Polymer, crosslinked body, medical composition, medical material, method for producing polymer, and method for producing crosslinked body

Abstract

The present invention provides: a polymer that can be used for preparing a composition and / or a medical material exhibiting excellent sealing properties; a crosslinked body of the polymer; and a composition and a medical material containing the polymer and the crosslinked body. The present invention also provides a method for producing a polymer that can be used for producing a composition and / or a medical material exhibiting excellent sealing properties, and a method for producing a crosslinked body of the polymer. According to the present invention, a polymer having weight average molecular weight of 3,000-50,000 and a molecular weight distribution of 1.0-1.5 is provided. The polymer includes at least one of a first repeating structural unit represented by formula (1) and a second repeating structural unit in which a carboxy group in the first repeating structural unit is converted into an active ester.

Description

Polymer, crosslinked body, medical composition, medical material, method for producing polymer, and method for producing crosslinked body

[0001] The present invention relates to a polymer, a crosslinked product, a medical composition, a medical material, a method for producing a polymer, and a method for producing a crosslinked product.

[0002] Various materials have been investigated as medical materials. For example, Patent Documents 1 and 2 describe medical materials made of active esterified poly-L-glutamic acid and gelatin.

[0003] JP-A-9-103479 JP-A-9-296039

[0004] However, some known materials have insufficient sealing properties, for example, depending on the severity of the wound. The present invention provides a polymer from which compositions and / or medical materials with excellent sealing properties can be prepared, a crosslinked product of the polymer, and a composition and medical material containing the polymer and the crosslinked product. The present invention also provides a polymer from which compositions and / or medical materials with excellent sealing properties can be prepared, and a method for producing a crosslinked product of the polymer.

[0005] According to the present invention, there is provided a polymer having a weight average molecular weight of 3,000 to 50,000 and a molecular weight distribution of 1.0 to 1.5, the polymer comprising at least one of a first repeating structural unit represented by the following formula (1) and a second repeating structural unit in which a carboxy group in the first repeating structural unit is converted into an active ester:

[0006] As a result of extensive research, the present inventors have found that by using a polymer having a specific weight-average molecular weight and molecular weight distribution and having specific repeating structural units, a composition and / or a medical material having excellent sealing properties can be obtained, leading to the completion of the present invention.

[0007] Various embodiments of the present invention are exemplified below. The embodiments shown below can be combined with each other. [1] A polymer having a weight average molecular weight of 3,000 to 50,000 and a molecular weight distribution of 1.0 to 1.5, the polymer comprising at least one of a first repeating structural unit represented by the following formula (1) and a second repeating structural unit in which a carboxy group in the first repeating structural unit is converted into an active ester: [2] The polymer according to [1], having the second repeating structural unit. [3] A crosslinked product of the polymer according to [2] and a polymer C having an amino group. [4] A medical composition comprising at least one of the polymer according to [1] and the crosslinked products, wherein the crosslinked product is formed by crosslinking a polymer B and a polymer C, wherein the polymer B is the polymer according to claim 1 and has the second repeating structural unit, and the polymer C has an amino group. [5] The medical composition according to [4], which is used as a bioadhesive, a hemostatic material, a vascular embolization material, or an aneurysm sealant. [6] A medical material having a first agent containing the polymer according to [2] and a second agent containing a polymer C having an amino group. [7] A method for producing a polymer A, comprising a synthesis step, wherein the polymer A has a weight-average molecular weight of 3,000 to 50,000, a molecular weight distribution of 1.0 to 1.5, and is composed of a first repeating structural unit represented by the following formula (1), and in the synthesis step, the polymer A is synthesized by reaction (I) catalyzed by a RimK protein: [8] A method for producing polymer B, comprising an active esterification step, in which at least a portion of the carboxy groups in polymer A obtained by the production method described in [7] is actively esterified with an active esterifying agent to obtain polymer B having an actively esterified functional group. [9] A method for producing a crosslinked body, comprising a crosslinking step, in which the crosslinked body is a crosslinked body of polymer B obtained by the production method described in [8] and polymer C having an amino group, in which the crosslinking step crosslinks the actively esterified functional group contained in polymer B with the amino group contained in polymer C to obtain the crosslinked body.

[0008] The polymer and crosslinked product of the polymer according to the present invention can be used to prepare a composition and / or medical material with excellent sealing properties. The production method according to the present invention can be used to produce a polymer and crosslinked product of the polymer that can be used to prepare a composition and / or medical material with excellent sealing properties.

[0009] FIG. 1 shows a schematic diagram of a test device for evaluating sealing properties in Examples and Comparative Examples.

[0010] The present invention will be described in detail below by illustrating embodiments of the present invention. The present invention is not limited by these descriptions. The features of the embodiments of the present invention described below can be combined with each other. Furthermore, each feature can be an invention independently. Any number of "0"s (for example, one or two) may be added to the end of the numerical values ​​disclosed in the following description. For example, one or two "0"s may be added after "1.4" to make it "1.40" or "1.400".

