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    • 专利摘要:
    BIODEGRADABLE MICROSPHERE, MATERIAL FOR USE IN CONNECTION WITH THE TREATMENT OF WOUNDS UNDERSTANDING MICROSPHERES, WOUND DRESSING, METHOD FOR CARRYING OUT 5 HEMOSTASIS, METHOD FOR CARING FOR A WOUND, METHOD OF CULTURE AND CULTURE OF VASCULAR EMBOLIZATION. The present invention is intended for biodegradable microspheres having a diameter of 10 - 2,000 (Mi) m comprising hydrolyzed starch with cross links in which at least one type of linker has been coupled through a carboxylic ester link. The ligand must be an endogenous molecule, charged with a molecular mass of less than 1,000 Da comprising at least one additional carboxylic acid function in addition to that used for coupling the ligand to the microsphere and / or at least one amine function. On average, 0.05 - 1.5 binders are attached to each glucose fraction in the hydrolyzed starch.
    公开号:BR112012012997B1
    申请号:R112012012997-0
    申请日:2010-11-17
    公开日:2020-12-01
    发明作者:Malin Malmsjo；Eddie Thordarson；Sten Peter Apell；Peter Fyhr
    申请人:Magle Ab；
    IPC主号:
    专利说明:

    [0001] The present invention is aimed at biodegradable microspheres of hydrolyzed starch with endogenous, charged ligands attached thereto. The invention is also intended for a material comprising such microspheres, and for the use of microspheres or material in hemostasis, wound treatment, cell culture or vascular embolization. BACKGROUND OF THE INVENTION
    [0002] Starch, a branched glucose polymer (α 4 -glucose chains with α6 branches), is a natural material, found in plants and animals where it functions as an energy store. The polymer consists of amylose (long chain and low branch) and amylopectin (highly branched and short chain).
    [0003] Degradable starch microspheres (DSM) are formed by cross-linked starch chains. Degradable starch microspheres have been used for many years for temporary vascular occlusion with and without co-administration of cytotoxic drugs (treatment of tumors and prevention of bleeding), but they are also used for topical and intraoperative hemostasis.
    [0004] Starch microspheres are degraded in vivo by plasma amylase to oligosaccharides, maltose and eventually to glucose that enters normal metabolism.
    [0005] Starch or modified starch microparticles have been shown in the prior art, for example in US 6,060,461 and WO 2009/091549 for example for biocompatible hemostasis.
    [0006] In addition, US 3,812,252 is assigned to US 6,060,461 and WO 2009/091549 for example for biocompatible hemostasis.
    [0007] In addition, US 3,812,252 is intended for hydrolyzed starch and its use for treating wounds, including those that are chronic.
    [0008] Wound treatment is the intricate process in which the skin or other organ heals after injury. The classic model of wound care is divided into four sequential phases, although they overlap: (1) hemostatic, (2) inflammatory, (3) proliferative and (4) remodeling.
    [0009] Hemostasis is the first phase in the treatment of wounds, which stops the bleeding process. Within minutes after the injury to the skin or another organ, platelets (thrombocytes) are activated and aggregated at the injury site to form a fibrin clot.
    [0010] When endothelial injury occurs, endothelial cells stop inhibiting coagulation and begin to secrete coagulation factors that induce hemostasis after the injury. Hemostasis has three main phases: 1) vasoconstriction, 2) temporary blockage through a platelet plug, and 3) blood clotting by converting fibrinogen to fibrin and forming a clot that seals the orifice until the tissues are repaired .
    [0011] In the inflammatory phase, bacteria and debris are phagocytosed and removed, and factors are released that cause the migration and division of the cells involved in the proliferative phase.
    [0012] In about 2 - 3 days, fibroblasts begin to enter the wound site, marking the beginning of the proliferative phase even before the inflammatory phase has ended. This phase is characterized by angiogenesis, collagen deposition, formation of granulation tissue, epithelialization and wound contraction. In angiogenesis, new blood vessels are formed, necessary for the supply of oxygen and nutrients to the wound site in order to support the later stages of wound treatment. Simultaneously, fibroblasts begin to accumulate at the wound site, reaching their peak 1 to 2 weeks after the trauma. At the end of the first week, fibroblasts are the main cells in the wound.
    [0013] In the first 2 or 3 days after the injury, fibroblasts mainly proliferate and migrate, while later, these are the main cells that establish the collagen matrix at the wound site. Initially, fibroblasts use the fibrin crust formed in the inflammatory phase to migrate through it, adhering to fibronectin. Then, fibroblasts deposit fundamental substance in the wound bed, and later collagen, which they can adhere to for migration. The granulation tissue, which grows from the base of the wound, begins to appear in the wound already during the inflammatory phase, and continues to grow until the wound bed is covered. Granulation tissue consists of new blood vessels, fibroblasts, inflammatory cells, endothelial cells, myofibroblasts and the components of a new, temporary extracellular matrix. The re-epithelialization of the epidermis occurs when epithelial cells proliferate and "crawl" at the top of the wound bed, providing coverage for the underlying newly formed tissue.
    [0014] Cell culture is the process by which cells are grown under controlled conditions. Historical developments and methods of cell culture are closely interrelated with those of tissue and organ culture. Animal cell culture became a common laboratory technique in the mid-twentieth century, but the concept of keeping living cell lines separate from their original tissue source was discovered in the 19th century. Tissue culture is the growth of tissues and / or cells separated from the organism. This is typically facilitated by using a liquid, semi-solid or solid growth medium, such as broth or agar. In this specification cell culture and tissue culture will be used interchangeably.
