Steam-sterilized modified starch for medical uses

Abstract

The invention provides a modified starch for medical uses and a method for preparing said 5 modified starch by steam-sterilization. The invention further provides said steam-sterilized modified starch for use in the inhibition of the formation of postoperative adhesions. Said steam-sterilized modified starch is preferably a gel comprising or consisting of carboxymethylated starch or etherified starch, which may optionally contain sodium citrate or citric acid. The invention also relates to a pharmaceutical composition comprising or consisting of a steam-sterilized gel comprising carboxymethylated starch or etherified starch and optionally sodium citrate and / or citric acid.

Description

[0001] STEAM- STERILIZED MODIFIED STARCH FOR MEDICAL USES

[0002] FIELD OF THE INVENTION

[0003] The invention provides a modified starch for medical uses and a method for preparing said modified starch by steam-sterilization. The invention further provides said steam-sterilized modified starch for use in the inhibition of the formation of postoperative adhesions. Said steam- sterilized modified starch is preferably a gel comprising or consisting of carboxymethylated starch or etherified starch, which may optionally contain sodium citrate or citric acid. The invention also relates to a pharmaceutical composition comprising or consisting of a steam- sterilized gel comprising carboxymethylated starch or etherified starch and optionally sodium citrate and / or citric acid.

[0004] BACKGROUND ART

[0005] Organs and tissues in the body that are covered with a mesothelium (heart, lungs and intraabdominal organs, parietal wall of the pericardium, thorax and abdominal cavity) are normally able to freely move and are separate from each other, even when their surfaces are in close contact. Articular cavities are covered with the synovium, which is essential there for the free movement of bones and joints. This also applies to the tendons and their tendon sheaths. After surgical interventions in the aforementioned body cavities or on the synovial structures, physiological wound healing starts and results in wound closure or covering of the wounds to protect them from infection. The processes that cause physiological wound healing are basically the same processes that lead to the formation of adhesions, which can cause a variety of problems. Adhesions may appear as slight accumulations that are easy to dissolve or as bands of fibrous, sometimes vascularized tissue (i.e. connective tissue containing blood vessels). They may even appear as complete adhesions, which abnormally fix the organs permanently.

[0006] Adhesions may cause chronic pain, disorders of the abdominal viscera such as obstruction of the intestinal passage up to total obstruction (ileus) and thus lead to further surgical interventions or even death. Furthermore, adhesions in the area of the internal genital organs of women are the most frequent cause of secondary female infertility. Adhesions increase the risk during surgery due to bleeding during removal of the adhesions. Moreover, the areas removed of adhesions are hearths for the development of new adhesions. Adhesions in synovial structures may cause chronic pain and movement impairment. Medical complications aside, the treatment of adhesions is a significant socioeconomic problem. For this reason, a safe and low-risk prevention and / or inhibition of adhesions is one of the main goals of modern surgery.

[0007] Surgeries are the most frequent cause for the formation of adhesions, and they may be a serious complication. The predisposing factors include mechanical injuries of mesothelial (mesotheliumlike such as, for example, synovial) surfaces or peritoneal ischemia due to manipulation and retraction of tissues during operation.

[0008] The incidence of postoperative adhesion formation is estimated today at 67% to 93%. At present, there is a variety of barriers on the market for reducing this problem. While the principle is invariably the physical separation of wound areas, the products differ considerably due to their application, structure and composition. There are barriers or rinse solutions produced from cellulose or polytetrafluorethylene, icodextrin, hyaluronic acid as well as carboxymethyl cellulose.

[0009] US 2002 / 0169144A1 discloses a pharmaceutical composition that comprises a cross-linked polyanionic polysaccharide, such as carboxymethyl amylose and a citrate buffer, the composition preventing the formation of surgical adhesions.

[0010] US 2004 / 0153040 Al discloses a wound dressing comprising a layer of hydrophilic polymer foam and a hydrogel layer used to contact the surface and as an anti-adhesion ingredient, wherein the hydrogel layer may contain carboxymethylated starch, wherein the polymer foam may be cross-linked with citric acid and the wound bandage is used during a postoperative treatment.

[0011] WO 2016 / 098057 Al discloses a chitosan hydrogel, which includes citric acid and carboxymethylated starch, the hydrogel being used to counteract postoperative adhesions.

[0012] DE 10 2013 211 316 Al discloses a hemostatic agent comprising carboxymethylated starch, which is chemically cross-linked by citric acid. WO 2016 / 100861 Al discloses a hemostatic composition comprising cross-linked modified amylopectin, icodextrin or maltodextrin, which includes carboxymethylated starch, carboxymethyl maltodextrin or carboxymethyl icodextrin, as well as sodium citrate as a constituent of an aqueous diluent.

[0013] US5807833A1 discloses a method for preparing modified starch formulations, specifically of hydroxyethyl starch (HES), and its use in reducing or preventing adhesion formation between tissue or organ surfaces. Prior to steam sterilization by autoclaving, undissolved particulate matter further had to be removed in a labor-intensive process.

[0014] In addition to commercial products for reducing or inhibiting postoperative adhesions, other medicinal materials are also conceivable. The most promising solutions appear to be the use of modified starches as mechanical barriers, as well as the use of citrate for inhibiting the formation of fibrin. Hoffmann et al. (2009), for example, reported that products for hemostasis may also reduce postoperative adhesions. Starch-based products for hemostasis, in particular, significantly reduce the degree of adhesions as compared to other “conventional” topical products for hemostasis. With Arista™ AH (disclosed by the patent family U.S. Pat. No. 6,060,461), consisting of microporous starch granules, it is possible to achieve a maximum 50% reduction of postoperative adhesions in the rat model. W02009 / 091549 discloses, in particular, a hemostatic material, which in this case is a modified starch having a water absorption capacity of no lower than the simple inherent weight, having a molecular weight of greater than 15,000 Daltons and having a starch granule diameter of 10 to 1,000 pm, at least 95% of the starch granules having a diameter between 30 pm and 500 pm. This modified starch is biocompatible, absorbable and is degraded by amylases and carbohydrases. Methods with regard to application are disclosed, in which the hemostatic material is applied to tissue, for example, in the form of powder or as a gel and is used, inter aha, for the purpose of hemostasis and adhesion prophylaxis. In two publications, Pope et al. (1914 and 1916) report on a reduction of postoperative adhesions due to the use of citrate. It was shown in 60 experiments on rabbits that 2% to 4% citrate in water or saline solution resulted in fewer adhesions. This was confirmed in clinical application in 400 abdominal surgeries and it was shown that a clear improvement with respect to the development of adhesions was evident as result of citrate. SUMMARY OF THE INVENTION

[0015] Alternative solutions to the problem of postoperative adhesions are always desirable. The fact, however, that adhesions may be reduced to a never before seen degree by modified starch (synergistically improved compared to modified starch as a mechanical barrier or citrate for inhibiting fibrin formation, in each case individually), which optionally contains citrate and citric acid (either from the manufacturing process or through admixture), applied to traumatized mesothelial (or mesothelium-like) surfaces (zones at risk for developing adhesions) and transformed from starch powder to a starch gel, is unique.

[0016] Usually, the raw material starch powder is sterilized by irradiation, such as gamma or beta rays. The liquids (isotonic saline solution, Ringer's solution, other isotonic solutions) for the production of the starch gel are also sterile. In general, there are two possibilities to generate a starch gel:

[0017] (i) The sterilized modified starch powder can be introduced into a body cavity and then transformed into a gel in the body cavity by adding sterile liquid; or

[0018] (ii) The sterilized starch powder can be mixed with sterile liquid in a sterile container to form a gel by means of a stirring process and then introduced into the body cavity as a gel.

[0019] The conventionally recommended mixing ratio is 1 g of radiation-sterilized starch and 8 to 12 ml of sterile saline solution.

[0020] The inventors have surprisingly found that after irradiation of the raw material starch powder, two microscopically different particle configurations are present in a gel produced from said irradiated starch powder:

[0021] - Particles as known from starch, swelling with water while retaining the particle structure; and

[0022] - Highly contrasted particles, internal structure highly refractive, non-swelling.

[0023] It was further found that these strongly contrasted particles are preserved in the starch gels after at last 3 months of ageing, and that the swollen particles in the starch gels are unstable after 3 months of ageing. After administration of gels produced from irradiated starch to a postoperative site, hematoxylinophilic spiral remnants were found in the healing connective tissue. These hematoxylinophilic spiral remnants are distributed across the entire area on which the starch gel has been applied. The hematoxylinophilic spiral remnants seem to attract cells corresponding to fibroblasts, which were found to be arranged in a circle around said hematoxylinophilic spiral remnants. This arrangement of cells and spiral remnants can lead to the development of microgranulomas. Microgranulomas are undesired after the administration of starch gel to a postoperative site, since they are known to trigger chronic infection and impair rapid and uneventful healing process.

