alu pouches containing <NUM> of silica and vacuum sealed.

Patches were cut into <NUM> × <NUM> pieces and tested in triplicate in the ex vivo liver perfused model. Time to haemostasis (TTH) was <NUM> (after <NUM> minute pressure) and no re-bleeding was observed during the <NUM> minutes observation time. The patch was also found to have great flexibility and bending properties.

The patches were also evaluated in the in vivo porcine heparinized model. They were found to have very good coagulation and adhesive properties. Active bleedings were efficiently stopped in resections of various organs: spleen, liver and kidney. A summary of the results is shown in Table <NUM>.

Hemostatic patches (Gelita Tuft-It®; <NUM> × <NUM>, appr. <NUM>) were impregnated with NHS-POx/NU-POx/P188 <NUM>%. One gram of the granulate was distributed throughout the patches as described in Example <NUM>. Next, the hemostatic patches were packed in alu-alu pouches containing <NUM> of silica and vacuum sealed.

The patches were evaluated in the in vivo porcine heparinized model. They were found to have very good coagulation and sufficient adhesive properties - the reduced adhesive properties enabled the patch to be removed in one piece as opposed to identical patches that did not include P188. Active bleedings were efficiently stopped in resections of various organs: spleen, liver and kidney. A summary of the results is shown in Table <NUM>.

Gelita Tuft-It® (<NUM> × <NUM>, appr. <NUM>) was impregnated with different reactive polymer powders. The haemostatic properties of the patches so obtained were tested in the ex-vivo and in-vivo porcine bleeding models described herein before.

The fibrous carrier structures were impregnated with <NUM> of powder using a pneumatic shaking device. The sheet of fibrous carrier was vibrated vertically. The engine of the long stroke type (NTK <NUM> AL L, ex Netter Vibration GmbH) was operated at <NUM> bar, <NUM> and an amplitude of <NUM>. Four cycles of <NUM> seconds were used to disperse the powder into the sheet. The granulates were distributed through the complete thickness of the sheets. Also the distribution over the surface of the sheets was homogeneous.

Seven different reactive granulates were tested. These granulates contained NHS-POx in combination with a water-soluble nucleophilic polymer. The preparation of these granulates has been described herein before.

The granulates that were tested are listed below:.

The different combinations of fibrous carrier structure and reactive polymer powders that were tested are shown in Table <NUM>.

The results obtained with these patches in the ex-vivo and in-vivo porcine bleeding models are summarised in Table <NUM>.

<NUM> of NHS-POx were dispersed in <NUM> dichloromethane (DCM). A turbid mixture was formed and IPA (<NUM>) was added slowly to obtain a clear solution; <NUM> of pre-dried gelatin (Gelita-SPONO, Gelita Medical GmbH) were dispersed in DCM/IPA (<NUM>/<NUM>) using a high shear mixer operating at <NUM>,<NUM> rpm at room temperature for <NUM> minutes. The prepared NHS-POx solution was added and the dispersion was stirred at <NUM>,<NUM> rpm for <NUM> minutes. Subsequently, all volatiles were removed under reduced pressure. The formed granulate was milled using a coffee grinder, dried under reduced pressure and vacuum sealed in an alu-alu bag.

The granulate was analysed using <NUM>H-NMR spectroscopy and size exclusion chromatography. The results so obtained showed that no NHS-POx was present in the gelatin / NHS-POx granulate, indicating that complete crosslinking between NHS-POx and gelatin had occurred.

Experiments were conducted to determine the effect of NU-POx content of the reactive NHS-POx/NU-POx granulate on in-vivo performance of the haemostatic patch.

Hemostatic patches (Gelita Tuft-It®; <NUM> × <NUM>, appr. <NUM>) were impregnated with reactive NHS-POx / NU-POx granules made via acetone granulation in molar ratios of <NUM>:<NUM>, <NUM>:<NUM> and <NUM>:<NUM>, said molar ratio referring to the ratio of the number of NHS groups provided by NHS-POx to the number of amine groups provided by the NU-POx. The same hemostatic patches were also impregnated with reactive NHS-POx powder.

One gram of the granulate/powder was distributed throughout the patches using the Fibroline SL-Preg laboratory machine. Next, the hemostatic patches were fixated, dried and packed in alu-alu pouches containing <NUM> of silica and vacuum sealed.

The Fibroline SL-Preg laboratory machine moves particles between electrodes by applying voltages up to <NUM> kV at frequencies of up to <NUM> for a period of up to <NUM> seconds. The two electrode plates have a size of about 50x40 cm. The top plate is grounded.

The following standard settings were used: <NUM> kV, <NUM>, <NUM> seconds.

Powders were dosed gravimetrically into a 3D printed PMMA array after the array had been mounted onto the bottom electrode plate. The array was filled with reactive polymer powders using a scraping carton or metal spatula. The array measured <NUM> × <NUM> × <NUM> and contained 22x33 = <NUM> square wells (inner dimensions of each well: <NUM> × <NUM> × <NUM>). The combined volume of the <NUM> wells was approximately <NUM>.