[0011] 1. Polymer A polymer according to one embodiment of the present invention has a weight-average molecular weight of 3,000 to 50,000 and a molecular weight distribution of 1.0 to 1.5. The polymer also contains at least one of a first repeating structural unit represented by the following formula (1) and a second repeating structural unit in which a carboxy group in the first repeating structural unit is converted into an active ester:

[0012]

[0013] The polymer has a weight average molecular weight (Mw) of 3,000 to 50,000, preferably 4,000 to 45,000, and more preferably 5,000 to 40,000. The weight average molecular weight of the polymer is, for example, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 2800 The values ​​are 0, 29000, 30000, 31000, 32000, 33000, 34000, 35000, 36000, 37000, 38000, 39000, 40000, 41000, 42000, 43000, 44000, 45000, 46000, 47000, 48000, 49000, and 50000, and may be within a range between any two of the values ​​exemplified here.

[0014] When the weight-average molecular weight of the polymer is within the above range, it is easy to adjust the molecular weight distribution (weight-average molecular weight (Mw) / number-average molecular weight (Mn)) to a narrow range, and the cohesive strength of a crosslinked body using the polymer can be improved. In other words, when a crosslinked body is produced, the gel fraction can be increased, and a gel with high strength can be obtained.

[0015] The molecular weight distribution of the polymer is 1.0 to 1.5, preferably 1.0 to 1.4, more preferably 1.0 to 1.3, and even more preferably 1.0 to 1.2. The molecular weight distribution of the polymer may be, for example, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5, and may be within a range between any two of the values ​​exemplified here. When the molecular weight distribution is within the above range, the cohesive strength of a crosslinked body using the polymer can be improved; that is, when a crosslinked body is produced, the gel fraction can be increased, and a gel with high strength can be obtained. Furthermore, such a gel has excellent water resistance. The weight average molecular weight and molecular weight distribution can be measured by the method described in the examples.

[0016] A polymer according to one embodiment of the present invention comprises at least one of a first repeating structural unit represented by the following formula (1) and a second repeating structural unit in which a carboxy group in the first repeating structural unit is converted into an active ester:

[0017]

[0018] The first repeating structural unit is the same as the repeating structural unit constituting poly-α-glutamic acid. The second repeating structural unit can have a structure in which the carboxy group in the first repeating structural unit is actively esterified, and can have a structure in which the carboxy group in the first repeating structural unit is actively esterified by reacting with an active esterifying agent (preferably in the presence of a dehydration condensation agent).

[0019] Examples of the active esterifying agent include N-hydroxyamine-based active esterifying agents, such as N-hydroxysuccinimide, 1-hydroxybenzotriazole, N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide, 2-hydroxyimino-2-cyanoethyl acetate, and 2-hydroxyimino-2-cyanoacetic acid amide. The active esterifying agent preferably contains at least one of the above-mentioned agents. Among these, N-hydroxysuccinimide is preferred from the viewpoint of safety. The active esterifying agent can be used alone or in combination of two or more.

[0020] As an example, the second repeating structural unit can be represented by the following formula (2).

[0021] In formula (2), R １ and R ２ can be any organic group, and R １ and R ２ are more preferably linked to form a cyclic structure.

[0022] As a more specific example, when N-hydroxysuccinimide is used as the active esterifying agent, the second repeating structural unit can be represented by the following formula (3).

[0023] As described above, the first repeating structural unit has an ethylene group in its side chain and a carboxy group connected via the ethylene group. Similarly, the second repeating unit has an ethylene group in its side chain and an active esterified structure connected via the ethylene group. On the other hand, poly-γ-glutamic acid, which is the main component of the viscosity of natto, is generally well known and widely available as polyglutamic acid, and the repeating structural unit constituting poly-γ-glutamic acid is shown below, with the carboxy group being closer to the main chain.

[0024]

[0025] In the first repeating structural unit and the second repeating structural unit according to one embodiment of the present invention, the carboxy group or the active esterified structure is located away from the main chain, and therefore can react without being restricted by steric hindrance or the like, and gelation proceeds in a short time, and a crosslinked product with a high crosslinking density can be obtained, which is presumed to result in a medical composition and medical material with high sealing properties.

[0026] A polymer according to one embodiment of the present invention preferably contains a total of 70% by mass or more of the first repeating structural unit and the second repeating structural unit, based on 100% by mass of the polymer, more preferably 80% by mass or more, and even more preferably 90% by mass or more. The total content of the first repeating structural unit and the second repeating structural unit may be, for example, 70, 75, 80, 85, 90, 95, or 100% by mass, and may be within a range between any two of the values ​​exemplified here. When the total content of the first repeating structural unit and the second repeating structural unit is within the above range, gelation occurs in a short time, a high gel fraction is easily obtained, and excellent cohesive strength and water resistance are obtained, which is preferable. The total content of the first repeating structural unit and the second repeating structural unit can be measured, for example, by amino acid analysis and nuclear magnetic resonance (NMR) analysis of the hydrolysate.