    [0015] Some cells live naturally in suspension, without being attached to a surface, such as cells in the bloodstream. These cells can be grown in suspension. However, most cells derived from solid tissues are dependent on binding, called adherent cells. Adherent cells require a surface, such as tissue culture plastic or a microcarrier, to grow on it. Microcarriers are available for the growth of adherent cells, for example the dextran microspheres. When adherent cells are harvested or passed (subculture transport), the cells need to be disconnected from the surface on which they grew. This is usually done by adding a trypsin-EDTA mixture to the culture.
    [0016] Vascular embolization (occlusion) is used as an alternative to minimally invasive surgery. The purpose of embolization is to prevent blood flow to an area of the body, creating ischemia, which can effectively shrink a tumor or block an aneurysm.
    [0017] The procedure is performed as an endovascular procedure, by a radiologist specialized in an interventional site. It is common for most patients to have treatment performed with little or no sedation, although this depends largely on the organ to be embolized.
    [0018] Access to the organ is achieved by means of a guidewire and catheter (es). The artificial plunger used is generally one of the following methods: gel or hydrogel, particles, foam or puncture.
    [0019] The agents used in embolization therapy are, for example, liquid embolic agents that are able to flow through complex vascular structures. Examples of such are ethiodol, made from iodine and poppy seed oil which is a highly viscous agent and which is generally used for chemical embolization, especially for hepatomas; sclerosing agents, which will harden the endothelial lining of the vessels and ethanol.
    [0020] Particular embolic agents are also used to embolize precapillary arterioles or small arteries. Gelfoam® temporarily occludes the vessels for 5 weeks. Microspheres are agents commonly used for both mild and chemical embolization. Polyvinyl alcohol (PVA) and acrylic gelatin microspheres are not degradable in vivo, so they remain permanently in the patient. Depending on the situation, different sizes of microspheres are used, ranging from about 50 µm to about 1.2 mm in diameter. SUMMARY OF THE INVENTION
    [0021] In some cases it may be interesting to change the properties of the biodegradable starch microspheres. The present invention provides ways to alter the biodegradability of the biodegradable starch microspheres; the affinity of the biodegradable starch microspheres with biological systems and / or their components; the degree of swelling of the biodegradable starch microspheres; the swelling rate of the biodegradable starch microspheres; the compressibility / elasticity of the biodegradable starch microspheres and / or the selectivity of the chemical interaction with ions and molecules in and on the biodegradable starch microsphere. The biological system and / or its components described above can for example constitute an organ or cell or any of its components; bacteria; virus; proteins and enzymes; polysaccharides; lipids; small molecules and / or ions.
    [0022] Thus, the present invention is intended for a biodegradable microsphere having a diameter of 10 - 2,000 µm comprising hydrolyzed starch with cross-links in which at least one type of linker has been coupled through a carboxylic ester link, wherein said linker is an endogenous molecule, charged with a molecular mass of less than 1,000 Da comprising at least one additional carboxylic acid function and / or at least one amine function, and on average 0.05 - 1.5 ligands have been coupled each fraction of glucose in the hydrolyzed starch.
    [0023] The present invention is also intended for different uses and applications of this microsphere. DESCRIPTION OF THE INVENTION
    [0024] The microspheres according to the invention comprise cross-linked acid hydrolyzed starch. Microspheres can be made from acid hydrolyzed starch by emulsifying a solution of starch in an organic solvent, such as toluene or ethylene dichloride. The poly-glucose chains have cross-links with a cross-linked reagent such as epichlorohydrin, forming glycerol (1,3-oxy-propan-2-ol) ether bonds, as shown below, forming degradable starch microspheres (DSM) .
    [0025] DSMs are degraded in vivo through amylase to oligodextrins and, eventually, to glucose. The transverse bonds remain as oligosaccharides of varying size. The fate of these in vivo is currently unknown, but it is likely that they will either be excreted in the urine or be filtered out of the reticuloendothelial system and degraded.
    [0026] Microspheres are biodegradable, defined as a material that is degraded and / or metabolized and excreted under physiological conditions (in vivo). In this case, physiological (in vivo) comprises animals, more specifically, vertebrates and more specifically mammals.
    [0027] Essentially, the biodegradable starch microspheres are totally degraded and eliminated from their physiological environment, such as the human body. Depending on the application, the microspheres are adapted to be degraded in a certain period of time appropriate for their intended use. This time can vary from minutes to 3 months, more preferably up to 1 month.
    [0028] The size of the biodegradable microsphere according to the invention is on the micro scale, and more particularly from 10 µm to 2,000 µm.
    [0029] The properties of the DSM can be altered by connecting the binders with the DSM and more particularly with the hydroxyl groups of glucose. The properties of DSM are affected by the choice of binders and also by the number of binders connected with the starch.
    [0030] The ligands are connected to the DSM by their coupling through a carboxylic ester bond with the DSM glucose monomers. In order to allow the connection of the binders to the hydrolyzed starch through this ester bond, the binders must include at least one carboxylic acid function, that is, at least one -COOH group, capable of forming an ester bond. The ester bond is hydrolyzable, through chemical or enzymatic hydrolysis in vivo, and the use of such an ester bond results in a bio-removable liquid.
