Patent Application
20180243465
The present invention relates to hemostatic material and methods for producing and using such materials.
Uncontrolled bleeding is still the leading cause of mortality in traumatic and surgical injuries.[1] Developing effective therapeutic approaches to control bleeding is therefore of paramount clinical and social values. In the last decades, a number of hemostatic products[2] have been developed, including fibrin-based glue or sealants,[3 a,b] zeolite powders,[4 a,b] crosslinked gelatin matrix[5 a,b] and so forth. However, each of these products has its respective limitations. Fibrin products suffer from high cost, short shelf-life and weak mechanical strength.[6] Zeolite minerals are prone to cause severe burns and are not degradable.[6] Crosslinked gelatin matrix could halt bleeding within minutes only when combined with high doses of thrombin.[7] However thrombin is unstable in solution due to autoproteolysis. Highly concentrated thrombin is known to induce apoptosis of human keratinocytes and can cause impairments in wound healing.[8] Hence, there exists a strong need to design alternative hemostatic materials with improved safety. In particular, designing an effective strategy that avoids the use of highly concentrated thrombin is a desirable solution.
Thrombin is a serine protease that plays important roles in blood clotting (coagulation).[9 a,b] As the key coagulation protease, thrombin converts soluble fibrinogen into fibrin networks crosslinked by a transglutaminase (FXIII).[10] In addition, thrombin is the most potent activator of platelets by stimulating protease-activated receptors (PAR).[11, 12] Upon activation by thrombin, platelets physically alter the conformation of GP IIb/IIIa receptors and provide high-affinity binding sites for fibrinogen, providing fibrinogen-crosslinked platelet aggregation. Both PAR-1 and PAR-4 are present on human platelets, yet activation of human platelets by thrombin is primarily mediated by PAR-1.[13] The molecular mechanism of PAR-1 activation by thrombin is depicted in
WO 96/40033 A1 discloses a hemostatic material with hemostatic agents, including epsilon aminocaproic acid and a thrombin receptor activating peptide, wherein the hemostatic agents are sprayed or coated on a hemostatic matrix so as to obtain a matrix wherein the agent is physically (but not covalently) adsorbed on the matrix. Such patches have the drawback that the hemostatic agents provided therewith easily release from the patch when contacted to a bleeding area of a patient. Such there is the potential danger of inducing systemic thrombotic events, especially since there are no circulating antagonists in the circulation.
WO 03/057072 A2 discloses hemostatic compositions comprising cellulose and a polysaccharide covalently linked thereto.
It is an object of the present invention to provide improved hemostatic material with thrombin receptor activating agents for controlling bleeding.
Therefore, the present invention provides a hemostatic material, wherein a thrombin receptor activating agent is covalently coupled to a biocompatible matrix.
With the present invention it is shown for the first time that a thrombin receptor activating agent can be covalently immobilized on a biocompatible matrix so as to obtain an improved hemostatic material suitable for administration to human patients in need thereof. As a preferred embodiment, covalent coupling of TRAP6, TRAP7 or TRAP8 to a synthetic hydrogel matrix (e.g. a polyvinyl alcohol based polymer) resulted in a suitable hemostatic material, maintaining the activity for platelet activation in a safe, localized manner over a considerable period of time so as to enable an improved hemostasis, especially via an induced platelet aggregation.
The biocompatible matrix according to the present invention may be any matrix that is useable for being administered to human patients, especially for wound coverage or filling of volumetric defects (e.g. in organs) of a human patient. According to the present invention, it is preferred to use the matrix materials that have been suggested in the prior art for such purposes. In general, a “biocompatible” matrix is a matrix that may be administered to human patients and that does not induce a negative effect in the course of this administration and contact with the patient. A “biocompatible” matrix is a matrix that does not contain materials or components that threaten, poison, impede, or adversely affect living tissue (e.g. human tissue that is exposed to the surface in wounds). Examples for such matrices are “classical” wound coverages, such as patches, sponges, but also flowable or sprayable matrices, powders, etc. such as FloSeal™ (a cross-linked gelatin matrix), Surgiflo™ (a bovine gelatin paste). Whereas patches are advantageous for general wound coverages, non-material hemostats, most prominently flowable matrices, can be delivered within the same phase as opposed to liquid/solid approaches or can be used to flexibly fill cavities or provide a flexible scaffold (“volumetric defects”). The matrix should be chemically active or chemically activated so that the thrombin receptor activating agent can be covalently coupled to the matrix according to the present invention. For example, the matrix may have hydroxyl groups, vinyl groups, carboxyl groups, or amino groups to allow covalent attachment of the thrombin receptor activating agent to the matrix.
Preferably, the matrix is a hemostatic matrix, i.e. the matrix material as such has already hemostatic properties. Such materials are well available in the art and comprise e.g. collagen, gelatin or chitosan.
A preferred biocompatible matrix is selected from the group consisting of a biomaterial, preferably a protein, a biopolymer or a polysaccharide matrix, especially a collagen, gelatin, fibrin, starch or chitosan matrix; and a synthetic polymer, preferably a polyvinyl alcohol, polyethylene glycol, poly(N-isopropylacrylamide), etc.
Preferably, the matrix of the present invention is biodegradable, i.e. it is naturally absorbed by the patient's body after some time. In any way, the material (including the matrix) must be biocompatible, i.e. have no harming effect to the patient to whom the material is administered. Such biodegradable materials are specifically suitable in situations where hemostasis is achieved inside the body, i.e. in the course of surgery and the site is closed after surgery.