[0024] Accordingly, it is the purpose of the present invention to provide a starch for medical use, in particular for the prevention and / or inhibition of postoperative adhesions, which does not show undesired side effects, such as the formation of microgranulomas. This problem is solved by a gel comprising a modified starch comprising, an aqueous liquid according to claim 1 and a pharmaceutical composition comprising said gel of a modified starch comprising an aqueous liquid, wherein said gel of a modified starch comprising an aqueous liquid is preheated to achieve complete homogenization and thereafter subjected to steam-sterilization.

[0025] The inventors now have surprisingly found that the appearance of starch gels made from a modified starch can be altered by treatment with two process steps, preheating and steamsterilization. Firstly, the appearance of starch gels made from a modified starch after preheating is different. Unsterilized starch gels made from modified starch that have been preheated at a temperature range between at least 60 °C and 80 °C at maximum, preferably at a temperature of 62.5°C for 30 minutes after gel formation, have different characteristics than unsterilized unheated starch gel, manifesting in two different configurations of particles detectable under a light microscope:

[0026] 1. The starch particles in preheated gels of modified starch are swollen by absorption, weakly contrasted, irregularly bounded and showing an internal structure resulting from the layering of the starch as known from swollen modified starch. The proportion of these particles is at least 99 %. 2. In contrast to unheated suspensions of modified starch, which contain about 5 to 10 % of particles exhibiting refraction similar to crystals, in the preheated gels of modified starch only a few particles are strongly contrasted with refraction similar to crystals. The particles of unheated suspensions of modified starch do not appear swollen. Compared to unheated suspensions of modified starch, the number of those particles in the preheated gel of modified starch is 1 % or less.

[0027] Secondly, the appearance of preheated starch gels made from a modified starch after sterilization is different, depending on the method applied for sterilization of the modified starch. Starch gels made from modified starch that has been preheated and steam-sterilized after gel formation have different characteristics than starch gel made from irradiated starch powder:

[0028] The starch particles are uniformly swollen and uniformly configured.

[0029] In contrast to irradiated starch, there are no particles that appear refractive like crystals and do not swell.

[0030] Under the light microscope, the uniformly swollen and uniformly configured starch particles remain unchanged after natural ageing for over 3 months. They are also unchanged after repeated freezing and thawing.

[0031] Histologically, after a week, irritation-free healing without spiral, hematoxylinophilic residues and correspondingly without granuloma formation can be observed.

[0032] The starch gel and the pharmaceutical composition according to the invention have excellent utility for reducing and / or inhibiting postoperative adhesions and is suitable for reducing and / or inhibiting postoperative adhesions in an unprecedented way, since the starch gel and the pharmaceutical composition according to the invention do not show the side effects of gels made from unheated and / or irradiated modified starch.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 : Microscopic image of a gel consisting of unsterilized modified starch (Medication 1) and sterile solution (isotonic saline solution). Mixing ratio 1 g powder and 10 ml sterile saline solution. No ageing.

[0035] Two different configurations of particles can be recognized in the gel: (1) About 5 to 10% of the particles are strongly contrasted, refraction similar to crystals. These particles do not appear swollen.

[0036] (2) Starch particles that are swollen by absorption of liquid. They are weakly contrasted, irregularly bounded and show an internal structure resulting from the layering of the starch, as known from swollen modified starch.

[0037] FIG. 2: Microscopic image of a gel consisting of unsterilized modified starch (Medication 1) and sterile solution (isotonic saline solution). Mixing ratio 1 g powder plus 10 ml solution. Gel after gelatinization for 30 minutes at 62.5°C. No ageing.

[0038] Two different configurations of particles can be recognized in the gel:

[0039] (1) Starch particles that are swollen by absorption of liquid. They are weakly contrasted, irregularly bounded and show an internal structure resulting from the layering of the starch, as known from swollen modified starch.

[0040] (2) Few particles are strongly contrasted, refraction similar to crystals. These particles do not appear swollen. Compared to unheated suspensions of modified starch, the number of those particles is minimized (< 1%).

[0041] FIG. 3: Microscopic image of a gel consisting of irradiation-sterilized modified starch (Medication 1) and sterile solution (isotonic saline solution). Mixing ratio 1 g radiation-sterilized starch and 10 ml sterile saline solution. No ageing.

[0042] Two different configurations of particles can be recognized in the gel:

[0043] (1) Starch particles that are swollen by absorption of liquid. They are weakly contrasted, irregularly bounded and show an internal structure resulting from the layering of the starch, as known from swollen modified starch.

[0044] (2) About 5 to 10% of the particles are strongly contrasted, refraction similar to crystals. Particles do not appear swollen. FIG. 4: Microscopic image of a gel consisting of irradiation-sterilized modified starch (Medication 1, irradiated with gamma rays (17-25 kGy)) and sterile solution (isotonic saline solution). Mixing ratio 1 g radiation-sterilized starch and 10 mL sterile saline solution. No ageing.

[0045] Greater enlargement of the gel from irradiation-sterilized starch. (1) The majority of the starch particles are swollen by absorption of liquid. These particles are weakly contrasted, irregularly bounded and show an internal structure resulting from the layering of the starch, as known from swollen modified starch. (2) Two of the particles are strongly contrasted with a clear internal structure. The refraction of light is partly similar to that of crystals. Particles do not appear swollen.

[0046] FIG. 5: Microscopic image of a gel consisting of irradiation-sterilized modified starch (Medication 1, irradiated with gamma rays (17-25 kGy)) and sterile solution (isotonic saline solution). Mixing ratio 1 g of radiation-sterilized starch and 10 mL of sterile saline solution. Aged naturally for 3 months at room temperature. Two different configurations of the starch particles can be recognized:

[0047] (1) Compared to the non-aged gel, an internal structure resulting from the layering of the starch is hardly recognizable in the swollen particles. The particles appear dissolved.

[0048] (2) Particles that are highly refractive, similar to crystals and not swollen, appear unchanged compared to the unaged gel.

[0049] FIG. 6: Microscopic image of a gel of modified starch powder (Medication 1) mixed with isotonic saline solution. Mixing ratio 1g powder plus 10 mL solution. Sterilized by autoclaving at 121 °C for 20 min. No ageing.

[0050] The starch particles are uniformly swollen and uniformly configured by absorbing liquid. They are irregularly bounded and show an internal structure resulting from the stratification of the starch, as known from swollen modified starch. The contrast of the particles appears stronger than with the radiation-sterilized particles. In contrast to irradiated starch, there are no particles that appear refractive like crystals and are not swollen.

[0051] FIG. 7: Microscopic image of a gel of modified starch powder (Medication 1) mixed with isotonic saline solution. Mixing ratio 1g powder plus 10 mL solution. Sterilized by autoclaving at 121 °C for 20 min. Aged naturally for 3 months at room temperature.

[0052] After ageing, the autoclaved starch particles are unchanged compared to unaged starch in terms of the external and internal structure of the particles. They are irregularly bounded and have an internal structure similar to that of swollen modified starch.

[0053] In contrast to irradiated starch, in which the swollen particles are only vaguely recognizable after ageing and appear dissolved, the structure of the particles is completely preserved. Particles with refraction reminiscent of crystals, as with irradiated starch, are still not present.

[0054] FIG. 8: Microscopic image of a gel of modified starch powder (Medication 1) mixed with isotonic saline solution. Mixing ratio 1g powder plus 10 mL solution. Sterilized by autoclaving at 121°C for 20 min. No ageing. Frozen twice (-18 “Celsius) and thawed again.

[0055] After freezing and thawing twice, the autoclaved starch particles are unchanged compared to non-aged starch and also aged starch in terms of the external and internal structure of the particles. They are irregularly bounded and have an internal structure similar to that of swollen modified starch. In contrast to irradiated starch, there are no particles that appear refractive like crystals and are not swollen.

[0056] FIG. 9: Histological section of abdominal wall healing according to OP AM model in the rat, treatment with radiation-sterilized starch gel.

[0057] Abdominal wall healing after OP AM: (1) Degradation of Medication 1 particles with preserved water absorption, degradation by macrophages and giant cells, small residues of starch in the connective tissue. (2) Degradation of Medication 1 particles without preserved water absorption, degradation by macrophages and giant cells, hematoxylinophilic spiral remnants in the connective tissue. (3) Regenerating muscle of the abdominal wall.

[0058] FIG. 10: Histological section of abdominal wall healing according to OPAM model in the rat, treatment with radiation-sterilized starch gel.

[0059] Abdominal wall healing after OPAM: (1) Degradation of Medication 1 particles with preserved water absorption, degradation by macrophages and giant cells, small residues of starch in the connective tissue. (2) Degradation of Medication 1 particles without preserved water absorption, degradation by macrophages and giant cells, hematoxylinophilic spiral remnants in the connective tissue. Delayed resorption of the particle with circular arrangement of fibroblasts, precursor of a microgranuloma. (3) Regenerating muscle of the abdominal wall.