    [0031] In addition, the binders must be endogenous substances that are charged at a physiological pH, that is, at pH 6 - 8. In addition to the carboxylic acid function used to allow the ligand to bond with the hydrolyzed starch via an ester bond, the linkers should include at least one additional carboxylic acid function and / or at least one primary, secondary, tertiary or quaternary amine function. Since the ligands are endogenous compounds, DSM thus degrades into endogenous compounds that are metabolized and / or excreted.
    [0032] The ligand can thus be positively charged, negatively charged, or amphoteric ion, that is, positively and negatively charged at the same time. The binders can also have non-polar (hydrophobic) parts in order to also modify the properties of the DSM. It is also possible to use a mixture of different binders.
    [0033] The charged binders require a counterion. When the ligand is positively charged, the counter ion will be negatively charged and when the ligand is negatively charged, the counter ion will be positively charged. This counterion can be a physiologically active counterion.
    [0034] When the ligand is amphoteric ionic, it constitutes its own counterion.
    [0035] Endogenous ligands can also be small molecules with a molecular weight of less than 1,000 Da.
    [0036] For each glucose portion in the DSM, on average, 0.05 - 1.5 ligands according to the invention can be coupled. The binder's molar ratio to glucose is therefore 1.5: 1 to 1:20 in the DSM,
    [0037] The linker can be selected from the group consisting of amino acids, another nitrogen containing organic acids and dioic acids.
    [0038] The binders that may be preferred for some embodiments of the invention are listed in Table 1.
    [0039] Table 1 shows the preferred binders. R in the structures represents a glucopyranosyl monomer, shown below, in the hydrolyzed starch. R2 represents a linker, in any of its possible positions 2, 3 and / or 6, in the glucose fraction of the DSM as shown below.
    [0040] The microsphere described above can be used in hemostasis, in the treatment of wounds, in cell culture in vitro and in vascular embolization. The microsphere described above can also be used to produce a biodegradable material suitable for use in wound care.
    [0041] These different applications are discussed below. HEMOSTASIA
    [0042] In some modalities for use in hemostasis, the ligands coupled to the microspheres are preferably positively charged or amphoteric ions.
    [0043] In some embodiments, the binders coupled to the microspheres are preferably positively charged. The counterion used can then be ellagic acid.
    [0044] For hemostasis, the microspheres according to the invention should preferably have an average diameter of 10 µm to 200 µm.
    [0045] When used for hemostasis, the microspheres according to the invention can be added to / on the wound as a powder, in a solution or attached to a support structure, such as gauze. WOUND CARE
    [0046] For wound care, microspheres can be used to produce a material. This material should have a three-dimensional structure consisting of microspheres and voids between the microspheres.
    [0047] Due to the voids, the material will be permeable to both gases and liquids and, therefore, non-gelling when in contact with liquids.
    [0048] The fact that the material is a non-gelling material means that it is possible to avoid a film-forming layer when using the material on / over a wound, and thus it is possible to prevent edema from accumulating under the layer; facilitate the efficient transport of oxygen and nutrients and in addition the migration of unobstructed cells and the efficient transduction of pressure to or from the underlying tissue are permitted.
    [0049] The microspheres in the material can be a fraction of homogeneous size. In order to establish empty spaces between the microspheres, it is in many cases preferred that the microspheres in the material have a fairly uniform size. If the microspheres are to be non-uniform in size, the voids must be filled with smaller microspheres, thereby creating a more solid structure that will be detrimental to the intended effect of the material. When the microspheres are part of a homogeneous size fraction, the size of the microspheres should, at least for some modalities, not vary by more than ± 15% from the median. For example, in a fraction of microspheres of 300 μm, the individual microspheres can be from 255 to 345 μm. The size of the voids, that is, the space between the uniformly sized round spheres packaged together, can be calculated as ((2 / square root of 3) - 1) ≈ 0.155 times the diameter of the microspheres.
    [0050] The material can consist of a one-piece, solid, porous and three-dimensional network.
    [0051] The microspheres can be attached to a supporting substrate, thereby immobilizing the microspheres. Such support can be a normal gauze or a polymeric foam material.
    [0052] At least for some modalities for use in the treatment of wounds, the ligands coupled to the microspheres are preferably positively charged.
    [0053] At least for some modalities for use in the treatment of wounds, the binders coupled to the microspheres are preferably positively charged and hydrophobic.
    [0054] For the treatment of wounds, the microspheres according to the invention preferably have an average diameter of 200 μm to 2,000 μm.
    [0055] Preferably the voids in the material have a diameter of 30 μm to 3 00 μm, and more preferably 100 μm to 3 00 μm. The voids should be at least 30 μm, as this allows the passage of tissue cells and bundles of nerve cells that are typically 20 - 30 μm in diameter.
    [0056] In addition, the material's surface characteristics stimulate cell adhesion and proliferation. This involves the affinity of the cell with the surface of the material and a material elasticity that is appropriate for adhesion.
    [0057] The biodegradable material suitable for the treatment of wounds according to the invention especially highlights cell connection, migration and proliferation, both in the management of standard wound treatment and in the NPWT (Negative Pressure Wound Treatment) procedures. Negative) specifically for the third and fourth phases of the wound treatment process, namely the proliferation and remodeling phases.
    [0058] The three-dimensional structure of the biodegradable material suitable for the treatment of wounds according to the invention decreases the formation of scar tissue. Realizing that the scar tissue is characterized by a very unidirectional deposition of collagen, a matrix capable of forcing a disorganized collagen deposition is possible to reduce the scar. Collectively, the material according to the present invention permanently stimulates and facilitates the growth of the new and healthy granulation tissue.