[0027] The method for producing the polymer is not particularly limited, and specific examples of the production method will be described later. In one embodiment of the present invention, a polymer having a first repeating structural unit (polymer A) is produced, and then at least a portion of the carboxy groups in the polymer having the first repeating structural unit (polymer A) are actively esterified with an active esterifying agent to obtain a polymer (polymer B) having a structure in which the carboxy groups in the first repeating structural unit are actively esterified. Hereinafter, a polymer having the first repeating structural unit but not the second repeating structural unit, i.e., a polymer before active esterification, will also be referred to as polymer A. Furthermore, a polymer having the second repeating structural unit, i.e., a polymer after active esterification, will also be referred to as polymer B.

[0028] A polymer according to one embodiment of the present invention may be referred to as polymer A. Polymer A is a polymer having a first repeating structural unit but not a second repeating structural unit, a polymer prior to active esterification, and a precursor of polymer B. Polymer A preferably contains 70% by mass or more of the first repeating structural unit, more preferably 80% by mass or more, and even more preferably 90% by mass or more, relative to 100% by mass of polymer A. The content of the first repeating structural unit in polymer A may be, for example, 70, 75, 80, 85, 90, 95, or 100% by mass, or may be within a range between any two of the values ​​exemplified here. Polymer A may contain a structural unit other than the first repeating structural unit, for example, a repeating structural unit constituting poly-γ-glutamic acid. It may also have a structure derived from an amino acid other than glutamic acid. Polymer A may also be composed of the first repeating structural unit, i.e., poly-α-glutamic acid. Glutamic acid exists in the L-form and the D-form, and the glutamic acid constituting polymer A according to one embodiment of the present invention may be either the L-form or the D-form. For example, polymer A may be poly-α-glutamic acid consisting of only L- or D-form glutamic acid, or may be poly-α-glutamic acid containing a mixture of L- and D-form glutamic acid.

[0029] A polymer according to one embodiment of the present invention may be Polymer B. Polymer B is a polymer having a second repeating structural unit and is a polymer after active esterification. Polymer B preferably contains the first repeating structural unit and the second repeating structural unit in a total amount of 70% by mass or more, more preferably 80% by mass or more, and even more preferably 90% by mass or more, relative to 100% by mass of Polymer B. The total content of the first repeating structural unit and the second repeating structural unit may be, for example, 70, 75, 80, 85, 90, 95, or 100% by mass, and may be within a range between any two of the values ​​exemplified here.

[0030] Polymer B may have a first repeating structural unit. The content of the second repeating structural unit in Polymer B is preferably 5 to 90 mol%, more preferably 30 to 80 mol%, relative to the total 100 mol% of the first repeating structural unit and the second repeating structural unit contained in Polymer B. The content of the second repeating structural unit in Polymer B may be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 mol%, and may be within a range between any two of the values ​​exemplified here. That is, Polymer B can be one in which, among the first repeating structural units contained in Polymer A, carboxy groups within the above numerical range are actively esterified. By adjusting the content of the second repeating structural unit, the crosslinking density and gel fraction of the crosslinked product can be adjusted. The content of each repeating structural unit in the polymer can be measured, for example, by amino acid analysis and nuclear magnetic resonance (NMR) analysis of the hydrolyzate.

[0031] When a polymer B solution and a gelatin solution are prepared by the method described in the Examples and the gel fractions are measured, the polymer B preferably has a gel fraction of 50% or more. The gel fraction may be, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, or may be within a range between any two of the values ​​exemplified here.

[0032] When a polymer B solution and a gelatin solution are prepared by the method described in the Examples and the gel generation time is measured, the polymer B preferably has a gel generation time of 20 minutes or less, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 minutes, or may be within a range between any two of the values ​​exemplified here.

[0033] When a polymer B solution and a gelatin solution are prepared by the method described in the Examples, a membrane is formed, and the sealing property is measured by applying pressure with saline solution. Preferably, saline does not leak even when a pressure of 10 kPa or more is applied, and more preferably, saline does not leak even when a pressure of 15 kPa or more is applied. The pressure at which saline leaks is, for example, 10, 15, 20, 25, or 30 kPa, and may be within a range between any two of the values ​​exemplified here.

[0034] Polymer B was prepared in a polymer B solution and a gelatin solution by the method described in the Examples, and the adhesive strength to a living body was measured. The adhesive strength was 5 N / m ２ The adhesive strength is preferably, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 N / m. ２ and may be in a range between any two of the values ​​given here.

[0035] The gel fraction, gel generation time, sealing property, and adhesive strength, which are evaluated by preparing a solution of polymer B and a specific gelatin, can be controlled by adjusting the type and amount of repeating structural units constituting the polymer, the weight-average molecular weight of the polymer, and the molecular weight distribution.

[0036] 3. Crosslinked Product A crosslinked product according to one embodiment of the present invention is a crosslinked product of a polymer having a second repeating structural unit and a polymer C having an amino group. A crosslinked product according to one embodiment of the present invention can be a crosslinked product of a polymer B and a polymer C having an amino group.

[0037] Examples of the polymer C having an amino group include polymers containing multiple amino groups, such as gelatin, albumin, casein, collagen, polylysine, polyornithine, and chitosan. The polymer C having an amino group preferably includes at least one of the above groups. Among these, gelatin is preferred from the viewpoint of safety. The polymer C having an amino group can be used alone or in combination of two or more.