[0060] FIG. 11 : Histological section of abdominal wall healing according to OPAM model in the rat, treatment with autoclaved starch gel.

[0061] Abdominal wall healing after OPAM: (1) Degradation of (Medication 1) with preserved water absorption, degradation by macrophages and giant cells, small residues of starch in the connective tissue. Hematoxylinophilic spiral remnants in the connective tissue are not found or precursors of a microgranuloma are not found. (2) Muscle of the abdominal wall in regeneration. (3) The unicellular layer of the mesothelium is regenerated.

[0062] FIG. 12: Microscopic image of a gel consisting of irradiation-sterilized modified starch (Medication 2) and sterile solution (isotonic saline solution). Mixing ratio 1 g radiation-sterilized starch and 10 ml sterile saline solution. No ageing.

[0063] Two different configurations of the starch particles can be seen.

[0064] (1) Starch particles that have swollen due to the absorption of liquid. They are rounded, partially irregularly bordered. (2) Particles, light-refracting similar to crystals, which have absorbed little or no liquid and make up about 5 % of the particles.

[0065] FIG. 13: Microscopic image of a gel consisting of gamma-ray sterilized, epichlorohy drin- modified etherified starch (Arista® AH, Medication 3) and sterile solution (isotonic saline solution). Mixing ratio 1 g radiation-sterilized starch and 10 ml sterile saline solution. No aging.

[0066] Two different configurations of the starch particles can be seen.

[0067] (1) Starch particles that are swollen by absorption of liquid. They are weakly contrasted, irregularly bounded and show an internal structure resulting from the layering of the starch, as known from swollen modified starch.. (2) About 5 to 10% of the particles are strongly contrasted, refraction similar to crystals. These particles do not appear swollen.

[0068] FIG. 14: Microscopic image of a gel consisting of steam-sterilized, epichlorohydrin-modified etherified starch and aqueous solution (SmartPAN, Medication 4). Aged naturally for 6 months at room temperature. Starch particles have swollen due to absorption of aqueous liquid. They are rounded, irregularly bordered. In contrast to irradiated or unsterilized starch, there are no particles that appear refractive like crystals and are not swollen.

[0069] FIG. 15: Histological section of abdominal wall healing according to OP AM model in the rat, treatment with radiation-sterilized starch gel (medication 1).

[0070] Abdominal wall healing after OP AM:

[0071] (1) Degradation of irradiated modified starch particles with preserved water absorption, degradation by macrophages and giant cells, small residues of starch in the connective tissue.

[0072] (2) Degradation of irradiated modified starch particles without preserved water absorption, degradation by macrophages and giant cells. Delayed resorption of the particles with circular arrangement of fibroblasts, showing formation of three microgranulomas .

[0073] FIG. 16: Histological section of abdominal wall healing according to OP AM model in the rat, treatment with starch gel sterilized by autoclaving (medication 1).

[0074] Abdominal wall healing after OP AM:

[0075] (1) Intact mesothelium

[0076] (2) Dense area of starch being degraded uniformly, no formation of microgranulomas

[0077] (3) Striated muscle cells

[0078] FIG. 17: Histological section of abdominal wall healing according to OP AM model in the rat, treatment with starch gel sterilized by autoclaving (medication 1).

[0079] Abdominal wall healing after OP AM in higher magnification:

[0080] (1) Intact mesothelium

[0081] (2) Dense area of starch being degraded uniformly, no formation of microgranulomas

[0082] (3) Mononuclear cells

[0083] FIG. 18: Histological section of abdominal wall healing according to OP AM model in the rat, treatment with starch gel sterilized by autoclaving (medication 1).

[0084] Abdominal wall healing after OP AM in very high magnification:

[0085] (1) Starch remnants in mononuclear cells

[0086] (2) Starch remnants in the tissue

[0087] (3) Starch remnants in a giant cell

[0088] (4) Capillary in regenerated lamina propria of peritoneum

[0089] Starch being degraded uniformly without formation of microgranulomas. DETAILED DESCRIPTION OF THE INVENTION

[0090] The objective problem to be solved by the present invention is the provision of a starch gel for medical use, in particular for the prevention and / or inhibition of postoperative adhesions, which does not show undesired side effects, such as the formation of microgranulomas.

[0091] This problem is solved by the invention in a first aspect by the provision of gel comprising a modified starch comprising an aqueous liquid according to claim 1 and a pharmaceutical composition comprising said gel of said modified starch comprising an aqueous liquid, wherein said gel of said modified starch comprising an aqueous liquid is preheated to achieve complete homogenization and thereafter subjected to steam-sterilization.

[0092] The starch according to the invention may be for example derived from different types of plants, starch may be modified in order to adapt the properties, e.g. thickening of the ultrasonic couplant composition, starch is selected from the group comprising corn starch, rice starch, wheat starch, potato starch, cassava starch and modified starch.

[0093] Preferred in accordance with the invention is modified starch. The person of ordinary skills in the art knows modification of starch. He is furthermore aware of the modifications suited to reach the desired properties. A “modified starch” is a starch that has been chemically modified to allow the starch to function properly under conditions frequently encountered during processing or storage, such as high heat, high shear, low pH, freeze / thaw and cooling. Thus, in a preferred embodiment of the present invention the modified starch is selected from the group consisting of acid-treated starch, alkaline-treated starch, bleached starch, oxidized starch, enzyme-treated starches, monostarch phosphate, distarch phosphate, phosphated distarch phosphate, acetylated distarch phosphate, starch acetate, acetylated distarch adipate, hydroxypropyl starch, hydroxypropyl distarch phosphate, hydroxypropyl distarch glycerol, starch sodium octenyl succinate, acetylated oxidized starch, dextrin, esterified starch, etherified starch, and cross-linked starch.

[0094] In a more preferred embodiment, the starch is a cross-linked starch. The cross-linked starch of the present invention includes, but is not limited to, at least one of epichlorohydrin cross-linked starch, cross-linked carboxymethyl starch (further named herein as carboxymethyl starch) crosslinked etherified starch. Most preferably, the modified starch according to the invention is carboxymethylated starch.

[0095] Accordingly, the invention provides a gel comprising a modified starch, preferably a gel comprising a carboxymethylated starch or an etherified starch which comprises an aqueous liquid and which is producible by a process comprising the steps of: a) Providing a modified starch, preferably a carboxymethylated starch or an etherified starch, as a powder; b) Premixing the powder of step a) with an aqueous liquid in an amount sufficient for forming a gel of said modified starch; c) Preheating the mixture obtained according to step b) at a temperature in the range of 60 °C to 80 °C; and d) Steam-sterilizing the gel of said modified starch comprising an aqueous liquid according to steps b) and c).

[0096] In a further embodiment, the invention relates to said process to produce a gel of a modified starch, preferably a gel of carboxymethylated starch of the invention.

[0097] The mixing ratio of said powder of a modified starch to said aqueous liquid in step b) is usually 0.5 - 20 g powder of a modified starch to 2 - 600 ml aqueous solution, preferably 1 g powder of a modified starch to 8 - 12 ml aqueous solution, most preferably 1 g powder of a modified starch to 10 ml aqueous solution.

[0098] The premixing of the powder of the modified starch and the aqueous liquid is step b) is usually performed at room temperature for a time sufficient to form the gel of the modified starch.

[0099] Preheating step c), is typically carried out on a hot plate or a heating mantle. At the beginning of the heating process, a thermometer is inserted into the vessel in which the heating is performed in order to measure the temperature inside the vessel. Steam sterilization according to step d) is a highly reliable sterilization method, which is preferable to all other sterilization methods wherever possible. Sterilization with steam does not require the use of chemicals or toxic substances, which also plays an important role in environmental compatibility.

[0100] Sterilization with steam is performed at high temperature and pressure in an autoclave - a thermally insulated and pressure-tight chamber. This chamber works like a pressure cooker. Water is heated until it turns into steam, which also increases the pressure of the water or steam. Since the pressure in the autoclave can exceed normal atmospheric pressure, the temperature of the steam can also rise to values of over 100°C, and sometimes even over 130°C such as up to 134°C. The hot steam reliably kills the microorganisms in the gel of the modified starch comprising an aqueous liquid.

[0101] In accordance with the present invention, the steam-sterilization of the gel of the modified starch comprising an aqueous liquid according to step d) is performed at a temperature in the range from 121 °C to 134°C for a duration of 10 min to 30 min, preferably at a temperature of 121 °C for a duration of 20 min.

[0102] In another embodiment, preheating the mixture obtained according to step b) is carried out at a temperature range selected from the group consisting of 61.0 °C to 75.0 °C; preferably 62.0 °C to 68.0 °C; more preferably 62.0 °C to 63.0 °C; even more preferably 62.25 °C to 62.75 °C , most preferably at 62.5°C.