    [0059] In the treatment of the wound, it may be advantageous to delay the biodegradability of the material by between 2 days and 2 weeks, selecting the appropriate binders (s). This allows for appropriate treatment without the need to change the dressing if it is not necessary for other reasons.
    [0060] When used in wound care or wound management, the material according to the invention can be added to / on the wound as a powder, in a solution, adhered to a support structure, such as gauze or a solid mesh. one piece.
    [0061] The material according to the invention can also be part of a wound dressing.
    [0062] It has been shown that when a 2 mm layer of non-gelling biodegradable starch spheres with an average diameter of 200 µm is applied, having a positively charged surface to a wound bed, very good granulation is obtained with cell growth up to 500 µm in four days. IN VITRO CELL CULTURE
    [0063] For use in cell culture in vitro, preferably the microspheres have an average diameter from 200 µm to 1,000 pm, more preferably between 200 pm and 500 µm.
    [0064] For some modalities for in vitro cell cultures, the ligands are preferably positively charged.
    [0065] The voids are important for cell cultures since they allow an effective passage for the adhesion and growth of cells and also allow an efficient transport of the growth matrix and larger molecules within the culture. VASCULAR EMBOLIZATION
    [0066] For vascular embolization, the microsphere according to the invention preferably has an average diameter from 10 pm to 1,200 µm.
    [0067] For use in vascular embolization, the ligands coupled to the microspheres are preferably negatively charged, at least in some embodiments.
    [0068] The negative charge can be used to ionically bind a cytostatic cationic drug, which then forms the counterion, for the treatment of tumors. Such cytostatic drugs include doxorubicin, irinotecan, topotecan, epirubicin, mitomycin, cisplatin and sorafenib.
    [0069] Microspheres according to any of the modalities of the invention as described above and as specified in the claims can be used in methods to improve, facilitate or carry out hemostasis, wound treatment and / or vascular embolization. Likewise, the material according to any of the embodiments of the invention as described above and as specified in the claims can be used in a method to facilitate or carry out wound treatment.
    [0070] The microspheres or the material are then respectively administered in an effective amount to a mammal, such as a human being, in need of hemostasis, wound treatment and / or vascular embolization. It may be a human suffering from a bleeding wound or some other type of wound, both internally and externally, such as on the skin.
    [0071] By "administration" is meant that the microspheres or the material according to the invention are placed in contact with the area where hemostasis, wound treatment and / or vascular embolization is required. In the case of a wound, for the purpose of hemostasis or wound treatment, the material can, for example, be placed in the wound cavity or on the wound surface. In the case of wound treatment, DSM can be formulated as a powder, suspension or ointment. In the case of hemostasis, DSM can be applied as a dry powder or incorporated into a gauze pad or pad. In the case of embolization, the DSMs are preferably suspended in an appropriate medium, such as saline.
    [0072] In this context, "effective amount" means an amount that will have a positive effect on hemostasis, wound treatment and / or vascular embolization.
    [0073] The microspheres according to the invention can also be used in methods to improve, facilitate or carry out cell culture in vitro. The microsphere according to the invention can then be added to an appropriate culture medium. The cells to be cultured are also added to this culture medium. Microspheres can be added to the culture medium simultaneously with the cells, before adding cells or after adding cells. The cells are then allowed to propagate. As explained above, cell culture in this specification also includes tissue culture.
    [0074] Microspheres according to any of the modalities of the invention as described above and as specified in the claims can also be used to improve, facilitate or perform hemostasis, wound treatment and / or vascular embolization.
    [0075] Microspheres according to any of the embodiments of the invention as described above and as specified in the claims can also be used for the production of a medical device or a pharmaceutical composition.
    [0076] Microspheres according to any of the embodiments of the invention as described above and as specified in the claims can also be manufactured specifically for use to improve, facilitate or perform hemostasis, wound treatment and / or vascular embolization.
    [0077] Throughout the description and claims, the words "comprises" and "contains" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and are not intended to exclude other fractions , additives, components, integers or phases.
    [0078] Throughout the description and claims of the present specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood as covering plurality as well as singularity, unless the context requires otherwise.