[0038] The number of amino groups in polymer C is preferably 0.2 to 7.0 mmol / g, more preferably 0.4 to 4.0 mmol / g. The number of amino groups in polymer C is, for example, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 mmol / g, and may be within a range between any two of the values ​​exemplified here. When the proportion of amino groups in polymer C having amino groups is within the above range, a higher gel fraction is easily obtained upon crosslinking with a polymer having a second repeating structural unit, and excellent cohesive strength and water resistance are achieved, which is preferable. The number of amino groups was calculated by a colorimetric test using 2,4,6-trinitrobenzenesulfonic acid.

[0039] The weight average molecular weight (Mw) of the polymer C having an amino group is not particularly limited, but is preferably 5,000 to 1,000,000, more preferably 8,000 to 500,000, and even more preferably 10,000 to 100,000. The weight average molecular weight (Mw) of the polymer C having an amino group is, for example, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, or 500,000, and may be within a range between any two of the values ​​exemplified here. An Mw within the above range is preferable because it provides excellent handleability and facilitates the attainment of sufficient cohesive strength by crosslinking with the polymer having the second repeating structural unit.

[0040] The crosslinked product can have an active esterified structure and a peptide bond (—CONH—) crosslinked by an amino group.

[0041] The proportions of polymer B and polymer C in the crosslinked product are not particularly limited, but the proportion of polymer B relative to 100% by mass of the crosslinked product is preferably 5 to 95% by mass, more preferably 15 to 85% by mass, and even more preferably 25 to 75% by mass. The proportion of polymer B relative to 100% by mass of the crosslinked product is, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% by mass, and may be within a range between any two of the values ​​exemplified here. Furthermore, the proportion of polymer C relative to 100% by mass of the crosslinked product is preferably 5 to 95% by mass, more preferably 15 to 85% by mass, and even more preferably 25 to 75% by mass. The ratio of polymer C to 100% by mass of the crosslinked body is, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% by mass, and may be within a range between any two of the values ​​exemplified here.

[0042] The crosslinked product preferably has a gel fraction of 50% or more when measured according to the method described in the Examples. The gel fraction may be, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, or may be within a range between any two of the values ​​exemplified here.

[0043] When a membrane is formed from the crosslinked product according to the method described in the Examples and pressure is applied using saline to measure sealing ability, preferably, saline does not leak even when a pressure of 10 kPa or more is applied, and more preferably, saline does not leak even when a pressure of 15 kPa or more is applied. The pressure at which saline leaks is, for example, 10, 15, 20, 25, or 30 kPa, and may be within a range between any two of the values ​​exemplified here.

[0044] The crosslinked product can be in the form of a film or a sheet. The thickness of the film or sheet can be adjusted depending on the application, and is, for example, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mm, or may be within a range between any two of the values ​​exemplified here. The size of the sheet can also be adjusted depending on the application, and is, for example, 1, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 cm ２ and may be within a range between any two of the numerical values ​​exemplified here. The crosslinked body according to one embodiment of the present invention can have sufficient strength and sealing properties when formed into a sheet of the above size and thickness, for example.

[0045] 4. Composition A composition according to one embodiment of the present invention includes at least one of the above-described polymers and crosslinked products. The composition according to one embodiment of the present invention may include at least one of polymer A, polymer B, and crosslinked products, and preferably includes at least one of polymer B and crosslinked products.

[0046] A composition according to one embodiment of the present invention can be used as a medical composition. Specifically, it is preferably used as any one of a bioadhesive, a hemostatic material, a vascular embolization material, an aneurysm sealant, an adhesion inhibitor, a scaffold material for tissue regeneration, and a drug carrier, and more preferably as any one of a bioadhesive, a hemostatic material, a vascular embolization material, and an aneurysm sealant. The polymer according to one embodiment of the present invention has a specific weight-average molecular weight and molecular weight distribution, and has a specific repeating structural unit in which the functional group of the side chain is appropriately spaced from the main chain. Therefore, when a crosslinked product is produced, gelation can be promoted in a short time, and the gel fraction can be increased, resulting in a gel with high strength and excellent water resistance and excellent sealing properties, making it suitable for medical use, particularly the applications specifically mentioned above.

[0047] Specifically, a composition containing polymer B according to one embodiment of the present invention can be used as a medical material such as a bioadhesive, a hemostatic material, a vascular embolization material, or an aneurysm sealant. A crosslinked product having excellent sealing power can be obtained by mixing a composition containing polymer B with a composition containing polymer C having an amino group. A composition containing polymer B can be preferably used in combination with a composition containing polymer C for the above-mentioned applications. A composition containing polymer A according to one embodiment of the present invention can be used as a precursor for polymer B, and can be used as a precursor for preparing a medical material. A composition containing a crosslinked product according to one embodiment of the present invention has excellent sealing power and can be used as a bioadhesive, a hemostatic material, a vascular embolization material, or an aneurysm sealant, and can also be used, in particular, as an adhesion inhibitor, a scaffolding material for tissue regeneration, or a drug carrier.