[0103] Thus, in a preferred embodiment, the invention provides a gel of a modified starch, preferably a gel of a carboxymethylated starch, which comprises an aqueous liquid and which is producible by a process comprising the steps of: a) Providing a modified starch, preferably a carboxymethylated starch, as a powder; b) Premixing the powder of step a) with an aqueous liquid at room temperature in an amount sufficient for forming a gel of said modified starch; c) Preheating the mixture obtained according to step b) at a temperature at a preferred temperature of 62.5 °C; and d) Steam-sterilizing said gel of the modified starch comprising an aqueous liquid according to steps b) and c) in the range from 121°C to 134°C for a duration of 10 min to 30 min, preferably at a temperature of 121 °C for a duration of 20 min. In a further embodiment, the invention relates to said process to produce a gel of the modified starch, preferably a gel of a carboxymethylated starch of the invention. The gel of the modified starch comprising an aqueous liquid according to the invention is further characterized by the features that starch particles after preheating are swollen because of liquid absorption. They are weakly contrasted, irregularly bounded and show an internal structure resulting from the layering of the starch, as known from swollen modified starch. Only a few particles exhibit strong contrast and crystal-like refraction, and these do not appear swollen. Compared to unheated suspensions of modified starch, the proportion of such particles is minimized (< 1%).”

[0104] After steam-sterilization of step d), the particles are uniformly swollen and uniformly configured, and that, in contrast to irradiated starch, there are no particles that appear refractive crystals (when examined under the light microscope) and do not swell. Accordingly, the gel of the modified starch comprising an aqueous liquid of the invention, such as a gel of a carboxymethylated starch comprising an aqueous liquid of the invention is free of refractive crystals, preferably is free of refractive crystals that do not swell in the aqueous liquid.

[0105] Furthermore, when examined under the light microscope, the uniformly swollen and uniformly configured starch particles of the gel of the modified starch comprising an aqueous liquid of the invention, such as a gel of a carboxymethylated starch comprising an aqueous liquid of the invention, which has been steam-sterilized, remain unchanged after natural ageing for over 3 months.

[0106] The gel of the modified starch, preferably the gel of the carboxymethylated starch of the invention may in a further embodiment comprise 1 to 20 percent by weight citric acid and / or a salt of citric acid relative to the weight of the carboxymethylated starch.

[0107] In this embodiment, the powder of modified starch, such as a carboxymethylated starch, comprises an easily soluble citrate salt, wherein easy solubility is defined as a solubility of 100 g / 1 to 1000 g / 1 citrate in water at a temperature of 25 °C. The presence of free citrate ions in the gel of the modified starch such as the carboxymethylated starch after the addition of an, preferably aqueous, liquid strongly supports the pharmaceutical effect of the gel of the modified starch. By adding an aqueous liquid, the modified starch, such as the carboxymethylated starch forms a gel, which optionally comprises citrate ions from citric acid or from a highly soluble citrate salt. The gel of the modified starch acts as a barrier during the healing process of mesenchymal organs or tissues, of organs in serous cavities or of epithelial tissue after a surgery by mechanically preventing contacts between organ or tissue surfaces. The dissolved citrate inhibits the formation of polyfibrin during blood coagulation. Polyfibrin is a basic building block of adhesion formation.

[0108] The citric acid and / or the citrate salt may be included in the gel of the modified starch such as the gel of the carboxymethylated starch in an amount of 1% to 20% of the total weight of the carboxymethylated starch (weights in powder form). The content of citric acid and / or a salt of citric acid relative to the weight of the gel of the modified starch such as the gel of the carboxymethylated starch is preferably 1 to 10 percent by weight, more preferably 1 to 8 percent by weight, most preferably 2 to 5 percent by weight.

[0109] In a preferred embodiment, the gel of the modified starch such as the gel of the carboxymethylated starch of the invention comprises 1 to 20 percent by weight of a salt of citric acid relative to the weight of the carboxymethylated starch (weights in powder form), wherein said salt of citric acid has a solubility in water of greater than 100 g / 1, preferably greater than 200 g / 1, more preferably greater than 300 g / 1, most preferably greater than 400 g / 1, at a temperature of 25°C.

[0110] Said salt of citric acid is preferably selected from the group consisting of a sodium citrate, a lithium citrate, a potassium citrate, a calcium citrate and a magnesium citrate. Most preferably, said salt of citric acid is trisodium citrate, which is biocompatible, and which has a solubility of 425 g / 1 at a temperature of 25°C.

[0111] Further most preferably, said citrate from citric acid or a citrate salt, especially trisodium citrate is located on the surface of the particles of the modified starch, such as the carboxymethylated starch.

[0112] The high solubility of the citrate salt and the location on the surface of the starch particles enable a fast dissolution of the citrate salt and a fast delivery of the dissolved citrate ions for unfolding of the pharmacological effect, namely the inhibition of the formation of polyfibrin during blood coagulation.

[0113] In addition, gel of the modified starch such as the gel of the carboxymethylated starch according to the invention advantageously further comprises water and / or at least one salt, for example and in particular sodium chloride or potassium chloride.

[0114] In a preferred embodiment, the gel of the modified starch of the invention comprises carboxymethylated starch and 1 to 20 percent by weight citric acid and / or a salt of citric acid relative to the weight of the carboxymethylated starch (weights in powder form), and, optionally water.

[0115] In a further preferred embodiment, the gel of the modified starch of the invention comprises carboxymethylated starch and 1 to 20 percent by weight citric acid and / or a salt of citric acid relative to the weight of the carboxymethylated starch (weights in powder form), water and / or at least one salt, for example and in particular sodium chloride or potassium chloride.

[0116] In practice, it has proven beneficial for the carboxymethylated starch to have a molecular weight of 500,000 daltons to 11,000,000 daltons, and / or for the starch particles, the carboxymethylated starch have a particle diameter in the range of 30 pm to 100 pm.

[0117] Accordingly, the invention provides a gel of the modified starch comprising or consisting of carboxymethylated starch and 1 to 20 percent by weight trisodium citrate relative to the weight of the carboxymethylated starch (weights in powder form), and optionally water, wherein said carboxymethylated starch has a molecular weight in the range of 500,000 daltons to 11,000,000 daltons, and / or wherein the starch particles of the carboxymethylated starch particles have a particle diameter of 30 microns to 100 microns.

[0118] Preferably, said carboxymethylated starch has a molecular weight in the range of 750,000 daltons to 10,000,000 daltons or 1,000,000 daltons to 9,000,000 daltons.

[0119] More preferably, said carboxymethylated starch has a molecular weight in the range of 2,000,000 daltons to 8,000,000 daltons or 3,000,000 daltons to 7,000,000 daltons. Most preferably, said carboxymethylated starch has a molecular weight in the range of 4,000,000 daltons to 6,000,000 daltons.

[0120] The carboxymethylated starch of the invention has the following, further preferred characteristics: It is a crosslinked, carboxymethylated starch containing approximately 20 wt % amylose and approximately 80 wt % amylopectin in a relatively constant ratio. The amylose is rather amorphous, and the amylopectin represents the crystalline portion of the starch. The modified starch is present as a salt-starch glycolate, and is generally the sodium salt of a carboxymethyl ether of the starch.

[0121] A starch particle of the carboxymethylated starch is made up of about 4.5xlO10to 2.3xl012amylose molecules and 5.6xl07to 1.3xl010amylopectin molecules. The degree of crosslinking of the carboxymethylated is preferably in the range of 25% to 45%.

[0122] The carboxymethylated starch may further contain at least one salt, which is selected from the group consisting of sodium chloride and potassium chloride, preferably sodium chloride, in an amount between 0 weight % and 10 weight %, preferably between 2 weight % and 5 weight % calculated on the basis of the dry mass of the carboxymethylated starch.

[0123] After addition of water, or when the carboxymethylated starch is in gel form, the pH value of the gel is in the range of 3.0 and 7.5, preferably from 5.0 to 7.5, most preferably from 6.5 to 7.5.

[0124] The carboxymethylated starch is present as a white or almost white fine, free-flowing powder. The powder is very hygroscopic and is practically insoluble in methylene chloride. It forms a translucent suspension in water.

[0125] The powder is made up of irregularly shaped, oval or pear-shaped particles in a size range from 30 to 100 pm, or 10 to 35 pm with rounding.

[0126] The starch particles have typically a surface of approximately 0.2 m2 / g. Occasionally occurring clusters of particles are made up of two to four particles. The particles have an eccentric hilus and clearly visible concentric grooves. The particles exhibit a distinct black cross on the hilus, between intersecting Nicol prisms. Small crystals are discernible at the surface of the particles. The particles exhibit significant swelling, up to approximately 40 times their own weight, upon contact with water or salt solutions.

[0127] The carboxymethylated starch comprised in the gel of the modified starch of the invention can be produced with the following method:

[0128] The synthesis of the carboxymethylated starch generally takes place in three steps, in each case as a suspension, wherein step 1 comprises crosslinking of the starch, step 2 comprises carboxymethylation of the starch, and step 3 is a neutralization step.