    [0079] Functions, integers, characteristics, compounds, chemical fractions or groups described together with a particular aspect, the modality or example of the invention should be understood as being applicable to any other aspect, modality or example described in this document unless incompatible with him. BRIEF DESCRIPTION OF THE DRAWINGS
    [0080] The invention is described in greater detail below in the Examples, which refer to the attached drawings in which; Fig. 1 is a schematic picture of a degradable starch microsphere (DSM) and the chemical modifications carried out in this study. Fig. 2 illustrates that the swelling of the microspheres can be assumed to follow Fick's diffusion with a rapid initial swelling rate that decreases exponentially: y = yco (1 - e-kt) where k = the first order swelling constant and Y = the increase in volume at maximum swelling. Fig. 3 illustrates platelet adhesion. Fig. 3A shows phase contrast and fluorescent micrographs showing the DSM and the platelets adhered to the DSM according to the different batches of modification. Fig. 3B shows approximations of the junction between two aggregated DSMs (lot 4) and the aggregated platelets coupled with the DSMs. Image created using the differential interference contrast microscope (DIC), Fig. 4 illustrates an in vivo study of the three DSM batches. Lots 5, 6 and 9 were evaluated in an experimental bleeding model (renal trauma) in anticoagulated rats. All animals treated with batch 9 obtained primary hemostasis, 29% bleed again within 20 min of observation. The other batches showed significantly less hemostatic efficiency with few animals reaching primary hemostasis. Fig. 5 illustrates a blood loss according to treatment batch in the experimental in vivo study. Blood loss was measured by weighing the excess blood collected on the gauze. There was a significant difference in blood loss between the different lots (p = 0.001), where lot 5 was DSM without changes, lot 6 proved activation of coagulation and DSM in lot 9 adsorbed platelets. EXAMPLES
    [0081] The degradable starch microspheres (DSM) were prepared by emulsion, crosslinking the hydrolyzed starch with epichlorohydrin in toluene. The DSMs are subsequently repeatedly washed with ethanol followed by distilled water and finally successively dehydrated with increased concentrations of ethanol and finally dried overnight at 60 ° C. DETAILS IN PREPARING DSM
    [0082] 2 g of sodium hydroxide are dissolved in 280 ml of purified water and 2 g of sodium borohydride are added and dissolved. 153 g of hydrolyzed starch are dissolved by slowly stirring for at least 2 hours. 20 g of surfactant (Rhodafac PA17) are dissolved in 450 g of toluene. The starch solution is then added and emulsified in the toluene solution, the temperature is increased to 70 ° C and the emulsion is stirred until the desired drop size distribution is obtained. 22 g of epichlorohydrin are added and cross-linking is carried out for 5 hours. The mixture is cooled to room temperature and allowed to settle so that the supernatant is then decanted. The DSM are washed three times with 95% ethanol, one wash with 0.8% acetic acid, followed by 4 washes with purified water and finally dehydrated with absolute ethanol before drying at 60 ° C in a drying oven ventilated. DETERMINATION OF THE DEGREE OF SUBSTITUTION (DS)
    [0083] The degree of substitution is defined as the average number of substitutes per glucose monomer.
    [0084] The alkaline saponification method, followed by titration of excess alkalis, was used to determine the degree of substitution. For a 250 mg sample of DSM, 10 mL of 0.50 M NaOH was added and allowed to stand at room temperature for 72 h with occasional stirring. The excess NaOH was titrated with 0.50 M HCl using phenolphthalein as an indicator. DETERMINATION OF DEGRADABILITY WITH AMYLASE
    [0085] A DSM sample (3 - 6 mg) was diluted with phosphate buffer, pH 7 (5 ml) and then 400 μΐ of human saliva was added, followed by incubation at 37 ° C for 4 h. The sample was allowed to stand for 20 minutes or was centrifuged, and then a small sample was taken from the bottom and analyzed under a microscope to determine the presence or absence of microspheres. GENERAL PROCEDURE FOR REPLACING DSM WITH DIOIC ACIDS (EXAMPLES LISTED IN TABLE 1)
    [0086] DSM (1 g) was suspended in DMF (10 ml), succinic anhydride (154 mg, 1.54 mmol) and pyridine (124 μΐ, 1.60 mmol) were added to this mixture. The mixture was stirred and heated to 90 ° C overnight, and then the material was washed three times with 40 ml of ethanol followed with 5 ml of saturated NaHC03 and then three times with 30 ml of water. The material was dehydrated with ethanol and dried in an oven at 60 ° C. The material was subsequently analyzed with FTIR showing carbonyl ester at 1,730 cm-1. DS: 0.25 (determined as described above).
    [0087] Degradable by a-amylase (determined as described above). GENERAL PROCEDURE FOR REPLACING DSM BY ESTERS Betaine modification
    [0088] Betaine (1.66 g, 10.8 mmol) and CDI (1.75 g, 10.8 mmol) were mixed with 50 ml of DMF and heated to 80 ° C for 2 h. Then, the DSM (5 g) was added and the temperature was raised to 90 ° C and the mixture was stirred overnight. The mixture was washed twice with ethanol (250 ml), diluted with hydrogen chloride (250 ml) and twice with water (250 ml). The material was dehydrated with ethanol and dried overnight at 60 ° C. FTIR showing carbonyl ester at 1,751 cm-1. DS: 0.23 (determined as described above).