[0048] The composition according to one embodiment of the present invention may contain other ingredients depending on the intended use, such as distilled water, physiological saline, buffer solutions such as sodium bicarbonate, boric acid, and phosphate, and organic solvents (DMF, DMSO, and ethanol), which are non-toxic.

[0049] 5. Medical Material A medical material according to one embodiment of the present invention may contain the above-described composition, and may contain at least one of the above-described polymers and crosslinked bodies. The composition according to one embodiment of the present invention may contain at least one of polymer A, polymer B, and crosslinked bodies, and preferably contains at least one of polymer B and crosslinked bodies.

[0050] A medical material according to one embodiment of the present invention comprises a first agent containing a polymer B having a second repeating structural unit and a second agent containing a polymer C having an amino group. This medical material can be used, among other things, as a bioadhesive, a hemostatic material, a vascular embolization material, and an aneurysm sealant. For example, the first agent and the second agent can be applied (coated) directly to the affected area of ​​a living body, causing a crosslinking reaction in the living body, thereby achieving hemostasis, etc.

[0051] As another example, a medical material according to one embodiment of the present invention can include the above-mentioned crosslinked body, and the crosslinked membrane or sheet can be applied (attached) to an affected area of ​​a living body to stop bleeding, etc.

[0052] 6. Method for Producing Polymer A The method for producing polymer A, i.e., a polymer having a first repeating structural unit, is not particularly limited. The method for producing polymer A according to one embodiment of the present invention may include a synthesis step, which may be at least one of a chemical synthesis method, a synthesis method using an enzyme (enzymatic method), and a fermentation method using a microorganism. From the viewpoint of easily obtaining a narrow molecular weight distribution, the synthesis step is preferably a step using either an enzymatic method and / or a fermentation method, and more preferably a step using an enzymatic method.

[0053] When a chemical synthesis method is used, the method for producing a polymer according to one embodiment of the present invention may include a fractionation step in which the polymer obtained by the chemical synthesis method is subjected to a known fractionation method such as GPC to adjust the weight-average molecular weight and molecular weight distribution, particularly the molecular weight distribution, of the polymer. As an example, in the fractionation step, polymer A having a specific weight-average molecular weight and molecular weight distribution can be obtained by hydrolyzing the polymer A by adding an acid, treating the obtained hydrolyzate by GPC, and collecting and freeze-drying an eluate having a specific retention time range.

[0054] When an enzymatic method is used, polymer A can be synthesized by reaction (I) catalyzed by RimK protein in the synthesis step. Here, polymer A has a weight-average molecular weight of 3,000 to 50,000, a molecular weight distribution of 1.0 to 1.5, and includes a first repeating structural unit represented by the following formula (1):

[0055]

[0056] When polymer A is synthesized by an enzymatic method, polymer A is preferably composed of the repeating structural unit represented by formula (1), and polymer A is more preferably poly-α-glutamic acid. In the enzymatic method, polymer A can be synthesized, for example, by reaction (I) catalyzed by a RimK protein. Specifically, poly-α-glutamic acid (αPGA) composed essentially of the repeating structural unit represented by formula (1), derived from glutamic acid, as its only constituent unit can be synthesized by dissolving a RimK protein, a glutamic acid source (e.g., sodium L-glutamate), and adenosine triphosphate (ATP) in a buffer solution and incubating the solution. Here, the RimK protein is an enzyme having αPGA synthetase activity that synthesizes αPGA using simple glutamic acid as a substrate. The RimK protein disclosed in JP 2011-229416 A can be used. The buffer solution preferably contains a magnesium salt, and one example is magnesium sulfate.

[0057] Preferably, the reaction system further includes reaction (II) catalyzed by polyphosphate kinase (PPK). Specifically, by adding PPK and polyphosphate to the reaction system, ATP consumed in the synthesis of αPGA by the RimK protein is regenerated by the PPK, thereby reducing the amount of ATP used, which is a relatively expensive material, and lowering production costs.

[0058] As an example, in the synthesis step, sodium L-glutamate can be added at concentrations of 1 to 100 mM, RimK protein at 0.1 to 5 mg / mL, ATP at 1 to 100 mM, a magnesium salt such as magnesium sulfate at 1 to 100 mM, PPK at 0.01 to 1 mg / mL, and polyphosphate at 1 to 100 mM.

[0059] The pH of the reaction solution is preferably 8.0 to 10.0, for example, 8.0, 8.5, 9.0, 9.5, or 10.0, and may be within a range between any two of the values ​​exemplified herein. The incubation temperature is, for example, 20, 25, 30, 35, or 40°C, and may be within a range between any two of the values ​​exemplified herein. The incubation time is, for example, 5, 10, 15, 20, 25, 30, 35, or 40 hours, and may be within a range between any two of the values ​​exemplified herein.