[0129] The crosslinking-step uses hydroxyl groups attached to starch (present in the basic glucose building blocks of the starch). The reaction of these hydroxyl groups with multifunctional reagents results in crosslinked starches. In a granule of starch, many chains are found in closer proximity, and such reactions not only take place between single chains, but it links side by side chains as well. These crosslinking agents form either ether or ester inter-molecular linkages between hydroxyl groups on starch molecules A small amount of multifunctional reagent is enough to interconnect starch molecules by crosslinking reactions. Methods for crosslinking of starches are generally known to the person skilled in the art. An overview of starch crosslinking is e.g. given in Shah, Nimish & Mewada, Rajubhai & Mehta, Tejal. (2016). Crosslinking of starch and its effect on viscosity behavior. Reviews in Chemical Engineering. 32. 10.1515 / revce- 2015-0047, which is incorporated herein in entirety.

[0130] In the present invention, the crosslinking step 1 of the starch is preferably performed with NagPgOy (sodium trimetaphosphate).

[0131] Since the reactions take place in suspension, the substitution is inhomogeneous. Outer areas of the starch are more greatly affected by the modifications than the core areas of the starch structures. The reactions are influenced by diffusion within the starch structure and accessibility to the starch structure. Amorphous areas are more easily accessible than crystalline areas, which are less strongly modified. From a chemical standpoint, both reactions are nonspecific, and, provided that diffusion inhibition or steric hindrance is present, have no preferred reaction pattern for the individual hydroxyl groups of a glucose unit. Theoretically, the degree of substitution is 3 (with substitution of all three OH groups of a glucose unit as the monomeric building block of the starch). The actual degree of substitution appears to be in the range of about 0.2 to 0.4; i.e., one OH group is substituted in approximately every fourth glucose unit (not taking crosslinking into account). This degree of substitution results from the values for the bound salt.

[0132] After steps 1 and 2 have been performed, neutralization is carried out as step 3, using an acid. Preferably, said acid is selected from the group consisting of succinic acid, phosphoric acid, hydrochloric acid, sulfuric acid, and citric acid. The selection of the acid has an effect on inorganic and organic substances that can be detected as residues in the modified starch. Most preferred according to the invention is citric acid.

[0133] Accordingly, the gel of the modified starch of the invention may further comprise an organic or inorganic salt resulting from the neutralization step, wherein said organic or inorganic salt is selected from the group consisting of succinate, phosphate, chloride, sulphate, and citrate.

[0134] To reach the content of 1 to 20 percent by weight of citric acid and / or a citrate salt relating to the weight of the carboxymethylated starch, citric acid or a citrate salt is added in the respective amount to the carboxymethylated starch in suspension and the components are intimately mixed.

[0135] In a preferred embodiment, when the gel of the modified starch is in gel form, the invention therefore provides an aqueous gel comprising crosslinked carboxymethylated starch, citrate ions and water, wherein the citrate ions are present in a concentration in the range of 1 mmole / liter to 500 mmoles / liter of the water, and wherein the crosslinked carboxymethylated starch has a degree of substitution in the range of 0.2 to 0.4 and a degree of crosslinking in the range of 25% to 45%.

[0136] In a second aspect, the invention provides a pharmaceutical composition, which comprises the steam-sterilized and gel of the modified starch comprising an aqueous liquid, such as the gel of the carboxymethylated starch comprising an aqueous liquid which optionally comprises citric acid or a citrate salt, and which has the features of the embodiments described for the first aspect herein.

[0137] Medical use and mode of administration to a subject In a further embodiment, the invention provides a gel of the modified starch or a pharmaceutical composition as described in aspects 1 and 2 hereinabove for use in a method for reducing and / or preventing and / or inhibiting postoperative adhesions.

[0138] In a further embodiment, the invention provides a method for reducing and / or preventing and / or inhibiting postoperative adhesions, said method comprising administering to a subject in need thereof a gel of the modified starch or a pharmaceutical composition as described in aspects 1 and 2 hereinabove.

[0139] In the simplest form of the use or method according to the invention the gel of the modified starch comprising an aqueous liquid or the pharmaceutical composition is administered directly on the postoperative area or site.

[0140] More specifically, the use or method of the invention comprises the following steps: i) Providing a gel of a modified starch such as a gel of a carboxymethylated starch which comprises an aqueous liquid, or a pharmaceutical composition which comprises a gel of a modified starch such as a gel of a carboxymethylated starch comprising an aqueous liquid, wherein said gel of the modified starch such as a gel of the carboxymethylated starch optionally further comprises citric acid and / or a dissolved salt of citric acid; ii) Administering the gel of the modified starch or pharmaceutical composition of step i) on the postoperative area or site in a subject.

[0141] The aqueous liquid is preferably selected from the group consisting of water, isotonic saline solution, or hypotonic, isotonic, or hypertonic saline solution as the liquid phase, which comprises one or more cations selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, iron(II), iron(III), aluminum; and / or further comprising one or more anions selected from the group consisting of fluoride, chloride, bromide, iodide, oxide, sulfide, carbonate, sulfate, phosphate, nitrate, chromate, permanganate, hexacyanoferrate(II). Ringer's solution, Ringer's acetate solution, and Ringer's lactate solution may also be used as the liquid phase.

[0142] Accordingly, the gel of the modified starch such as the gel of the carboxymethylated starch may further comprise one or more cations selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, iron(II), iron(III), and aluminum; and / or further comprising one or more anions selected from the group consisting of fluoride, chloride, bromide, iodide, oxide, sulfide, carbonate, sulfate, phosphate, nitrate, chromate, permanganate, hexacyanoferrate(II), acetate and lactate.

[0143] Most preferably, the aqueous liquid is water or isotonic saline.

[0144] The subject is preferably a mammal. Accordingly, the gel of the modified starch or the pharmaceutical composition according to aspects 1 and 2 of the invention is applicable in veterinary and human medicine. Most preferably, the subject is a human and the gel of the modified starch or the pharmaceutical composition according to aspects 1 and 2 of the invention is applied in human medicine.

[0145] Mode of action of the pharmaceutical composition of the invention

[0146] The gel of the modified starch or the pharmaceutical composition according to aspects 1 and 2 of the invention preferably comprises carboxymethylated and cross-linked starch, and optionally 1% to 20% percent by weight of which is citrate, as detailed hereinabove. This combination offers never before seen positive results for reducing and / or inhibiting postoperative adhesions. This applies, in particular, to the reduction or prevention or inhibition of postoperative adhesions, in particular, traumatized mesothelial (or mesothelium-like) surfaces (zones at risk for developing adhesions) such as, for example, the pleura in the chest cavity, peritoneum in the belly and pelvic cavity or pericardium serosum in the pericardial cavity, as well as synovial structures of bones, joints, tendons or nerves.

[0147] Accordingly, the invention provides a method, wherein said postoperative adhesions are reduced or prevented on mesothelial or mesothelium-like surfaces selected from the group consisting of mesothelial surfaces of the peritoneum, pericardium, pleura, synovium and tendon sheaths.

[0148] Advantageously, the method of the invention further comprises the treatment of a disorder associated with a surgery of mesenchymal organs or tissues, of organs in serous cavities or of epithelial tissue. Said disorder associated with a surgery of mesenchymal organs or tissues is preferably selected from the group consisting of impaired wound healing, epithelial defects and hemorrhage.

[0149] Said mesenchymal organs or tissues are preferably selected from the group consisting of loose connective tissue, tight connective tissue, reticular connective tissue, bones, cartilage, smooth muscles, heart muscle, kidneys, adrenal cortex, hematopoietic system, blood vessels and lymphatic vessels.

[0150] The never-before-seen positive properties of the pharmaceutical composition of the invention for reducing and / or inhibiting postoperative adhesions is assumed to be based on the following mechanisms:

[0151] An efficient tissue separation during wound healing is guaranteed by the gel of the modified starch or the pharmaceutical composition according to aspects 1 and 2 of the invention, applied in gel form or transformed from powder into gel form, on traumatized mesothelial (or mesotheliumlike) surfaces (zones at risk for developing adhesions). The application of the gel of the modified starch or the pharmaceutical composition according to aspects 1 and 2 of the invention in gel form or powder form or, for example, as a film, leads to the formation of a complete, effective and reliable barrier between organ and / or tissue surfaces and reaches every corner of the traumatized area after a surgery. Since carboxymethylated and cross-linked starch and citrate are absorbed within a few days, there is no need for a second surgery to remove the mechanical barrier. This makes it possible to prevent adhesions because the traumatized areas are covered with the starch-gel barrier and are thus physically separated from adjacent or other surfaces. The effect is synergistically reinforced by the effect of citrate, which inhibits fibrin formation, in particular polyfibrin formation.

[0152] Moreover, as shown herein, due to the steam-sterilization, the administration of the gel of the modified starch or the pharmaceutical composition according to aspects 1 and 2 of the invention is safe and does not cause microgranulomas.