A spacer mask was placed on top of the array. The spacer was used to allow particles to move up and down when subjected to the alternating electric field. If no spacer is used, penetration and distribution through the carrier is limited. For TUFT-IT this was a mask of <NUM>. This results in <NUM> + <NUM> = <NUM> distance of the electrodes.

The in vivo performance of haemostatic patches containing NHS-POx:NU-POx granulate (<NUM>, <NUM>, <NUM> and <NUM> percent amine groups from NU-POx, the percentage being calculated on the basis of the number of NHS groups provided by the NHS-POx) or NHS-POx powder was evaluated in a-non heparinised in-vivo porcine model. The details of the patches that were tested are shown in Table <NUM>.

Tests were carried out on adult female domestic pigs (<NUM>-<NUM>) No anticoagulation agent was applied. Patch performance was tested on both spleen and liver. The spleen or liver were located and externalized as needed as the testing period progressed and their natural humidity was kept by covering them with saline soaked sponges.

Different types of injuries were created:.

An appropriately sized section of the liver parenchyma was abraded/punched to cause moderate to severe bleeding. The liver abrasions were created by surgical scalpel and a template of <NUM> × <NUM> cm2 and the circular punches using a <NUM> circular biopsy punch. Liver and spleen resections were created using a surgical knife.

The patch was applied immediately after the tissue resection or scarification:.

The tested patches were applied on the bleeding tissue and gently pressed down by compression using a pre-wet gauze with saline solution. Tamponade was applied for an initial period of <NUM> seconds followed by subsequent <NUM> seconds intervals up to a total of <NUM> minutes.

A TUFT-IT patch that had not been impregnated was used as a reference (referred to as TUFT-IT).

The results of the in vivo tests are summarised in Table <NUM>.

Patches <NUM> to <NUM> showed very strong tissue adhesion, whereas only mild adhesion was observed for the TUFT-IT patch.

Patches <NUM> and <NUM> showed no more than very limited swelling after application. Patches <NUM> to <NUM> showed more, but still acceptable, swelling.

Hemostatic patches (Gelita Tuft-It®; <NUM> × <NUM>, appr. <NUM>) were impregnated with either a solution of NHS-POx, NHS-POx powder or NHS-POx / NU-POx granulate. The NHS-POx / NU-POx granulate used was made via acetone granulation in a molar ratio of <NUM>:<NUM> (see Example <NUM>).

A spraying solution containing NHS-POx was prepared by dissolving NHS-POx in a <NUM>:<NUM> mixture of isopropyl alcohol and dichloromethane (<NUM>/L). The patches were impregnated with <NUM> of this spraying solution using a glass laboratory sprayer and pressurized air in a single spraying cycle. The total amount of NHS-POx delivered in this way was <NUM> gram per patch. After impregnation the patches were allowed to dry inside an oven at <NUM> for <NUM> hours, following which they were stored in a desiccator for <NUM> days before being packing in alu-alu pouches containing <NUM> of silica and vacuum sealing.

In addition, patches were impregnated with <NUM> gram of NHS-POx powder or <NUM> gram of the NHS-POx / NU-POx granulate using the procedure described in Example <NUM>.

The performance of the patches so prepared was tested in triplicate in the ex vivo liver perfused model under mild (<<NUM>/min) and severe bleeding (><NUM>/min) conditions. With a flat, round, rotating abrasion tool a circular bleeding wound (<NUM> diameter) was created on the liver surface, with a rubber onlay so that the depth of the punched bleeding was always <NUM>. The results are shown in Table <NUM>.

Haemostatic powders were prepared by mixing NHS-POx / NU-POx granulate with a haemostatic starch powder (Arista™ AH, ex BARD). The NHS-POx / NU-POx granulate used was made via acetone granulation in a molar ratio of <NUM>:<NUM> (see Example <NUM>).

The NHS-POx / NU-POx granulate was mixed in a pestle and mortar with the starch powder in a <NUM>/<NUM> and a <NUM>/<NUM> w/w ratio.

The gel formation capacity of these powder blends, the pure starch powder and the pure NHS-POx / NU-POx granulate was evaluated as follows:.

Claim 1:
A biocompatible, flexible, haemostatic sheet comprising:
• a cohesive fibrous carrier structure comprising a three-dimensional interconnected interstitial space; and
• distributed within the interstitial space, a haemostatic powder comprising at least <NUM> wt.% of particle agglomerates, said particle agglomerates having a diameter in the range of <NUM>-<NUM> and comprising:
(a) electrophilic polyoxazoline particles containing electrophilic polyoxazoline carrying at least <NUM> reactive electrophilic groups that are capable of reacting with amine groups in blood under the formation of a covalent bond; and
(b) nucleophilic polymer particles containing a water-soluble nucleophilic polymer carrying at least <NUM> reactive nucleophilic groups that, in the presence of water, are capable of reacting with the reactive electrophilic groups of the electrophilic polyoxazoline under the formation of a covalent bond between the electrophilic polyoxazoline and the nucleophilic polymer.