[0060] The enzymatic method may include a step of inactivating the protein by heating at a high temperature after synthesis. It may also include a step of separating the product by centrifugation and a purification step of removing low-molecular-weight contaminants. Furthermore, the method for producing polymer A may include a step of desalting the resulting product, for example, a step of desalting sodium poly-α-glutamate to obtain poly-α-glutamic acid.

[0061] 7. Method for Producing Polymer B The method for producing polymer B, i.e., the polymer having the second repeating structural unit, is not particularly limited. The method for producing polymer B according to one embodiment of the present invention may include an active esterification step. In the active esterification step, at least a portion of the carboxy groups in polymer A are converted into active esters with an active esterifying agent, thereby obtaining polymer B having an actively esterified functional group. The structure of polymer B is as described above.

[0062] Specifically, in the active esterification step, polymer A (e.g., poly-α-glutamic acid) is dissolved in a solvent, and an active esterification agent and preferably a dehydration condensation agent are added. At least a portion of the carboxy groups in polymer A are actively esterified with the active esterification agent, thereby obtaining polymer B having actively esterified functional groups. The active esterification agent may be any of the active esterification agents described above. The dehydration condensation agent may be a carbodiimide. Examples of carbodiimides that may be used include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate, and dicyclohexylcarbodiimide. Examples of solvents that may be used include N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).

[0063] The amount of active esterification agent added is preferably adjusted appropriately so that the content of the second repeating structural unit in the resulting polymer B is within the above-mentioned numerical range. In the active esterification step, the active esterification agent can be added in an amount of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 mol% relative to 100 mol% of polymer A, and may be within a range between any two of the numerical values ​​exemplified here. Furthermore, in the active esterification step, the dehydration condensation agent can be added in an amount of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 mol% relative to 100 mol% of polymer A, and may be within a range between any two of the numerical values ​​exemplified here.

[0064] The active esterification step can be carried out by shaking for 12 to 48 hours at 15 to 35° C. The method for producing polymer B can include a step of reprecipitating the reaction solution obtained in the active esterification step with a solvent such as isopropyl alcohol, washing by decantation, and freeze-drying the product to obtain polymer B.

[0065] 8. Method for Producing Crosslinked Product The method for producing a crosslinked product may include a crosslinking step. The crosslinked product is a crosslinked product of a polymer B containing a second repeating structural unit and a polymer C having an amino group. In the crosslinking step, the active esterified functional group contained in the polymer B is crosslinked with the amino group contained in the polymer C to obtain the crosslinked product.

[0066] Polymer B and polymer C are as described above. In the crosslinking step, the proportions of polymer B and polymer C that are crosslinked are not particularly limited, but the proportion of polymer B is preferably 5 to 95 mass%, more preferably 15 to 85 mass%, and even more preferably 25 to 75 mass%. The proportion of polymer C is preferably 5 to 95 mass%, more preferably 15 to 85 mass%, and even more preferably 25 to 75 mass%.

[0067] The crosslinking temperature may be 10 to 45° C., for example, 10, 15, 20, 25, 30, 35, 40, or 45° C., or may be within a range between any two of the values ​​exemplified herein. The crosslinking time may be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes, or may be within a range between any two of the values ​​exemplified herein.

[0068] According to one embodiment of the present invention, by using polymer B having a specific weight-average molecular weight and molecular weight distribution and a specific structure, gelation proceeds sufficiently at the above-mentioned crosslinking temperature and for the above-mentioned crosslinking time, resulting in a crosslinked product with a high gel fraction and high strength, and a medical composition and medical material with high sealing properties.

[0069] The present invention will be described in more detail below based on examples, but the present invention should not be construed as being limited to these examples.

[0070] Example 1 Production of Polymer A (Poly-α-glutamic Acid (αPGA)) by Enzymatic Method 340 mg of sodium L-glutamate, 120 mg of magnesium sulfate, 40 mg of ATP, 50 mg of polyphosphate (10-15 mer), 10 mg of purified RimK protein (disclosed in JP 2011-229416 A), and 10 mg of purified polyphosphate kinase (PPK) (Pa0141) protein were dissolved in 100 mL of 100 mM Tris-HCl (pH 9) (the concentrations of each component in the reaction solution were: sodium L-glutamate 20 mM, magnesium sulfate 10 mM, ATP 0.8 mM, polyphosphate (10-15 mer) 1.6 mM, purified RimK protein 0.7 mg / mL, and purified polyphosphate kinase (PPK) (Pa0141) protein). The resulting solution was incubated at 30°C for 20 hours (0.1 mg / mL) to obtain sodium poly-α-glutamate. The protein was then inactivated by heating at 90°C for 10 minutes, followed by centrifugation at 10,000 G for 10 minutes. The supernatant was desalted and then treated on a PD-10 column (fraction range 1,000-5,000 Mw) to obtain polymer A, enzymatically synthesized αPGA. The molecular weight was measured by GPC to find Mw of 6,800 and Mw / Mn of 1.1.