[0153] Highly soluble salts of citric acid are used in medicine to prevent the coagulation of blood, e.g. donated blood (so-called citrated blood). Blood coagulation requires calcium ions as essential cofactors for blood clotting. Calcium ions are present in the blood plasma in a concentration of 46 to 54 mg / 1. In the presence of citrate, the calcium ions form a very poorly soluble salt with the citrate (very poor solubility is defined here as a solubility of 0.1 to 1 g / 1 in water at 25 °C). The solubility of calcium citrate with 0.85 g / 1 is in this range). This salt precipitates immediately, so that free calcium ions are no longer available as a cofactor for blood clotting. This in turn inhibits the formation of polyfibrin. The end product of blood clotting, polyfibrin, is a basic building block of adhesion formation between the organs and tissues discussed hereinabove.

[0154] Citrate forms insoluble chelate complexes with calcium, as a result of which a formation and stabilization of fibrin polymerization sites in all subsequent stages of fibrin polymerization is achieved. Citrate limits the deposition of fibrin through the binding of calcium ions, in order in this way to render the subsequent formation of adhesions impossible. Additionally calcium ions are also co-factors for other steps of the coagulation cascade before the formation of fibrin: they are part of the prothrombinase complex and the extrinsic tenase complex, both of which would not function without it and are responsible for platelet activation as well as the activation of several coagulation factors. As a result, the pharmaceutical composition of the invention offers two different antiadhesive effects, physical barrier and fibrin polymerization inhibition.

[0155] Advantageously, in the gel of the modified starch or the pharmaceutical composition according to aspects 1 and 2 of the present invention, citric acid and / or a citrate salt, such as trisodium citrate, is located on the surface of the particles of the carboxymethylated starch. Trisodium citrate is highly soluble, i.e. it dissolves immediately in the presence of water or another aqueous liquid. The dissolved citrate is immediately available, which, together with calcium ions, immediately forms the very poorly soluble calcium citrate. The calcium citrate precipitates immediately and is no longer available for the deposition of fibrin.

[0156] As the result of administering the gel of the modified starch or the pharmaceutical composition according to aspects 1 and 2 of the invention, a gel is acting locally on the postoperative area or site. The presence of the swollen starch powder particles in starch gel is associated with an increased invasion of cellular components of early healing. Activated macrophages come into contact with the surfaces of the swollen starch particles quite rapidly, forming a framework, followed by other cellular components of early healing Although the starch framework / matrix is degraded within days, an increased number of cellular components remain in the healing area, resulting in improved wound healing, epithelial defects and hemorrhage, wherein in particular an increased number of macrophages for the macrophagocytosis of bacteria, apoptopic cells, necrotic cells, cells with foreign protein, and polysaccharide surfaces are observed in the anastomotic area. In addition, an increased number of fibroblasts and other cellular components of wound healing, epithelial defects and hemorrhage are also observed. The presence of cells for early healing and the subsequent wound healing processes are accelerated by these factors.

[0157] Histological analyses in an area have shown improved submesothelial healing after one week due to application of the above-mentioned starch gel, i.e., in a form of the gel of the modified starch or the pharmaceutical composition according to aspects 1 and 2 according to the invention showing remnants (residues) of hydrobeads, macrophages and giant cells, fibroblasts, and emerging connective tissue.

[0158] The improvement in healing results from the presence of the gel of the modified starch or the pharmaceutical composition according to aspects 1 and 2 of the invention, which activates the early cellular healing processes, i.e., the attraction of macrophages, followed by the attraction of fibroblasts. The latter represent stabilizing factors for improving wound healing, epithelial defects and hemorrhage.

[0159] The subject matter according to the invention is explained in the following embodiment by way of two examples and in a nonlimiting manner.

[0160] Example 1: Application of gels made from irradiated or steam-sterilized modified starch and sterile solution under the conditions of a surgical procedure:

[0161] Medications

[0162] The following medications comprising carboxymethylated starch were used:

[0163] Medication 1 (comprising citrate)

[0164] Chemical name: Starch, carboxymethyl ether, sodium salt, present as sodium glycolate

[0165] Appearance: white or almost white fine, free-flowing powder Odour: neutral

[0166] Molecular weight: in the range of 500,000 daltons to 11,000,000 daltons

[0167] Particle size: in the range of 30 gm to 100 gm

[0168] Water content: 3.9 % (w / w)

[0169] Sodium chloride: 4.0 (w / w)

[0170] Sodium glycolate: < 2.0 (w / w)

[0171] Sodium citrate: 3.2 % (w / w)

[0172] Degree of crosslinking: 34 %

[0173] Crosslinking agent: NagPgOy (sodium trimetaphosphate)

[0174] The carboxymethylated starch without sodium citrate (Medication 2) had the following composition:

[0175] Medication 2 (w / o citrate)

[0176] Chemical name: Starch, carboxymethyl ether, sodium salt, present as sodium glycolate

[0177] Appearance: white or almost white fine, free-flowing powder

[0178] Odour: neutral

[0179] Molecular weight: in the range of 500,000 daltons to 11,000,000 daltons

[0180] Particle size: in the range of 30 pm to 100 pm

[0181] Water content: 3.9 % (w / w)

[0182] Sodium chloride: 4.0 (w / w)

[0183] Sodium glycolate: < 2.0 (w / w)

[0184] Degree of crosslinking: 34 %

[0185] Crosslinking agent: NagPgOy (sodium trimetaphosphate)

[0186] Medication 3

[0187] Arista® AH (epichlorhydrin-modified starch, see Lybarger, K.S., Review of Evidence Supporting the Arista® Absorbable Powder Hemostat. Medical Devices: Evidence and Research 2024: 17 173-188) Medication 4:

[0188] SmartPAN (Degradable starch microspheres (DSM; Magle Chemoswed AB, Malmo, Sweden) are produced from potato starch crosslinked with epichlorohydrin, see Pausch TM, Mitzscherling C, Abbasi S, Cui J, Liu X, Aubert O, Weissenberger M, Johansson H, Schuisky P, Busch C, Bruckner T, Golriz M, Mehrabi A, Hackert T. SmartPAN: A novel polysaccharide-microsphere-based surgical indicator of pancreatic leakage. J Biomater Appl. 2020 Jul;35(l):123-134. doi: 10.1177 / 0885328220913057. Epub 2020 Mar 17. PMID: 32183581.)

[0189] Comparative mass spectrometry and IR spectroscopy measurements of Medications 1 and 2 were performed. IR spectroscopy results show that both samples are constituent with carboxymethyl starch sodium salt. The IR spectroscopy analysis did not find any differences between the two samples except for the five signals for trisodium citrate in the sample of the pharmaceutical composition of the invention (Medication 1). Results of the mass spectrometry measurements further confirm that the pharmaceutical composition of the invention (Medication 1) contains sodium citrate while Medication 2 does not. Mass spectrometry did not reveal any further differences as well.

[0190] Sterilization by irradiation

[0191] The raw material powder made from modified starch (Medication 1 or Medication 2 or Medication 3) was sterilized by irradiation (gamma or beta rays). The liquids (isotonic saline solution, Ringer's solution, other isotonic solutions) were also sterile.

[0192] Gel by mixing irradiated sterile powder and sterile liquid

[0193] The irradiated modified starch powder was either introduced into the body cavity and then transformed into a gel in the body cavity by adding sterile liquid; or the sterilized starch powder was mixed with sterile liquid in a sterile container to form a gel by means of a stirring process and then introduced into the body cavity as a gel.

[0194] The mixing ratio was 1 g of radiation-sterilized modified starch and 8 to 12 ml of sterile saline solution. Gel by mixing powder of modified starch and sterilization of resulting gel by autoclaving A starch was gel prepared by mixing modified starch (Medication 1 or Medication 2 or Medication 3), which has not been irradiated, with aqueous solution (0.9% saline solution), mixing ratio 1g powder and 10 ml 0.9% saline solution, preheated on a hot plate at 62.5 °C for 30 minutes and then sterilized by autoclaving at 121 °C for 20 min.

[0195] Ready-to-use sterile gel by mixing radiation-sterilized starch and sterile solution, microscopy Figures 1 and 2 show gels made from unsterilized powder of a modified starch and aqueous solution (0.9% saline solution) on the day of preparation, mixing ratio 1g powder and 10 ml solution. The gels were analyzed on the day of preparation and after three months of natural aging at room temperature. Figure 1 was captured under the microscope after steps a) and b) were carried out, while figure 2 was captured after steps a), b), and c) were carried out.

[0196] Conclusion

[0197] In modified starch gels, which were preheated after mixing, the starch particles retained their structure. In contrast to unheated unsterilized starch, which exhibited about 5 to 10 % of particles with refraction similar to crystals, particles with morphological changes in the particle structure were calculated to count less than 1 %. The starch particles in gels with or without preheating remained morphologically unchanged after 3 months.