Accordingly, the matrix is preferably a biomaterial selected from biopolymers such as a protein, or a polysaccharide. Especially preferred is a biomaterial selected from the group consisting of collagen, gelatin, fibrin, a polysaccharide, e.g. hyaluronic acids, chitosan, and a derivative thereof, more preferred collagen and chitosan, especially preferred collagen. Such collagen matrix used for the present invention can be derived from any collagen suitable to form a gel, including a material from liquid, pasty, fibrous or powdery collagenous materials that can be processed to a porous or fibrous matrix as well as particles. The preparation of a collagen gel for the production of a sponge or sheet may include acidification until gel formation occurs and subsequent pH neutralisation. To improve gel forming capabilities or solubility the collagen may be (partially) hydrolyzed or modified, as long as the property to form a stable sponge or sheet when dried is not diminished. The matrix used for coupling the thrombin receptor activating agent can be a biopolymer, i.e., a naturally occurring polymer or a derivative thereof, or can be a synthetic polymer. Examples of biopolymers useful in a hemostatic material according to the present invention include polypeptides such as collagen, collagen derivatives such as gelatin, elastin, and elastin derivatives, and polysaccharides such as hyaluronic acids, starch, cellulose, or a derivative thereof, for example, oxidized cellulose. Preferably, the biopolymer is a human biopolymer, which can be isolated from an individual or can be a synthetic biopolymer, e.g., a recombinantly produced biopolymer.
In various embodiments, the matrix comprises a recombinant human polymer. In particular, the recombinant human polymer can be a recombinant human collagen, such as, for example, recombinant human collagen type I, recombinant human collagen type III, or a combination thereof. In one embodiment, the matrix comprises recombinant human collagen type III. In another embodiment, the matrix comprises recombinant human collagen type I. For example, the recombinant human gelatin can be derived from recombinant human collagen type III. In yet another embodiment, the matrix comprises recombinant gelatin derived from recombinant human collagen type I. In further embodiments, the matrix comprises recombinant gelatin produced directly by expression of encoding polynucleotide
The polysaccharide used as a matrix in the present invention is preferably selected from the group consisting of cellulose, alkyl cellulose, methylcellulose, alkylhydroxyalkyl cellulose, hydroxyalkyl cellulose, cellulose sulfate, salts of carboxymethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, chitin, carboxymethyl chitin, hyaluronic acid, salts of hyaluronic acid, alginate, alginic acid, propylene glycol alginate, glycogen, dextran, dextran sulfate, curdlan, pectin, pullulan, xanthan, chondroitin, chondroitin sulfates, carboxymethyl dextran, carboxymethyl chitosan, chitosan, heparin, heparin sulfate, heparan, heparan sulfate, dermatan sulfate, keratan sulfate, carrageenans, chitosan, starch, amylose, amylopectin, poly-N-glucosamine, polymannuronic acid, polyglucuronic acid, polyguluronic acid, derivatives of said polysaccharides, or combinations thereof.
The present matrix may also be based on a synthetic polymer. The synthetic absorbable polymer can be an aliphatic polyester polymer, an aliphatic polyester copolymer, or combinations thereof.
The present matrix may also be provided in the form of a woven or non-woven fabric made of fibers. Such fibers are preferably made of a biocompatible and/or biodegradable material. A number of such fibers have been used so far to provide hemostatic fabrics. In some embodiments, such nonwoven or woven fibers may comprise one or more polysaccharides such as pectin, acetylated pectin, hyaluronic acid and derivatives of thereof, and the like. In some embodiments, the pectin and/or acetylated pectin may be derived from sugar beets. In other embodiments, the polysaccharide may be a non-cellulosic polysaccharide. The woven or nonwoven fibers may also include fibers comprising other biodegradable polymers including polyglycolide, polylactide, poly(lactide-co-glycolide), poly(t-caprolactone), poly(dioxanone), polycaprolactone, poly(3-hydroxybutyric acid), poly(3-hydroxybutyric acid-co-3-hydroxy valeric acid), alginates, collagen, chitosan, gelatin, fibrinogen, elastin, polyethers, polyanhydrides, polyesters, polyorthoesters, polyphosphazenes, polyvinyl alcohol, polyvinylpyrrolidone, polytrimethylene carbonate, and the like. In addition, natural protein fibers such as cotton, silk and wool may also be used.
The matrix material according to the present invention may preferably be provided as granules of various morphologies, including powder or matrices for flowable hemostats. For example, the granules may have a (median particle) size of 1 to 1.000 μm, preferably from 10 to 1.000 μm, especially from 200 to 800 μm. Suitable matrices as flowable hemostats are disclosed e.g. in WO 98/08550 A or WO 2003/007845 A.
According to a specifically preferred embodiment, the present invention uses polyvinyl alcohol (PVA) as a matrix material. PVA is a water-soluble polymer originated from partial hydrolysis of polyvinyl acetate. PVA-based hydrogels used as a matrix according to the present invention have been widely used in tissue engineering and drug delivery systems because of their superior biocompatibility (FDA-approved).[16 a,b] In addition, PVA hydrogels are well known for being uniquely stronger than most other synthetic hydrogels.
The present invention uses thrombin receptor activating agents covalently bound to a hemostatically suitable matrix. Preferred embodiments of thrombin receptor activating agents are thrombin receptor activating peptides (TRAPs). TRAPs are a family of peptides of varying amino acid length which correspond to the new N-terminal region of the thrombin receptor. These synthetic or recombinant peptides mimic the activated form of the extracellular portion of the thrombin receptor protein and function as thrombin agonists.
U.S. Pat. No. 5,256,766 A and WO 96/40033 A1 describe pharmaceutical compounds and hemostatic patches containing TRAPs or “agonists” as useful to encourage blood clotting, for example, in localized application at internal bleeding sites of hemophiliacs. The agonists are disclosed as mimicking thrombin's ability to stimulate fibroblast proliferation and, concomitantly, platelet aggregation. TRAPs thus can be useful in promoting hemostasis and wound healing. With the use of TRAPs, an effective hemostatic material and hemostatic bandage can be provided which can be completely free of biological compounds such as thrombin and fibrinogen and the concomitant dangers of viral contamination.