    [0089] Α-amylase degradable (determined as described above). Modification with dimethylglycine
    [0090] As in the example with betaine above, but DSM (2 g), N hydrochloride, N-dimethylglycine (430 mg, 3.1 mmol) and CDI (500 mg, 3.1 mmol) were used. FTIR showing ester carbonyl at 1,753 cm-1. DS: 0.24 (determined as described above). Α-amylase degradable (determined as described above). Modification with Na-acetyl-L-arginine
    [0091] As in the example with betaine above, but DSM (2 g), Na-acetyl-L-arginine (623 mg, 2.5 mmol), CDI (400 mg, 2.5 mmol) were used. FTIR showing ester carbonyl at 1,748 cm-1. DS: 0.24 (determined as described above). Α-amylase degradable (determined as described above), Modification with proline
    [0092] As in the example with betaine above, but DSM (1 g), Boc-Pro-OH (266 mg, 1.2 mmol), CDI (200 mg) were used followed by deprotection of tert-butoxycarbonyl with TFA. FTIR showing ester carbonyl at 1,743 cm-1. Degradable by α-amylase (determined as described above). Modification with glycine
    [0093] As in the example with betaine above, but DSM (1 g), Boc-Gly-OH (216 mg, 1.2 mmol), CDI (200 mg) were used followed by deprotection of tert-butoxycarbonyl with TFA. FTIR showing ester carbonyl at 1,748 cm-1. Degradable by α-amylase (determined as described above). Modification with phenylalanine
    [0094] As in the example with betaine above, but DSM (1 g), Boc-Phe-OH (327 mg, 1.2 mmol), CDI (200 mg) were used followed by deprotection of tert-butoxycarbonyl with TFA. FTIR showing ester carbonyl at 1,743 cm-1. Degradable by α-amylase (determined as described above). NON-DISCONNECTABLE SURFACE MODIFICATIONS USED IN THE INVESTIGATION OF LOAD EFFECTS
    [0095] The surface changes are illustrated in Fig. 1. Octenyl succinate (negative and hydrophobic)
    [0096] 80 g of DSM were suspended in purified water, N-octenyl succinic anhydride (Pentagon) was added to 0.08 g / g of dry DSM and the reaction was maintained for 3 h. A pH above 7.4 was maintained by adding 0.75 M NaOH. The resulting material was washed 8 times with 2,000 ml of purified water and then dehydrated with increasing concentrations of ethanol and finally dried overnight at 60 ° C (Hui Rea. Preparation and properties of octenyl succinic anhydride modified potato starch. Food Chemistry 2009; 114: 81-6). Carboxymethylation (negative)
    [0097] 50 g of DSM were suspended in purified water; chloroacetic acid was added to 0.1 g / g dry DSM and the reaction was maintained for 5 h at 70 ° C. Before adding chloroacetic acid, it was dissolved in water and neutralized with 1 M NaOH. The resulting material was washed 6 times with 2,000 ml of purified water and then dehydrated with increasing concentrations of ethanol and finally dried overnight at 60 ° C (Tomaski P, Schilling, CH Chemical modification of starch. Adv Carbohydr Chem Brochem 2004; 59: 175-403). Acetylation (hydrophobic)
    [0098] 50 g of DSM were suspended in purified water, acetic anhydride was added to 0.05 g / g of dry DSM. Acetic anhydride was added dropwise and a pH between 7.3 and 7.8 was maintained through the addition of 0.75 M NaOH. The resulting material was washed 7 times with 2,000 ml of purified water and then dehydrated with increasing concentrations of ethanol and finally dried overnight at 60 ° C (Sathe SK, Salunkhe, DK Isolation, Partial Characterization and Modification of the Great Northern Bean (Phaseolus vulgaris L.) Starch, J Food Sci 1981; 46: 617-21). Diethylaminoethyl chloride, Aldrich (positive)
    [0099] 50 g of DSM were suspended in purified water, 0.375 mol of DEAE hydrochloride was added and the temperature was increased to 60 ° C. 250 ml of 3 M sodium hydroxide solutions were added and the reaction was maintained at 60 ° C for one hour. The DSM were then washed with 20 L of purified water in a Büchner funnel. The DSM were then dehydrated and dried as above (Manousos M, Ahmed M, Torchio C, Wolff J, Shibley G, Stephens R, and others. Feasibility studies on oncornavirus production in microcarrier cultures. In Vitro 1980 Jun; 16 ( 6): 507-15). Ellagic acid (negative adsorbed / absorbed)
    [0100] Ellagic acid (Alfa Aesar) was passively adsorbed using two different methods. Method 1: 0.1 mM ellagic acid was dissolved in water and then mixed with the DSM. Method 2: 0.1 mM ellagic acid was dissolved in ethanol and then mixed with DSM (Ratnoff OD, Saito H. Interactions among Hageman factor, prekallikrein plasma, high molecular weight kininogen, and previous thromboplastin plasma. Proc Natl Acad Sci USA 1979 Feb; 76 (2): 958-61). Washing and drying as above. El ellagic acid was adsorbed / absorbed passively and was not applicable for the measurement of loads.