[0071] <Production of Polymer B (Succinimidated PGA)> 258 mg of the obtained PGA was dissolved in 0.6 mL of DMSO, and 20 mg of molecular sieves 4A, 161 mg of N-hydroxysuccinimide, and 268 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride were added, followed by shaking at 25°C for 24 hours (the concentrations of each component were PGA 2 mM, N-hydroxysuccinimide 1.4 mM, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride 1.4 mM). The reaction solution was then reprecipitated with isopropyl alcohol, washed twice more by decantation, and the product was freeze-dried to obtain succinimidated PGA, which was Polymer B.

[0072] <Weight-average molecular weight and molecular weight distribution> The weight-average molecular weight Mw and molecular weight distribution Mw / Mn of the obtained PGA, calculated in terms of standard polystyrene, were determined by gel permeation chromatography (GPC) under the following conditions: Measurement device: HLC-8320GPC (Tosoh Corporation) GPC column configuration: the following triple columns (all Tosoh Corporation): (1) TSKgel PWXL (2) TSKgel GMPWXL (3) TSKgel G2500PWXL Sample concentration: 1.0 mg / mL Mobile phase solvent: 0.2 M NaNO3 aqueous solution Flow rate: 0.6 mL / min Column temperature: 40°C

[0073] <Gel fraction> 0.1 g of the obtained succinimidated PGA was dissolved in 7 w / v % NaHCO ３ 0.2 g of bovine bone gelatin (amino group content: 0.77 mmol / g) was dissolved in 1 mL of aqueous solution to prepare a 10 w / v % succinimidated PGA solution. ３ The gelatin solution was dissolved in 0.8 mL of aqueous solution to prepare a 25 w / v% gelatin solution. 200 μL each of the prepared succinimidated PGA solution and gelatin solution was placed in a screw cap vial with an inner diameter of 10 mm and shaken in a thermostatic water bath at 37°C for 30 minutes to obtain a crosslinked product. The obtained crosslinked product was freeze-dried and its mass was measured (W1). Next, the mass of the mesh was measured in advance (W2), and the crosslinked product was wrapped in the mesh and immersed in 37°C water. After one week, it was removed, freeze-dried, and its mass was measured (W3). The gel fraction was then calculated according to the following formula: Gel fraction (%) = (W3 - W2) / W1 x 100

[0074] <Gel generation time> A succinimidated PGA solution and a gelatin solution were prepared in the same manner as in the above <Gel fraction>. 200 μL of the prepared gelatin solution was placed in a screw cap bottle with an inner diameter of 10 mm, immersed in a thermostatic water bath at 37°C, and stirred at a speed of 60 rpm / min with a 5 mm stirrer tip. 200 μL of succinimidated PGA solution was added thereto, and gel generation was confirmed visually. The time until it was confirmed that the gelled gelatin began to rotate together with the stirrer tip was measured as the gel generation time.

[0075] <Sealability> A succinimidated PGA solution and a gelatin solution were prepared in the same manner as in the above <Gel Fraction>. The succinimidated PGA solution and the gelatin solution were applied sequentially to the top surface of a collagen casing having a 3 mm diameter hole at a volume ratio of 1:1 to form a membrane with a diameter of 150 mm and a thickness of 1 mm, centered on the hole. Next, the circumference of a 300 mm diameter circle centered on the hole was fixed with a metal jig and allowed to stand for 10 minutes in a 37°C environment. Then, saline at 37°C was injected into the space below the collagen casing from the bottom side at a rate of 2 mL / min, gradually increasing the water pressure on the membrane. A schematic diagram of the test device for evaluating sealability is shown in Figure 1. The pressure applied when saline leakage to the top side occurred after the start of saline injection was measured, and the sealability was evaluated according to the following criteria. ○: The pressure applied when leakage of saline solution occurred was 15 kPa or more. △: The pressure applied when leakage of saline solution occurred was 10 kPa or more and less than 15 kPa. ×: The pressure applied when leakage of saline solution occurred was less than 10 kPa.

[0076] <Adhesive strength> A succinimidated PGA solution and a gelatin solution were prepared in the same manner as in the above <Gel fraction>. Then, two 5 cm x 2 cm pieces of fresh beef were prepared, and 20 μL of each of the succinimidated PGA solution and the gelatin solution was applied to one end of a 2 cm x 2 cm area. The other piece of beef was placed on top of the other piece, and a 200 g load was applied for 5 minutes. Then, using a tensile tester, the piece was pulled in the shear direction at a pulling rate of 10 mm / min, and the peel force was measured.

[0077] Example 2 Evaluation was carried out in the same manner as in Example 1, except that the following PGA was used instead of the enzymatically synthesized αPGA. 1 g of commercially available chemically synthesized αPGA (Peptide Institute, Inc.) was added to 10 mL of 1N hydrochloric acid and treated at 80°C for 6 hours to hydrolyze. The hydrolyzate was subjected to GPC, and the eluate with a retention time of 25 to 26 minutes was collected and freeze-dried to obtain PGA with a Mw of 7000 and a Mw / Mn of 1.2. The GPC conditions were the same as those for <Molecular weight> above.

[0078] Comparative Example 1 Evaluation was carried out in the same manner as in Example 1, except that commercially available αPGA (Peptide Institute, Inc.) (Mw 23,000, Mw / Mn 1.6) prepared by chemical synthesis was used instead of the enzymatically prepared αPGA.