Representative TRAPs which may be incorporated into a material according to the present invention include peptides capable of activating thrombin receptor, such as the agonists identified in the U.S. Pat. No. 5,256,766 A by the formula AAx--AAy--(AAi)n--z.
Other TRAPs which have been disclosed which activate fibroblasts and are implicated in wound healing include peptides TRAP 508-530, amino acids AGYKPDEGKRGDACEGDSGGPFV (SEQ ID NO:2); and TRAP 517-530, amino acids RGDACEGDSGGPFV (SEQ ID NO:3). Further suitable TRAPs are disclosed by Carney et al. J. Clin Invest. 89:14691477 (1992); Furman et al. PNAS 95 (1998), 3082-3087 and Cromack et al., J. Surg. Res. 53: 117 (1992). Accordingly, suitable TRAPs useful in the present invention, for example, include peptides SFLLRNPNDKYEPF (SEQ ID NO:4), SFLLRNPNDKYEP (SEQ ID NO:5), SFLLRNPNDKYE (SEQ ID NO:6), SFLLRNPNDKY (SEQ ID NO:7), SFLLRNPNDK (SEQ ID NO:8), SFLLRNPND (SEQ ID NO:9), SFLLRNPN (TRAP8 (SEQ ID NO:10)), SFLLRNP (TRAP7 (SEQ ID NO:11)), SFLLRN (TRAP6), SFLLR, SFLL, and SFL, and the amidated forms thereof. Because TRAPs are small peptides, they are more stable than large proteinaceous platelet activating agents, such as thrombin. The stability of TRAPs contributes to the properties of the present material which permit it to be stored without refrigeration.
Preferably, the thrombin receptor activating agents (preferably the TRAP) is provided with a linker so as to covalently couple the TRAP to the matrix. Preferred linkers are amino acids (single amino acids, such as Cysteine, Arginine, Lysine, Serine, Glycine, etc. (preferably Cysteine), or short amino acid linkers with e.g. 2 to 5 amino acid residues, preferably comprising amino acids selected from the group of Cysteine, Arginine, Lysine, Proline, Asparagine, Glutamine, Serine and Glycine. Preferred dipeptidic linkers may be Cys-Gly- (the terminal “-” indicates the bond to the thrombin receptor activating agent), -Gly-Cys, Cys-Arg-, -Arg-Cys, -Asn-Cys, Cys-Asn-, -Pro-Cys, Cys-Pro-; preferred tripeptidic linkers may be Cys-Gly-Gly-, -Gly-Gly-Cys, -Pro-Asn-Cys, Cys-Asn-Pro-, Cys-Pro-Asn-, -Asn-Pro-Cys etc., or any other peptidic linker comprising 2 to 5 amino acid residues known for pharmaceutical peptide coupling to carriers or matrices.
According to a specifically preferred embodiment, the thrombin receptor activating agent of the present material is a thrombin receptor activating peptide (TRAP), preferably TRAP8, TRAP7, TRAP6, TRAP1-41, SLIGKV (for PAR-2 (human) (SEQ ID NO:12)), TFRGAP (for PAR-3 (human) (SEQ ID NO:13)), GYPGQV (for PAR-4 (human) (SEQ ID NO:14)), or amidated forms thereof, as well as mixtures of such agents.
The hemostatic material according to the present invention may have any suitable form that is usable for the treatment of human patients in need of a hemostatic material, i.e. as a flowable or sprayable form; as a two-dimensional form (where the third dimension extension is comparably small (e.g. less than 1/10 or 1/20) compared to the other two dimensions; or as a three-dimensional form (e.g. a sponge, a paste, a cavity implant, etc.). A preferred two- or three-dimensional embodiment of the material according to the present invention may, for example, be a sponge, a woven or non-woven fabric, a preformed shape, preferably as a cylinder or cone (e.g. for tooth extraction) or as being used as a flexible or non-flexible scaffold, a particulate or granulate material or a sheet. It is specifically preferred if the matrix is able to absorb fluid from the wound so as to attract further blood coagulation components from the wound once the material is applied to the wound to achieve platelet aggregation. Furthermore, the material is preferably flexible and suitable to be applied on diverse tissues and locations with various shapes.
Further preferred embodiments of the hemostatic material according to the present invention comprise further ingredients, such as anti-bacterial agents, coagulatively active agents, immunosuppressive agents, anti-inflammatory agents, anti-fibrinolytic agents, such as aprotinin or ECEA, growth factors, vitamins, cells, etc. In a preferred embodiment, however, the material according to the present invention may or may not have such further ingredients, provided that the material is free of components which could have negative impact on the storability or administration of the hemostatic material. Accordingly, the present hemostatic material is preferably essentially free of any protein degrading activity, especially free of protease activity, specifically free of thrombin activity. Thrombin is added frequently to hemostatic materials in order to promote fibrin cleavage and clot formation; however, it may also be proteolytic to the hemostatic material, which may be unwanted especially during production and storage of such material. With the present invention, the addition or presence of thrombin or comparable components is not required and may therefore be omitted without negative impact on the hemostatic properties of the hemostatic material.
Preferably, the hemostatic material according to the present invention is provided in a state wherein it is able to soak up liquid material, such as blood. The ability to soak up blood (and the components therein promoting clot formation, bleeding termination and wound closure) significantly enhances the overall efficacy of the hemostatic material. For example, the hemostatic material according to the present invention is provided in a dry form or in a wet form still allowing the material to take up further liquid material (i.e. being soaked with liquid in an amount which amount is still under its soaking capacity). This allows blood entering and/or passing through the hemostatic material so as to provide the blood components useful e.g. in the wound closure process in an enlarged volume or in the whole (or virtually the whole) volume of the material applied.
According to a further aspect, the present invention relates to a method for producing the hemostatic material according to the present invention. This method for producing a hemostatic material is characterised by the step of covalently coupling a thrombin receptor activating agent to a hemostatic matrix.