    [0101] The different surface modifications were produced with standard modification protocols (not optimized). The modifications were selected to prove the concept of a hemostatic effect in vitro and in vivo and have not been evaluated because they are toxicologically acceptable in humans, SURFACE LOAD
    [0102] The degree of surface charge was measured by a PCD 02, Particle Charge Detector (Mütek). DRAWING
    [0103] The nine different modified DSMs were random and blind. No information about the modifications was sent to the authors of the studies. CHARACTERIZATION OF DSM
    [0104] The morphology of the starch microspheres was determined by observation under the microscope (AxioObserver Zl, Zeiss), and the diameters of the sphere were measured for a minimum of five spheres in each of the nine batches. Absorption was determined by measuring the diameter before and at fixed time intervals (1, 3, 9, 15 and 30 s) after the addition of 100 pL and phosphate buffer. A minimum of five spheres from each batch was measured and then their volume was calculated, assuming that the DSM were completely spherical. The swelling of the microspheres occurs by diffusion of water in and hydration of the polymer, a process that continues towards the balance in the maximum relaxation of the starch chains with cross links. Thus, it can be assumed that the process follows Fick's diffusion with a rapid initial swelling rate that decreases exponentially. In this way, the data can be explained by: y = YCo (1 - e-kc) where k is the first order swelling constant and Y ~ is the swelling at maximum swelling. ADHESION OF PLATELETS IN VITRO
    [0105] To study the possible affinity / interaction between the various DSM batches and the factors of known importance for the coagulation process, platelet adhesion for the different DSM batches was investigated. 45 0 μl of heparinized platelet-rich plasma was added to test tubes containing 1 µg of DSM and subsequently shaken on an orbital shaker for 20 minutes at 500 rpm. After that, the DSM was completely washed in PBS, repeatedly allowing the DSM to settle at the bottom and changing the supernatant with fresh PBS and, subsequently, the vortex tube. The adhered DSM platelets were then fixed with 3.7% PFA in PBS and permeabilized using 0.1% Triton-X in PBS, and finally fluorescently labeled with Alexa 546-Phalloidin. Complete washing was carried out between each step of the procedure. The DSM and fluorescent platelet images were obtained with an AxioObserver Z1 fluorescence microscope (Zeiss) and AxioVision imaging software (Zeiss). IN VIVO PILOT STUDY IN AN EXPERIMENTAL MODEL OF RENAL BLEEDING
    [0106] The study was carried out according to the guidelines of good laboratory practices and approved by the Local University Ethics Committee for Animal Experiments. Three different batches of DSM were chosen based on the results of the in vitro studies described above. For the in vivo assay, a neutral lot was chosen, one that activated coagulation and finally a lot with platelet adhesion properties. The batches were blind and randomized to the investigator who conducted the study. Twenty-one adult male Sprauge-Dawley rats were anesthetized (medium weight 342 g, iqr: 314 - 360) with free food and water supply (Hynorm, Janssen Pharma, Belgium and Midazolam Hameln, Pharma Hameln, GmbH). A transverse laparotomy was performed after catheterization of the jugular vein (by IV injections). The left kidney was dissected and the renal vessels were stapled for two minutes after IV administration of unfractionated Heparin (UH, LEO Pharma A / S, Denmark) 200 IU / kg. A lateral third of the kidney was then removed and 1 mL of random DSM was applied to the surface of the raw kidney, manual compaction started (with a gauze pad between the researchers' finger and the starch powder) and the clamp of the vessel was removed . The compression was maintained for 2 minutes and then released to control hemostasis. If bleeding occurred, compression was continued with hemostatic controls every minute. Primary hemostasis was defined as no visible bleeding within 20 minutes of renal resection. The animals that obtained hemostasis were observed for another 20 minutes for possible re-bleeding. All animals were sacrificed with an IV injection of phenobarbituric acid and ethanol. The blood loss was collected and weighed. The end points of the study were: the ability to obtain primary hemostasis, the time of hemostasis, the frequency of bleeding and blood loss. STATISTICS
    [0107] Descriptive data are presented with median and individual values or interquartile range (iqr). Non-parametric tests were performed, since the data distribution was skewed. Χ2 tests were performed for contingency tables and Kruskal-Wallis analysis of variance was used when unpaired data were compared. A value of p <0.05 was considered significant.
    [0108] The SPSS 17.0 software for Mac and Windows (www.spss.com) was used. RESULTS DSM modifications
    [0109] The surface loads are given in Table 2. The synthetic procedure has not been optimized and the carboxymethylation does not result in an appreciable surface load. It is not expected that acetylation will alter the surface charge where other methods should lead to significant positive and negative surface charges. TABLE 2 The chemical modifications of the DSM and the result in the measured loads.
    [0110] There was a significant difference in dry diameter between batches (p = 0.006), with batch 6 having the smallest spheres (average diameter of 54 pm, iqr: 38 - 58) and batch 2 with the largest size (average diameter of 72 pm , iqr: 67 - 76). After the addition of phosphate buffer all batches rapidly increased in volume (Fig. 2) and after 30 s they had expanded by between 5 and 25 times their dry volume (Table 3). The amount of swelling was significantly different between batches (p = 0.001). TABLE 3 DSM dry volume and after 30 seconds in phosphate buffer solution.
    [0111] There was an evident adhesion of platelets to the DSM in three of the modified lots (No. 4, 7 and 9), in which the rest of the DSM lots did not affect the platelets in any way (Fig. 3). The results were confirmed using PRP from three different donors. The blind randomized in vivo pilot study
    [0112] All animals treated with batch 9 obtained primary hemostasis, compared with 14 - 43% of primary hemostasis with other batches (Fig. 4). The time for hemostasis also differed between groups (p = 0.044), animals treated with lot 9 were the fastest to stop bleeding (median 2 min: iqr: 2-3: 20) while lot 6 required a median of 6 min (n = 3) and batch 5 of 10 min (n = 1). Two animals treated with lot 9 were the only ones to bleed again (p = NS, compared to the other lots). Animals treated with lot 9 had less blood loss (median 1 g, iqr: 0.4 - 1.2) when compared to the other lots (lot 5: 5 g, 4.3 - 6.7, lot 6: 5.3 g, 2.2 -8.6), p = 0.001 (Fig. 5).
    [0113] The postulated hemostatic effect of DSM by absorbing fluid (and small molecules) from the blood and endogenous coagulation factors concentrating in the spheres, may be dependent on rapid and considerable swelling of the microspheres. All batches in this study rapidly increased in volume after the addition of phosphate buffer, but both the speed and the total amount of swelling differed between batches. The swelling depends on the relaxation of the poly-glucose chains as they are hydrated. This is restricted by many cross-links and facilitated by the repulsion of the binder load. However, we were unable to find any clear correlations with the measurement characteristics (for example, load). Low transverse connection and high and rapid swelling imply rapid degradation and, therefore, the increase in volume will not be dangerous even if applied intraoperatively in places where space may become limited at the end of the procedure. In this study, the rapid absorption of fluids and swelling of the DSM was not sufficient for hemostasis in vivo, only 1 of the 7 animals treated with unmodified microspheres obtained primary hemostasis.