[0198] Figures 3 to 5, 12 and 13 show gels made from radiation-sterilized powder of Medication 1, Medication 2, and Medication 3, respectively, and aqueous solution (0.9% saline solution) on the day of preparation, mixing ratio 1g powder and 10 ml solution. The gels were analyzed on the day of preparation and after three months of natural aging at room temperature.

[0199] Figures 3 and 4 show unaged gels on the day of manufacture. Particles of different configurations can be seen here. The majority of the gel particles have a configuration similar to that of swollen modified starch with a dense edge and internal structure due to the layering of the starch in the particles. A smaller part (5 to 10% of the particles) is strongly contrasted, appears more refractive and is not swollen. Figure 4 shows the differently configured particles at higher magnification. During natural ageing at room temperature for 3 months (Fig. 5), the strongly contrasted particles remain unchanged. In contrast, the swollen particles show a strong reduction in their microscopically visible structure after ageing and thus signs of dissolution. The low stability of the microscopic morphology is already evident after 3 months.

[0200] Conclusion

[0201] 1. After irradiation, two microscopically different particle configurations are present in a gel of modified starch:

[0202] Particles as known from modified starch, swelling with water while retaining the particle structure

[0203] Highly contrasted particles, internal structure highly refractive, non-swelling

[0204] 2. The strongly contrasted particles are preserved after three months of ageing

[0205] 3. The swollen particles are unstable after three months of ageing

[0206] 2,1,2 Ready-to-use sterile gel by mixing modified starch and aqueous solution, sterilized by autoclaving and subsequent microscopic analysis

[0207] Figures 6 to 8 show gels prepared by mixing modified starch of Medication 1 with aqueous solution (0.9% saline solution), preheated at a temperature of 62.5 °C for 30 minutes and then sterilized by autoclaving, mixing ratio 1g powder and 10 ml 0.9% saline solution.

[0208] Figure 6 shows an unaged gel on the day of production. The starch particles are uniformly swollen and uniformly configured by absorption of liquid. They are irregularly bounded and show an internal structure resulting from the stratification of the starch, as known from swollen modified starch. The contrast of the particles appears stronger than with the radiation-sterilized particles, after water absorption. In contrast to irradiated starch, there are no particles that appear refractive like crystals and are not swollen.

[0209] Figure 7 shows starch after a natural ageing period of 3 months. There are no microscopic changes compared to the unaged starch. In contrast to irradiated starch, in which the swollen particles are only vaguely recognizable after ageing and appear dissolved, the structure of the particles is completely preserved. Particles with refraction reminiscent of crystals, as with irradiated starch, are still not present.

[0210] Figure 8 shows the autoclaved starch after freezing and thawing twice. The freezing and thawing processes also have no influence on the microscopic structure of the autoclaved gel.

[0211] Conclusion

[0212] In modified starch gels, which were preheated first and then sterilized by autoclaving, the starch particles retain their structure. In contrast to radiation sterilization, there are no morphological changes in the particle structure that cancel out the swelling of the particles. The starch particles in gels sterilized by autoclaving remain morphologically unchanged after 3 months and remain stable during freezing and thawing.

[0213] Example 2: In vivo comparison of gels made from Medication 1:

[0214] Gel from powder (Medication 1), powder first sterilized by irradiation, then mixed with sterile liquid versus

[0215] Gel from powder (Medication 1) first mixed with liquid, then sterilized with steam sterilization

[0216] The pharmaceutical composition of the invention used in the treatment group was medication 1 as described above. Medication 1 is a starch powder which becomes a gel when mixed with an aqueous solution. The gel of starch powder Medication 1 mixed with 0.9% saline solution or Ringer' s solution and other similar isotonic solutions has been shown to be effective as medical device for adhesion prevention.

[0217] The local use of a medical product in the body requires sterility. A sterile gel can be prepared either by sterilization of starch powder Medication 1 by irradiation and mixing the sterile powder with sterile aqueous solution (gel method 1) or by mixing starch powder Medication 1 with aqueous solution followed by steam sterilization (gel method 2)

[0218] An in vivo study was conducted to compare gel method land gel method 2. The in vivo comparison was carried out on rats using the established optimized peritoneal adhesion model (OP AM). The adhesion inhibition efficacy and the histologically traceable degradation process of gel method 1 and gel method 2 were studied.

[0219] Thereby, an untreated control group where the animals only received sterile 0.9% Ringer’s solution was used as baseline.

[0220] Thirty Lewis rats were used for the study. They were housed under standard conditions, had continuous access to fresh water and were fed with standard nutrition. Their well-being was monitored through daily examinations of observed changes during application. In addition, they were weighed on a regular basis so that deviations from normal body weight development could be determined. All protocols with respect to quality of life of the animals were carried out in compliance with national and European laws.

[0221] Ten animals were each randomly assigned to one of the three groups:

[0222] 1. Control group (no adhesion inhibition measures were taken, animals only received sterile 0.9% Ringer's solution)

[0223] 2. Group with gel method 1 (starch powder medication 1 , powder first sterilized by irradiation, then mixed with sterile Ringer' s solution)

[0224] 3. Group with gel method 2 (starch powder medication 1, first mixed with Ringer's solution, then sterilized with steam sterilization)

[0225] Ketamine and xylazine were used for the anesthesia. The required level of narcosis was achieved once the flexor-reflexes were suppressed. After shaving, the abdomen was cleaned with ethanol and iodine solutions. Access to the abdomen was achieved by a median laparotomy of approximately 3.5 cm. The induction of adhesions was carried out in accordance with the OP AM. The cecum was exposed and kept moist. The visceral peritoneum of the cecum was then repeatedly rubbed with a watery gauze over an area 1x2 cm in size. A surface of the parietal peritoneum on the right abdominal wall 1x2 cm in size, including the inner muscle layer was sharply resected.

[0226] After both defects were created, gels were applied upon them. In group 2 and 3, a total of 3 mL of gel in each animal were applied. After application of the respective interventions, the two defects were drawn in spatial proximity to one another using a 4 / 0 prolene stitch in order to induce maximum adhesions. The abdomen was closed with a dual layer closing technique using a consecutive stitch. The animals were monitored postoperatively until they were fully awakened.

[0227] On the seventh postoperative day, the animals were sacrificed. In order not to destroy a potentially formed adhesion, the peritoneal cavity was opened through an incision to the left of the original laparotomy scar. During the autopsy, the adhesion formation between the defective abdominal wall and the cecum was macroscopically evaluated. For this purpose, the following system was utilized:

[0228] Score Description

[0229] 0 No adhesions

[0230] 1 Thin filmy adhesions

[0231] 2 More than one thin adhesion

[0232] 3 Thick adhesion with focal point

[0233] 4 Thick adhesion with planar attachment

[0234] 5 Very thick vascularized adhesions or more than one planar

[0235] Adhesion values are represented as arithmetic averages with standard deviations (SD). Since most of the data sets did not follow a Gaussian distribution (determined with the D'Agostino-Pearson normality test), the multiple comparison of the adhesion assessment was carried out using the Kruskal -Wallis -Test, followed by Dunn’s multiple comparison test for non-parametric data (which use the correction for multiple comparisons with the aid of statistical hypothesis testing). Groups were considered as significantly different from one another if p < 0.05.

[0236] Results

[0237] The tested animals all showed a comparable viability and body weight development. One animal of group 3 had to be prematurely killed due to complications; the other 29 animals completed the experiment.

[0238] Adhesions were scored in the four groups as follows: • Group 1: control (only sterile 0.9% Ringer's solution): 9 of 10 animals showed massive adhesions, each of which received the maximum number of points (five), whereas the tenth animal developed no adhesions at all and therefore received a zero. The arithmetic mean was 4.5 (SD: 1.6).

[0239] • Group 2: pharmaceutical composition of the invention (gel method 1): None of the animals developed any adhesions, so that all animals were scored with a zero. The arithmetic mean was 0.0 (SD: 0.0).

[0240] • Group 3: pharmaceutical composition of the invention (gel method 1): One animal did not receive a score as it had to be prematurely terminated due to complications. None of the other animals developed any adhesions, so that all animals were scored with a zero. The arithmetic mean was 0.0 (SD: 0.0).

[0241] In comparison to the control, both treatment groups were able to achieve the following adhesion reductions (calculated as (mean of the control group - mean of the treatment group) / mean of the control group * 100):

[0242] Group 2: 100%

[0243] Group 3: 100%

[0244] The statistical comparison using Dunn’s multiple comparison as describe above yielded:

[0245] Comparison p-value Significant?

[0246] Group 1 vs. group 2 <0.001 Yes

[0247] Group 1 vs. group 3 <0.001 Yes

[0248] Group 2 vs. group 3 >0.999 No

[0249] Group 2 (gel method 1) and group 3 (gel method 3) both showed a significant reduction in adhesion compared to controls. Groups 2 and 3 were not significantly different from one another.

[0250] The results of this study show that macroscopically there are hardly any to no postoperative adhesions apparent in previously traumatized mesothelial (or mesothelium-like) surfaces after one week of treatment as well with gel method 1 and gel method 2. Gel method 1 and Gel method 2 are equally suitable for preventing macroscopical adhesions.