Preferably, the thrombin receptor activating agent is a thrombin receptor activating peptide (TRAP), preferably TRAP8, TRAP7, TRAP6, TRAP1-41, SLIGKV (for PAR-2 (human)), TFRGAP (for PAR-3 (human)), GYPGQV (for PAR-4 (human)), or amidated forms thereof, as well as mixtures of these agents.
It is, of course, important for the present invention that the thrombin receptor activating agent keeps its thrombin receptor activating activity after covalent coupling to the biocompatible matrix. Not any biomolecule can be simply bound to a biocompatible surface and still retain its bio-functionality. In the course of generating the present invention, it turned out that the usage of conventional binding techniques to covalently couple C- or N-terminus of the thrombin receptor activating agents according to the present invention (e.g. TRAP6 peptides) usually results in the loss of said bioactivity. Accordingly, the provision of covalent immobilization approach that indeed retains full bio-functionality of the thrombin receptor activating agent (which is a peptide) is not trivial and needs to rely on the disclosure to obtain such functional embodiments contained herein.
For example, use of traditional coupling techniques for peptides, such as those using EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide; reaction of C-terminus, Asp and Glu) or NHS (N-hydroxysuccinimide; reaction of N-terminus, Lys), did not result in active immobilised thrombin receptor activating agent (because they are usually non-specific, prone to side reaction with acidic amino acids or lysine, deactivation of peptides that relies on N-terminus, etc.[29], [30], [31]). Techniques using cysteine and/or Michael addition also carry the risk of loss of function due to possible side reactions with amine groups[32]. Accordingly, such traditional coupling techniques result in loss of activity. While a variety of chemical approaches for peptide immobilization have been reported[34], most of these methods rely on reactions with carboxyl groups at the C-terminus (e.g. EDC)[29], or with primary amines at the N-terminus (e.g. NHS)[30], [31], or with cysteine residues via Michael-addition based on maleimide or vinyl sulfone groups[32]. Unfortunately, most reported reactions are non-specific and prone to side reactions, either with acidic/basic amino acids (for EDC/NHS) or with amine groups (for Michael addition), which induce unfavorable loss of peptide activity. For instance, N-hydroxysuccinimide (NHS) ester conjugation is the most often used approach to covalently immobilize bioactive peptides (e.g. cell-adhesive RGD) onto polymer substrates through reacting with the N-terminus of peptide[35]. Although primary amines are the most reactive groups for NHS esters, recent studies have demonstrated that a series of side reactions could occur with other peptide residues (e.g. —OH for tyrosine, serine, threonine, guanidinium for arginine)[30], [31]. Furthermore, previous studies on the structure-function relationship of TRAPs (SFLLRN-), highlighted the significance of the amino acids at the N-terminus to maintain peptide activity[36]. With these considerations in mind, it has been found that it is important for the present invention to develop a peptide immobilization approach that circumvents side reactions with the N-terminus of the thrombin receptor activating agent (being a peptide, such as TRAP6), in order to retain its bioactivity.
The present invention therefore provides a thrombin receptor activating active agent in immobilised form with retained activity. This turned out to be not feasible to be provided by conventional state-or-the-art approaches for peptide immobilization. The present invention therefore also provides a selection of specific coupling techniques (referred to above) with no risk of side reactions with N- or C-terminus or acidic or basic amino acids by using e.g. bioorthogonal reactions, such as photo-click conjugation of cysteine-containing TRAP6 onto PVA norbornenes, which offers a very high degree of conjugation efficiency (>95%), site-specificity and modularity. It is clear that the same conjugation approach is also applicable for other substrates bearing norbornene groups, such as naturally-derived molecules (gelatin, hyaluronan, alginate, etc.) and synthetic analogues such as PEG. In addition, the polymer-bound thrombin receptor activating active peptide conjugates have lower probability to be internalized by blood cells than soluble forms thereof through PAR-1 receptor signalling, as the size of polymer substrates applied as biocompatible solid matrix for the present invention, such as PVA substrates (hundreds of repeating units), is far larger than a short TRAP6 sequence. In all, the approach according to the present invention with coupling the thrombin receptor activating active agent with retained activity to a biocompatible solid matrix (e.g. by photo-click conjugation for TRAP6 peptide immobilization) provides significant practical values for local hemostasis as well as other medical applications.
According to a preferred embodiment of this method, the hemostatic matrix is functionalized with chemical groups (e.g., alkene, sulfhydryl, alkyne, azido, hydroazide, hydrazine), preferably groups that allow high-efficiency covalent binding of the thrombin receptor activating agent via bioorthogonal reactions (e.g., thiol-ene addition, alkyne-azide cycloaddition, Diels-Alder reaction, hydrazide-hydrazine reaction).
Preferably, the thrombin receptor activating agent is modified with peptide sequences that are engineered with bioorthogonal groups (e.g., alkene, sulfhydryl, alkyne, azido, hydroazide, hydrazine), especially with a cysteine moiety with an —SH group.
In a preferred embodiment of the method according to the present invention, the hemostatic material is functionalized with an ene group, such as norbornene, maleimide, allyl, vinyl ester, acrylate, vinyl carbonate, methacrylate, etc.
According to a specifically advantageous embodiment of the present method, the chemical coupling is performed by photo-induced reactions, especially by radical-mediated thiol-(norborn-)ene photo-click chemistry (reviewed in [18a]) or (other) photo-triggered click chemistry (reviewed in [18b]).
The present hemostatic material may be finished as a commercial product by the usual steps performed in the present field, for example by appropriate sterilisation and packaging steps. For example, the present material may be treated by UV/vis irradiation (200-500 nm), preferably with the help of photoinitiators with different absorption wavelengths (e.g. Irgacure 184, 2959), preferably water-soluble initiators (Irgacure 2959). Such irradiation is usually performed for an irradiation time of 1-60 min, but also longer irradiation times may be applied, depending on the specific method. The material according to the present invention may be finally sterile-wrapped so as to retain sterility until use and packaged (e.g. by the addition of specific product information leaflets) into suitable containers (boxes, etc.).