[0079] Comparative Example 2 Evaluation was carried out in the same manner as in Example 1, except that the following PGA was used instead of the enzymatically synthesized αPGA. 1 g of commercially available chemically synthesized αPGA (Peptide Institute, Inc.) was added to 10 mL of 1N hydrochloric acid and treated at 80°C for 6 hours to hydrolyze. The hydrolyzate was subjected to GPC, and the eluate with a retention time of 24 to 30 minutes was collected and freeze-dried to obtain PGA with a Mw of 7000 and a Mw / Mn of 2.1. The GPC conditions were the same as those for <Molecular weight> above.

[0080] Comparative Example 3 Evaluation was carried out in the same manner as in Example 1, except that commercially available γPGA (Mitan Enterprise Co., Ltd.) (Mw 600,000, Mw / Mn 2.3) produced by a fermentation method was used instead of αPGA produced by the enzymatic method.

[0081] Comparative Example 4 Evaluation was carried out in the same manner as in Example 1, except that the following PGA was used instead of the enzymatically produced αPGA. 1 g of commercially available fermentation-produced γPGA (Mitan Enterprise Co., Ltd.) was added to 10 mL of 1N hydrochloric acid and treated at 80°C for 6 hours to hydrolyze. The hydrolyzate was subjected to GPC, and the eluate with a retention time of 23 to 28 minutes was collected and freeze-dried to obtain PGA with a Mw of 25,000 and a Mw / Mn of 1.7. The GPC conditions were the same as those for <Molecular Weight> above.

[0082] Comparative Example 5 Evaluation was carried out in the same manner as in Example 1, except that the following PGA was used instead of the enzymatically produced αPGA. 1 g of commercially available fermentation-produced γPGA (Mitan Enterprise Co., Ltd.) was added to 10 mL of 1N hydrochloric acid and treated at 80°C for 6 hours to hydrolyze. The hydrolyzate was subjected to GPC, and the eluate with a retention time of 25 to 28 minutes was collected and freeze-dried to obtain PGA with a Mw of 6500 and a Mw / Mn of 1.7. The GPC conditions were the same as those for <Molecular weight> above.

[0083] Comparative Example 6 Evaluation was carried out in the same manner as in Example 1, except that the following PGA was used instead of the enzymatically produced αPGA. 1 g of commercially available fermentation-produced γPGA (Mitan Enterprise Co., Ltd.) was added to 10 mL of 1N hydrochloric acid and treated at 80°C for 6 hours to hydrolyze. The hydrolyzate was subjected to GPC, and the eluate with a retention time of 25 to 26 minutes was collected and freeze-dried to obtain PGA with a Mw of 7000 and a Mw / Mn of 1.2. The GPC conditions were the same as those for <Molecular weight> above.

[0084]

[0085] The results in Table 1 show that succinimidated PGA prepared using αPGA, which is a polymer having a repeating structural unit represented by formula (1), takes a short time to gel, and those with a low molecular weight distribution Mw / Mn tend to have a high gel fraction. Therefore, the present invention is expected to have better water resistance and cohesive strength than conventional materials, making it suitable as a medical material.

Claims

1. A polymer having a weight average molecular weight of 3,000 to 50,000 and a molecular weight distribution of 1.0 to 1.5, comprising at least one of a first repeating structural unit represented by the following formula (1) and a second repeating structural unit in which a carboxy group in the first repeating structural unit is converted into an active ester:

2. The polymer according to claim 1, having the second repeating structural unit.

3. A crosslinked product of the polymer described in claim 2 and a polymer C having an amino group.

4. A medical composition comprising the polymer described in claim 1 and at least one of the crosslinked products, wherein the crosslinked product is formed by crosslinking polymer B and polymer C, wherein polymer B is the polymer described in claim 1 and has the second repeating structural unit, and polymer C has an amino group.

5. The medical composition according to claim 4, which is used as a bioadhesive, a hemostatic material, a vascular embolization material, or an aneurysm sealant.

6. A medical material comprising a first agent containing the polymer described in claim 2 and a second agent containing polymer C having an amino group.

7. A method for producing polymer A, comprising a synthesis step, wherein the polymer A has a weight average molecular weight of 3,000 to 50,000, a molecular weight distribution of 1.0 to 1.5, and is composed of a first repeating structural unit represented by the following formula (1), and in the synthesis step, the polymer A is synthesized by reaction (I) catalyzed by a RimK protein:

8. A method for producing polymer B, comprising an active esterification step, in which at least a portion of the carboxy groups in polymer A obtained by the production method described in claim 7 are converted into active esters with an active esterifying agent, thereby obtaining polymer B having active esterified functional groups.

9. A method for producing a crosslinked product, comprising a crosslinking step, wherein the crosslinked product is a crosslinked product of polymer B obtained by the production method described in claim 8 and polymer C having an amino group, and in the crosslinking step, an active esterified functional group contained in polymer B is crosslinked with an amino group contained in polymer C to obtain the crosslinked product.

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