[0251] Histology, healing of abdominal wall defects (OP AM model, rat) with the support of radiation- sterilized modified starch (gel method 1)

[0252] The efficiency of the pharmaceutical composition of the present invention to prevent adhesion formation was tested in rats utilizing the Optimized Peritoneal Adhesion Model (OP AM). OP AM technique provides a superior, reproducible induction of severest adhesions of injured areas., i.e. agglutination. Due to the high reproducibility in incidence and extend of adhesion formation, the OP AM is ideal to determine the efficacy of adhesion inhibiting medical devices.

[0253] In addition, with OPAM healing processes in tissue defects can be analyzed, as in this model a defined abdominal wall defect is created.

[0254] Figures 9 and 10 show histological sections through the regeneration zone of an abdominal wall defect created according to the OPAM model, treated with radiation-sterilized starch. The abdominal wall defect is filled by the formation of connective tissue. The connective tissue is covered by a new mesothelial epithelium towards the abdominal cavity. This is essential to prevent the formation of adhesions. Hematoxylinophilic spiral remnants of the irradiated starch are found in the healing connective tissue.

[0255] Figure 9 shows that the spiral remnants are distributed across the regeneration zone. In Figure 10, cells corresponding to fibroblasts are arranged in a circle around the spiral remnant of the starch. This configuration can lead to a microgranuloma. The formation of microgranulomas can be seen in the histological section in Figure 15. Here, the presence of irradiated modified starch particles without preserved water absorption has led to delayed resorption of the particles with circular arrangement of fibroblasts, resulting in the formation of three microgranulomas.

[0256] Histology, healing of abdominal wall defects (OPAM model, rat) with the support of autoclaved modified starch (gel method 2)

[0257] Figure 11 shows the healing of the abdominal wall in an abdominal wall defect after OPAM treated with autoclaved starch. The autoclaved starch is broken down by macrophages and giant cells. Residues of starch are found in these cells, small residues of starch in the connective tissue outside the cells. There are no hematoxylinophilic spiral remnants in the connective tissue or circularly arranged fibroblasts in the sense of incipient granuloma formation. Further details of the healing process after treatment with starch gel sterilized by autoclaving can be found in Figures 16-18, containing histological images in low (Figure 16), medium (Figure 17) and high (Figure 18) magnification. Figure 16 shows a dense area of starch that is degraded uniformly without the formation of microgranulomas, as well as an intact mesothelial coverage. In Figure 17, a dense area of starch being degraded uniformly without microgranuloma formation and an intact mesothelium can also be detected, as well as the presence of mononuclear cells. Figure 18 indicates the presence of starch remnants within mononuclear cells, in the tissue and within a giant cell. Furthermore, it corroborates the observation of uniform starch degradation without microgranuloma formation. Figures 16-18 clearly evidence that starch gel sterilized by autoclaving is degraded uniformly without the formation of microgranulomas.

[0258] Summary and conclusion

[0259] In starch gel, which is made from radiation-sterilized particles of modified starch (i.e. gel method 1),

[0260] 90 to 95% of the particles have a microscopic structure like native, modified, liquidabsorbing starch. These particles have a limited shelf life after ageing.

[0261] 5 to 10% of the particles are microscopically strongly contrasted, have a light refraction similar to crystals and do not swell. These particles are more durable, but are insignificant for function. Histologically, spiral, hematoxylinophilic remnants of these particles can be seen in the tissue after a week, which can become the nucleus of microgranulomas .

[0262] In starch gel sterilized by autoclaving (i.e. gel method 2), the particles are uniformly swollen and uniformly configured.

[0263] In contrast to irradiated starch, there are no particles that appear refractive like crystals and do not swell Under the light microscope, they remain unchanged after natural ageing for over 3 months. They are also unchanged after repeated freezing and thawing. Histologically, after a week of irritation-free healing without spiral, hematoxylinophilic residues and correspondingly no granuloma formation can be seen.

[0264] Histologically, one week after implantation, spiral, hematoxylinophilic spiral remnants can be seen in radiation-sterilized gel particles, which may be the core of microgranulomas.

[0265] A ready-to-use gel of modified starch and aqueous solution, sterilized by autoclaving, is superior to a gel of radiation-sterilized modified starch particles.

Claims

Claims1. A gel of a modified starch, preferably a gel of a carboxymethylated starch or an etherified starch, which comprises an aqueous liquid and which is producible by a process comprising the steps of: a) Providing a modified starch, preferably a carboxymethylated starch or an etherified starch, as a powder; b) Premixing the powder of step a) with an aqueous liquid in an amount sufficient for forming a gel of modified starch; and c) Preheating the mixture obtained according to step b) at a temperature in the range of 60 °C to 80 °C, and d) Steam-sterilizing the preheated gel of the modified starch comprising an aqueous liquid according to steps b) and c).

2. The gel of the modified starch according to claim 1, wherein said gel of the modified starch is free of refractive crystals, preferably is free of refractive crystals that do not swell in the aqueous liquid.

3. The gel of the modified starch according to claim 1 or 2, further comprising 1 to 20 percent by weight of a compound selected from the group consisting of citric acid, a salt of citric acid, and a mixture thereof, relative to the weight of a carboxymethylated starch.

4. The gel of the modified starch according to any of claims 1 to 3, wherein said pharmaceutical composition comprises a carboxymethylated starch and 1 to 20 percent by weight of a salt of citric acid relative to the weight of a carboxymethylated starch, wherein said salt of citric acid has a solubility in water of greater than 100 g / 1, preferably greater than 200 g / 1, more preferably greater than 300 g / 1, most preferably greater than 400 g / 1, at a temperature of 25°C.

5. The gel of the modified starch according to any of claims 1 to 4, wherein said mixture obtained according to step b) is preheated at a temperature of 62.5 °C.

6. The gel of the modified starch according to any of claims 1 to 5, wherein said salt of citric acid is selected from the group consisting of a sodium citrate, a lithium citrate and a potassium citrate.

7. The gel of the modified starch according to any of claims 3 to 6, wherein said salt of citric acid is on the surface of carboxymethylated starch particles.

8. The gel of the modified starch according to any of claims 3 to 7, wherein said gel of the modified starch comprises carboxymethylated starch and 1 to 20 percent by weight trisodium citrate relative to the weight of the carboxymethylated starch, and water, wherein said carboxymethylated starch has a molecular weight in the range of 500,000 daltons to 11,000,000 daltons, and wherein particles of the carboxymethylated starch have a particle diameter of 30 microns to 100 microns.

9. The gel of the modified starch according to any of claims 1 to 8, wherein said gel of the modified starch is cross-linked carboxymethylated starch having a degree of crosslinking in the range of 25% to 45%.

10. The gel of the modified starch according to any of claims 3 to 9, wherein said gel of the modified starch comprises crosslinked carboxymethylated starch, citrate ions and water, wherein the citrate ions are present in a concentration in the range of 1 mmole / liter to 500 mmoles / liter of the water, and wherein the crosslinked carboxymethylated starch has a degree of substitution in the range of 0.2 to 0.4 and a degree of crosslinking in the range of 25% to 45%.

11. The gel of the modified starch according to any of claims 1 to 10, wherein said aqueous liquid is selected from the group consisting of water, isotonic saline solution, or hypotonic, isotonic, or hypertonic saline solution as the liquid phase, which comprises one or more cations selected from the group consisting of sodium, potassium, ammonium, magnesium, calcium, iron(II), iron(Ill), aluminum; and / or further comprising one or more anions selected from the group consisting of fluoride, chloride, bromide, iodide, oxide, sulfide, carbonate, sulfate, phosphate, nitrate, chromate, permanganate, and hexacyanoferrate(II).

12. The gel of the modified starch according to any of claims 1 to 11, wherein said aqueous liquid is selected from the group consisting of isotonic saline solution, Ringer's solution, Ringer's acetate solution, and Ringer's lactate solution.

13. The gel of a modified starch according to any of claims 1 to 12, which is producible by a process comprising the steps of: a) Providing a modified starch, preferably a carboxymethylated starch or an etherified starch, as a powder; b) Premixing the powder of step a) with an aqueous liquid at room temperature in an amount sufficient for forming a gel of a modified starch; and c) Steam-sterilizing the gel of the modified starch comprising an aqueous liquid according to step b) in the range from 121°C to 134°C for a duration of 10 min to 30 min, preferably at a temperature of 121 °C for a duration of 20 min.

14. A pharmaceutical composition comprising a gel of a modified starch according to any of claims 1 to 13.

15. A gel of a modified starch according to any of claims 1 to 14 or a pharmaceutical composition according to claim 13 for use in the inhibition of postoperative adhesions.

16. The gel of the modified starch or the pharmaceutical composition for use according to claim 15, wherein said gel of said modified starch or said pharmaceutical composition is administered on a postoperative area or site in a subject.

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