According to another aspect, the present invention also relates to the hemostatic material according to the present invention for use in surgery and/or in the treatment of injuries and/or wounds. The hemostatic material according to the present invention is specifically suitable and effective for increasing the release of platelet-derived growth factors and for accelerating wound healing.
This makes the hemostatic material according to the present invention an excellent tool for sealing of anastomosis, for suture line sealing and to safeguard hemostasis in resection sites.
According to another aspect of the present invention, the hemostatic material according to the present invention may also be provided in kit form combined with other components necessary for administration of the material to the patient. For example, if the hemostatic material may be provided in flowable dry form (e.g. as granules or as a powder) or as a flowable paste, it is preferred to provide such material with a suitable buffer solution which can be added shortly before administration to the patient. Such a buffer solution usually contains (besides the buffer components, such as phosphate, carbonate, TRIS, etc. buffer systems) divalent metal ions, preferably Ca2+ ions, or other functional components (if not already present on or in the matrix), such as anti-bacterial agents, coagulatively active agents, immunosuppressive agents, anti-inflammatory agents, anti-fibrinolytic agents, such as aprotinin or ECEA, growth factors, vitamins, cells, etc. The kit may further contain means for administering or preparing administering the hemostatic material, such as syringes, tubes, catheters, forceps, scissors, sterilising pads or lotions, etc.
Accordingly, the present invention relates to a kit, preferably for use in surgery and/or in the treatment of injuries and/or wounds, comprising
Preferably, the buffer solution further comprises a component selected from the group anti-bacterial agent, coagulatively active agent, immunosuppressive agent, anti-inflammatory agent, anti-fibrinolytic agent, especially aprotinin or ECEA, growth factor, vitamin, cell, or mixtures thereof. Alternatively, the kit may also further comprise a container with a component selected from the group anti-bacterial agent, coagulatively active agent, immunosuppressive agent, anti-inflammatory agent, anti-fibrinolytic agent, especially aprotinin or ECEA, growth factor, vitamin, cell, or mixtures thereof.
The present invention is further illustrated by the following examples and the figures, yet without being restricted thereto.
The present examples demonstrate the present invention by way of a water-soluble PVA-TRAP6 conjugate as model platelet-activating polymers as well as insoluble (crosslinked) PVA-TRAP6 hydrogel particulates (PVA-TRAP6-P) for safe and localized acceleration of hemostasis. In this work, it is demonstrated for the first time that TRAPs platelet-activating peptides, such as TRAP6, can be covalently immobilized in synthetic hydrogel matrices (
Several hemostatic strategies rely on the use of blood components such as fibrinogen and thrombin, which suffer from high cost and short shelf-life. In the present examples, a cost-effective synthetic biomaterial is developed for rapid local hemostasis. Instead of using thrombin, thrombin-receptor-agonist-peptide-6 (TRAP6) is covalently engineered in polyvinyl alcohol (PVA) hydrogels. Soluble PVA-TRAP6 was firstly prepared by covalent attachment of cysteine-containing TRAP6 onto the backbone of PVA-norbornenes (PVA-NB) through photo-conjugation. Cytotoxicity studies using C2C12 myoblasts indicated that PVA-NB and PVA-TRAP6 are nontoxic. Thromboelastography revealed that hemostatic activity of TRAP6 was retained in conjugated form, which was comparable to free TRAP6 solutions with equal concentrations. A 0.1% PVA-TRAP6 solution can shorten the clotting time (CT) to ˜45% of the physiological CT. High platelet-activating efficiency was further confirmed by platelet aggregation assay and FACS. For potential clinical applications, TRAP6-presenting hydrogel particulates (PVA-TRAP6-P) were developed for local platelet activation and hemostasis. PVA-TRAP6-P was prepared by biofunctionalization of photopolymerized PVA-NB hydrogel particulates (PVA-NB-P) with TRAP6. It was demonstrated that PVA-TRAP6-P can effectively shorten the CT to ˜50%. FACS showed that PVA-TRAP6-P can activate platelets to a comparable extent as soluble TRAP6 control. Altogether, PVA-TRAP6-P represents a promising class of biomaterials for safe hemostasis and wound healing.
All reagents were purchased from Sigma-Aldrich and used as received unless otherwise noted.
In a three-neck flask, 10 g of PVA (22 kDa) and 20 mg of p-toluenesulfonic acid were dissolved in 250 mL of anhydrous DMSO at 60° C. for 1 h under argon atmosphere. In a second flask, under argon atmosphere 2 g of cis-5-norbornene-endo-2,3-dicarboxylic anhydride (0.1 eq. to —OH groups) was dissolved in mL of anhydrous DMSO. The obtained solution was added dropwise into the first flask containing PVA. The reaction was maintained at 50° C. for 12 h. After reaction, the crude product was purified by dialysis against 10 mM NaHCO3 solution for 24 h and subsequently against deionized (DI) water for 12 h. After lyophilization, PVA-NB was obtained as colorless solid in 95% yield. 1H-NMR (D2O): δ (ppm): 6.2 (2H, s, —CH═CH—), 3.3 (2H, s, —C═CCH—CH—), 3.1 (2H, s, —C═C—CH—CH—), 1.3 (2H, s, —CH2-).
Degree of substitution (DS): 7.5%.
PVA-TRAP6 was prepared by covalent attachment of a cysteine-containing TRAP6 peptide (N-C: SFLLRNPNC (SEQ ID NO:15), China Peptide Co.) onto the backbone of PVA-NB through thiol-ene photo-click conjugation. Specifically, 60 mg of PVA-NB was dissolved in PBS solution of 0.5% Irgacure 2959 (I2959, BASF) to give a final macromer concentration of 5%. To this solution, 100 mg of TRAP6-Cys peptide (1.2 Eq. to NB groups) was added. The obtained solution was stirred under argon and irradiated with filtered UV-light (320-500 nm) for 300 s at 20 mW cm−2. The UV-light was guided from an Omnicure 52000 lamp.