    [0114] DSM with superior hemostatic capacity in vivo proved to be those with platelet-stimulating properties. Platelets adhered to positively charged DSM, batches prepared with diethylaminoethyl (DEAE) (4, 7 and 9), which are in accordance with the reported platelet adhesion to surfaces exposing positively charged groups (Lee JH, Khang G, Lee JW, Lee HB. Platelet adhesion onto chargeable functional group gradient surfaces. J Biomed Mater Res 1998 May; 40 (2): 180-6). There was no objective quantification of the amount of platelets that adhered to the respective batch of modified DEAE, but by visual assessment there was no obvious difference in the amount of platelets that adhered between batches 4, 7 and 9, even if there was a measured difference in load between lots 4 and 9. DEAE chloride reacts with hydroxyl groups on the surface of the DSM, generating DEAE groups that are positive at physical pH. DEAE binders have generated microspheres that are not biodegradable and are probably unsuitable for human use. However, as a proof of concept in order to distinguish whether the spheres can become platelet adherent and whether this has any clinical hemostatic significance, the DEAE modification was valuable. Rapid and efficient platelet stimulation is crucial for instant hemostasis produced by a physical attachment of aggregated platelets. Platelets are also required for efficient amplification and for the propagation of thrombin generation, a process strongly catalyzed by the stimulated platelet surface, resulting in a fibrin network that stabilizes the primary platelet bond.
    权利要求:
    Claims (9)
    [0001]
    BIODEGRADABLE MICROSPHERE, having a diameter of 10 - 2,000 μm, characterized by comprising crosslinked hydrolyzed starch, in which at least one type of ligand selected from the group consisting of amino acids and nitrogen containing organic acids was coupled through a carboxylic ester bond, in that said ligand is a positively charged endogenous molecule, with a molecular mass of less than 1,000 Da comprising at least one additional carboxylic acid function and / or at least one amine function, and on which 0.05 - 1.5 binders for each fraction of glucose in the hydrolyzed starch.
    [0002]
    MICROSPHERE according to claim 1, characterized in that said ligand is an amino acid selected from the group consisting of arginine, histidine, lysine, glycine, proline, alanine, isoleucine, leucine, phenylalanine, tryptophan, tyrosine, valine, serine, asparagine, glutamine, threonine, glutamic acid and aspartic acid; or nitrogen containing organic acid selected from the group consisting of betaine, carnitine, creatine, methylglycine and dimethylglycine
    [0003]
    MICROSPHERE according to either of claims 1 or 2, characterized in that the ligand has a physiologically active counterion.
    [0004]
    MICROSPHERE according to any one of claims 1 to 3, characterized in that it is for use in hemostasis.
    [0005]
    MATERIAL FOR USE IN CONNECTION WITH THE TREATMENT OF WOUNDS UNDERSTANDING MICROSPHERES, as defined in claims 1 to 3, characterized by said microspheres forming a three-dimensional structure comprising voids between the microspheres.
    [0006]
    MATERIAL according to claim 5, characterized in that the binder is hydrophobic.
    [0007]
    MICROSPHERE according to any one of claims 1 to 3, characterized in that it is for use in vascular embolization.
    [0008]
    WOUND DRESSING, characterized in that it comprises the material according to either of claims 5 or 6.
    [0009]
    METHOD FOR CULTURING IN VITRO CELLS, characterized in that at least one microsphere, as defined in claims 1 to 3, is added to a culture medium to which the cells to be cultured are also added, and then the cells are allowed to propagate.
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    同族专利:
    公开号 | 公开日
    CN102740862A|2012-10-17|
    CA2781593C|2018-01-23|
    ES2584319T3|2016-09-27|
    HUE030071T2|2017-04-28|
    US20180022833A1|2018-01-25|
    CA2781593A1|2011-06-09|
    RU2016122487A3|2019-11-29|
    PL2506859T3|2016-11-30|
    WO2011068455A1|2011-06-09|
    US20120244198A1|2012-09-27|
    UA110776C2|2016-02-25|
    SI2506859T1|2016-10-28|
    IL220069A|2017-02-28|
    RU2012125200A|2014-01-20|
    EP2506859A1|2012-10-10|
    EP2506859A4|2013-10-23|
    AU2010327380B2|2014-11-27|
    RU2725890C2|2020-07-07|
    HK1172268A1|2013-04-19|
    HRP20160835T1|2016-09-23|
    LT2506859T|2016-09-26|
    KR20120130166A|2012-11-29|
    BR112012012997A2|2017-03-01|
    ZA201203497B|2013-08-28|
    JP5767239B2|2015-08-19|
    PT2506859T|2016-08-12|
    US9708416B2|2017-07-18|
    EP2506859B1|2016-05-04|
    JP2013512895A|2013-04-18|
    RU2016122487A|2018-11-29|
    AU2010327380A1|2012-05-31|
    CN102740862B|2014-12-24|
    BR112012012997B8|2021-05-25|
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    法律状态:
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    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
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    优先权:
    申请号 | 申请日 | 专利标题
    SE0901521|2009-12-04|
    SE0901521-5|2009-12-04|
    PCT/SE2010/051268|WO2011068455A1|2009-12-04|2010-11-17|Microspheres of hydrolysed starch with endogenous, charged ligands|
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