PVA-NB (DS-7.5%) was dissolved in 0.5% 12959 solution, achieving a final concentration 10%. Then, aliquots of this solution was mixed with appropriate amount of dithiothreitol (DTT), providing —SH/-NB ratios as 0.4 (I), 0.8 (II), 1.0, and 1.2 (III), respectively. Hydrogel pellets were prepared by photopolymerization in a multi-well PDMS mold (well diameter: 6 mm). Specifically, 200 μL of macromer solutions were pipetted between two glass coverslips separated by the PDMS mold (thickness: 1.5 mm) and then exposed to filtered UV light (20 mW cm2) for 600 s. Pellets were detached from the slides and washed with sterile PBS.
Hydrogel precursor solutions (I-III) were prepared as aforementioned and photopolymerized at same conditions except using a 10 mL cylindrical glass vial as the mold. After photopolymerization, the hydrogel cylinders were transferred into a 100 mL beaker and washed with PBS (2 changes) for 12 h in order to remove unreacted polymer and PI. Afterward, the swollen hydrogels were frozen with liquid N2 and lyophilized. Finally, the dry PVA-NB matrix was grinded into fine powders using a RETSCH Cryomill RS232.
PVA-TRAP6-P was prepared by covalent attachment of TRAP6-Cys peptide onto the residual NB groups on PVA-NB-P. Specifically, 100 mg of PVA-NB was dispersed in PBS solution of 0.1% visible light PI (LAP)[19] to give a final polymer content of 5%. To this suspension, specific amounts of TRAP6-Cys peptide (1.2 Eq. to NB groups) were added. The obtained suspension was stirred under argon and irradiated with UV-light (365 nm) for 300 s at 20 mW cm−2. The UV-light was guided from an Omnicure LX400 LED lamp.
Photo-rheometry was performed on a modular photo-rheometer (Anton Paar MCR-302) as previously reported.[27] Specifically, MCR302 was integrated with filtered UV-light (320-500 nm) from a light guide (Omnicure 52000) to the bottom of the glass plate. Specifically, plate-to-plate oscillatory photo-rheometry was applied for real-time monitoring of the curing kinetics of hydrogel formulations during photopolymerization. Light intensity at the plate surface was ˜20 mW·cm−2 as determined by an Ocean Optics USB 2000+ spectrometer. Both storage moduli (G′) and loss moduli (G″) of the samples could be monitored as a function of irradiation time. Gel point was determined in the vicinity of the G′ and G″ crossover.
Mass swelling ratios of PVA-NB hydrogels were tested using a generic protocol.[28] Hydrogel pellets (n=3) were prepared as aforementioned and allowed to swell in DI H2O for 24 h at room temperature. The wet pellets were weighed to determine the equilibrium swollen mass (Ms) and then lyophilized to obtain the dry weight (Md). The equilibrium mass swelling ratio (Qm) was calculated as Ms/Md.
Cytotoxicities of PVA, PVA-NB and PVA-TRAP6 macromer solutions were evaluated via MTT assay. C2C12 cells were cultured in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 5% fetal calf serum (FCS), 1% L-Glutamine, 1% Penicillin/Streptomycin (all from Sigma-Aldrich, Austria). Macromer solutions with three concentrations (5%, 1% and 0.1%) were prepared in DMEM culture medium. C2C12 cells were then seeded in a 96-well plate at a density of 5×103 cells per well in 200 μL of culture medium. After 24 h incubation (37° C., 5% CO2), 100 μL of the respective macromer solutions were added to the cells in triplicates. After 24 h incubation, cells were washed twice with sterile PBS before the addition of 100 μL thiazolyl blue tetrazolium bromide (MTT) working solution (5 mg/mL in PBS). After 1 h incubation, the liquid was discarded and 100 μl of DMSO was added to dissolve the formazan crystals. Finally, the absorbance was measured at 540 nm using a microplate reader.
ROTEM (TEM Innovation, Germany) was applied to monitor the interactions of platelet-activating polymers and whole blood over time. ROTEM system contains an oscillating sensor pin that is immersed in a temperature-controlled cuvette containing the blood sample. Four measurements can be performed in parallel in the same device. Generally, coagulation of the citrated blood sample is initiated by re-calcification. The formation kinetics of a fibrin clot could be monitored mechanically and calculated by an integrated computer to the typical curves and numerical parameters.
Blood was collected from three un-medicated and healthy donors using minimal stasis from an antecubital vein through a 21-gauge needle. After discarding the first 3 ml, blood was collected in 3.5 ml tubes (Vacuette; Greiner Bio-One) containing 0.3 ml buffered 3.2% sodium citrate. The samples were kept on a pre-warming stage at 37° C. for at least 10 min prior to analysis and were processed within 3 h. ROTEM analysis of the whole blood sample was initiated by recalcification with the addition of 20 μl of CaCl2 (star-TEM®, 200 mM). Polymer solutions and/or polymer suspensions were added directly to the cuvette immediately after recalcification of the citrated blood and mixed by gently pipetting up and down as previously reported.[21] The final reaction volume per ROTEM cuvette was 370 μl, consisting of 300 μl citrated whole blood, 20 μl CaCl2 and 50 μl polymer solution or polymer suspension.
The following ROTEM parameters were calculated from the signal and included in the statistical analysis: clotting time (CT) in seconds (sec), i.e. the latency until the clot reaches a firmness of 2 mm, which is a measure of initial fibrin formation; clot formation time (CFT) in sec, i.e. the time from CT until the clot reaches a firmness of 20 mm, which indicates platelet function and fibrinogen quality; α-angle, the angle (°) between the x-axis and the tangent of the forming curve starting from CT point, which is comparable to CFT; maximum clot firmness (MCF) in mm, the maximum amplitude of the curve, which indicates the absolute strength of the clot; and A30 (mm), i.e. the clot firmness after 30 min.
The principle of Multiplate test is based on the fact that platelets become sticky upon activation, and thus prone to adhere and aggregate on the metal sensor wires in the Multiplate test cuvette. The sensor wires are made of highly conductive copper, which is silver-coated. As activated platelets adhere and aggregate on the sensor wires, the electrical resistance between the wires rises, which can be monitored in real time. For each measurement, 300 μL of saline and 300 μL of hirudinized whole blood was added sequentially to a Multiplate cuvette. After 3 min incubation at 37° C., 20 μL of sample solution was added and at the same time the program started to collect signals. The Multiplate device allows 4 measurements in parallel. Each measurement was performed for 6 min and in duplicate.
The blood of two unmedicated and healthy donors was collected and stabilized by sodium citrate. To 110 μL of whole blood (WB), 20 μL of PVA-TRAP6 solution (30 mg mL−1 and 3 mg mL−1 to obtain final TRAP6 concentrations in the WB of 2 mM and 0.2 mM) were added and incubated for 15 min at 37° C. Twenty microliter of TRAP6-C solution (14 mg mL−1) and/or 20 μL of NaCl solution (0.9%) were added as the positive control and the negative control, respectively. The incubated mixture was mixed with 300 μL of 1% paraformaldehyde for 15 min at room temperature. After washing and centrifugation a double immuno-staining was performed using 20 μL of phycoerythrin (PE) labeled monoclonal antibody directed against CD41 and 20 μL of allophycocyanin (APC) labeled monoclonal antibody directed against CD62P. Isotype controls were incubated with mouse IgG antibodies conjugated with APC or PE dye, respectively (all antibodies were purchased from BD Biosciences, Heidelberg, Germany). After 30 min incubation at room temperature the activation state of the platelets was determined using fluorescence flow cytometry. Expression of the platelet activation marker CD62P and the constitutively present platelet marker CD41 were detected using a Beckmann Coulter Cytomics FC-500 equipped with Uniphase Argon ion laser, 488 nm, 20 mW output. Overall 100 000 platelets were measured per sample and analyzed with the Cytomics CXP software. The experiment was repeated twice.
All error bars indicate the standard deviation. The statistical significance was determined by student's t-test, where ‘*’, ‘**’, ‘***’ indicate P<0.05, P<0.01, P<0.001, respectively.
In this study, a linear synthetic polymer polyvinyl alcohol (PVA) was selected as the substrate for covalent immobilization of the potent platelet-activating peptide (TRAP6) due to the following reasons. First, PVAs are FDA-approved polymers with superior cost-efficacy and cytocompatibility, therefore intriguing for many biomedical applications.[17] Second, the existence of a high number of hydroxyl groups in PVA offers significant freedom for tunable functionalization and presentation of bioactive ligands, which is not available with other synthetic substrates such as arm-dependent polyethylene glycol (PEG).
To introduce TRAP6 peptides onto PVA, we chose the robust thiol-ene photo-click chemistry as the conjugation approach. On one hand, norbornene group was selected as the ene functionality due to its ultrahigh reactivity towards thiol-ene reaction as well as low cytotoxicity.[18, 19] On the other hand, we engineered a cysteine moiety with a free thiol group into the C-terminus of a TRAP6 peptide sequence (SFLLRNPNC), since it is accepted that the N-terminus of TRAP6 sequence is critical for its ability to activate platelets.[15]
PVA-NB was synthesized through a facile esterification reaction between PVA and norbornene anhydride for 12 h at 50° C. in DMSO (
PVA-TRAP6 conjugates were prepared by photo-conjugation of cysteine-containing TRAP6 peptide with the NB groups of PVA-NB in PBS solution of 12959 as photoinitiator (PI). To confirm the conjugation efficiency, NMR model reactions were firstly performed in D2O. Based on the NMR reaction,
To prove the applicability of the prepared materials for biomedical applications, the in vitro biocompatibility of PVA, PVA-NB and PVA-TRAP6 solutions was investigated by MTT assay using C2C12 myoblasts. MTT assay showed that PVA and PVA-NB (
We next studied the hemostatic efficacy of PVA-TRAP6 in comparison with TRAP6 and PVA-NB using rotational thromboelastometry (ROTEM),[21, 22] which is a clinical diagnostic tool allowing in situ characterization of viscoelastic properties of blood clot during coagulation.
From the ROTEM results, it was observed that the CT of PVA-TRAP6 at 0.1 mM was very comparable to that of TRAP6 at 0.1 mM while the MCF of PVA-TRAP6 was relatively less than that of TRAP6 control. The lower MCF might be attributed to the decreased accessibility of TRAP6- to platelets after conjugation in PVA-TRAP6, which is a macromolecular conjugate (65 kDa) and significantly larger than TRAP6 (1 kDa). In comparison, the PVA-NB control (
To test whether the hemostatic activity of PVA-TRAP6 is dose-dependent, we tested PVA-TRAP6 solutions in comparison with TRAP6 solutions at three peptide concentrations (0.01, 0.1, 1 mM) in ROTEM (
In order to quantify the extent of platelet activation, we utilized Multiplate assay to investigate the influence of PVA-TRAP6, TRAP6 and PVA-NB on platelet aggregation. The principle of this method is based on the fact that platelets become sticky upon activation, and thus prone to adhere and aggregate on the metal sensor wires in the Multiplate test cuvette. As activated platelets adhere and aggregate on the sensor wires, the electrical resistance between the wires rises, which can be continuously monitored. A typical Multiplate curve (
We next utilized flow cytometry (FACS) to quantify the extent of platelet activation. Platelets can be distinguished from other blood cells by the constitutive expression of the surface antigen CD41, which recognizes the platelet membrane glycoprotein GpIIb which is non-covalently associated with GpIIIa (the integrin beta 3 chain) to form the GpIIb/IIIa complex. Importantly, the CD62p (P-selectin) membrane glycoprotein is exclusively expressed on activated platelets. The CD62p marker was used to identify the extent of activation in human platelets after incubation with the studied materials (PVA-TRAP6, TRAP6, PVA-NB, and NaCl). The measurement of the CD41/CD62p co-expression (
Since platelet-activating PVA-TRAP6 in soluble form or in releasable form, such as in WO 96/40033 A1 has the potential to cause thrombotic risks in the circulation, we further developed an insoluble PVA-TRAP6 system for localized hemostasis whereby TRAP6 peptide were covalently immobilized in photocrosslinked PVA hydrogel matrices. We selected radical-mediated thiol-NB photopolymerization as the approach to create PVA hydrogel matrices (
We utilized in situ photo-rheometry to test the photo-reactivity and mechanical properties of PVA hydrogels. It was hypothesized that the chemo-physical properties of PVA hydrogels could be easily adjusted by tuning the thiol to NB ratio. Four PVA-NB/DTT formulations with equal macromer content (10%) but varying thiol to NB ratios (0.4, 0.8, 1.0, 1.2) were screened in photo-rheometry (
We further analyzed the water-uptake properties of PVA hydrogels (I-IV). Photopolymerized PVA hydrogel pellets were soaked in PBS for 48 h to reach an equilibrium wet weight (mwet), which was compared to the polymer dry weight (mdry) after lyophilization and give the equilibrium mass swelling ratio (Qm). As shown in
In order to prepare appropriate hydrogel matrices for TRAP6-functionalization, photopolymerized PVA hydrogels (I, —SH:-NB=0.4) were sequentially lyophilized and cryo-milled into fine particulates (
We tested the hemostatic ability of PVA-TRAP6-P in comparison with PVA-NB-P in ROTEM. Prior to test, these particulates were carefully mixed with saline to form injectable slurries. As shown in
In order to quantify the ability of these particulated materials to activate platelets, we analyzed whole blood samples that were pre-incubated with PVA-TRAP6-P and/or PVA-NB-P in FACS. FACS analysis (
2.6. Comparison of Conventional Immobilization Techniques with Coupling Techniques Preserving Activity of the Peptidic Thrombin Receptor Activating Agent
To show the importance of diligently choosing the suitable immobilization technique, a comparison of conventional peptide immobilization methods (e.g. NHS conjugation) and methods which have been selected in the course of the present invention, such as the photo-click conjugation, was performed for the process of covalent binding of the TRAP6 peptide to polymer substrates. It turned out that conventional peptide immobilization methods lead to the loss of its bio-activity. This results in a lack of thrombin receptor activation and to no induction of blood coagulation, making the resultant materials not useful for local hemostasis.
To prepare polymer-TRAP6 via NHS conjugation, polyethylene glycol (10 kDa) with terminal NHS group (PEG-10k-NHS) was used as the polymer substrate. TRAP6 was linked to PEG-10k-NHS through reaction at the N-terminus, and unreacted NHS groups were blocked with glycine. Furthermore, PEG-10k-NHS reacted with excessive glycine was prepared as the negative control. After dialysis against distilled water and lyophilization, the conjugates were obtained in high yields and subsequently analyzed in standard rotational thromboelastometry (ROTEM) to study their effects on blood coagulation. In order to compare the bioactivity of polymer-TRAP6 conjugates prepared by different approaches, PVA-TRAP6 prepared by our claimed approach (i.e. photo-click conjugation) was included as the positive control. The samples consist of 1 mM TRAP6, 1 mM PEG-TRAP6, 1 mM PEG-Glycine, 1 mM PVA-TRAP6 and saline.
As shown in
To further demonstrate the ability of PVA-TRAP6 conjugates for platelet activation, we tested the samples in standard platelet aggregation assay (Multiplate) and flow cytometry (FACS). As shown in
In contrast to state-or-the-art approaches for peptide immobilization, the approach of the present invention is based on specific coupling techniques with no risk of side reactions with N- or C-terminus or acidic or basic amino acids by using e.g. bioorthogonal reactions, such as photo-click conjugation of cysteine-containing TRAP6 onto PVA norbornenes, which offers a very high degree of conjugation efficiency (>95%), site-specificity and modularity. The same conjugation approach is also applicable for other substrates bearing norbornene groups, such as naturally-derived molecules (gelatin, hyaluronan, alginate, etc.) and synthetic analogues such as PEG. In addition, the polymer-bound TRAP6 conjugates have lower probability to be internalized by blood cells than soluble TRAP6 peptide through PAR-1 receptor signaling, as the size of PVA substrates (hundreds of repeating units) is far larger than a short TRAP6 sequence. In all, the approach according to the present invention with coupling the thrombin receptor activating active agent with retained activity (e.g. by photo-click conjugation for TRAP6 peptide immobilization) provides significant practical values for local hemostasis as well as other medical applications.
In this work, we developed a synthetic hemostatic system that can efficiently activate platelets and accelerate hemostasis in a localized manner. The use of highly potent protease, thrombin, is avoided in this system. Instead, the thrombin receptor agonist peptide (TRAP) was covalently engineered on cytocompatible PVA hydrogels via highly efficient thiol-norbornene photo-conjugation. The presented TRAP6-functionalization approach is also applicable, but not restricted to other synthetic materials/hydrogels such as PEG as well as naturally-derived hydrogels such as gelatin and hyaluronic acid. From a biological point of view, activated platelets are known to release platelet-derived growth factors (PDNF), which regulate cell proliferation and play a significant role in blood vessel formation (angiogenesis). Therefore, we anticipate that these platelet-activating hydrogel matrices are versatile biomaterials not only for safe hemostasis but also for potential applications in tissue regeneration and wound healing.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15183295.3 | Sep 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/070619 | 9/1/2016 | WO | 00 |
