Compound, composition, biological tissue embrittlement agent, biological tissue surface peeling method and biological tissue peeling kit

Abstract

A compound (Z) represented by the general formula (1) or the general formula (2). [In the general formula (1) and the general formula (2), X is each independently a carboxyl group, a carboxylate group, or a monovalent group represented by the general formula (3); at least one of the multiple Xs in the general formula (1) and the general formula (2) is a carboxyl group or a carboxylate group; at least one of the multiple Xs in the general formula (1) and the general formula (2) is a monovalent group represented by the general formula (3). ]-C(O)-Y-(AO) n -R(3) [In the general formula (3), -Y- is -O-, -NH-, or -S-; A is an alkylene group having 2 to 4 carbon atoms; R is a hydrogen atom or a monovalent hydrocarbon group having 1 to 15 carbon atoms in which the hydrogen atom may be substituted by an alkoxy group having 1 to 10 carbon atoms; n is an integer from 4 to 1000; at least one of the n A's is an ethylene group; when there are a plurality of monovalent groups represented by the general formula (3) in the general formula (1) and the general formula (2), -Y-, A, R, and n are the same or different.]#imgabs0#

Description

Technical Field

[0001] The present invention relates to a compound, a composition, a biological tissue embrittlement agent, a biological tissue surface peeling method and a biological tissue peeling kit. Background Art

[0002] With the development of regenerative medicine technology, the processing of biological tissue surfaces and the transplantation of cells have become practical. This technology is useful for the removal of abnormal tissues and the repair of intractable inflammatory tissues. As one of the intractable diseases in the intestine, short bowel syndrome caused by extensive resection of the small intestine is an intractable disease that requires long-term total parenteral nutrition (TPN) or small intestine transplantation due to residual intestinal dysfunction. Small intestine transplantation treatment is sometimes performed, but postoperative rejection and donor shortage are important issues, and a practical treatment method has not yet been established.

[0003] Short bowel syndrome requires a revolutionary treatment, and extensive research is being conducted in the field of regenerative medicine. In recent years, the use of intestinal organoids in regenerating small intestinal function has garnered significant attention. For example, a method has been reported in which small intestinal organoids are transplanted into the mouse colon epithelium after exfoliation of the mouse colon epithelium using the chelating agent ethylenediaminetetraacetic acid (EDTA) to induce tissue embrittlement and subsequent mechanical stimulation, resulting in long-term maintenance of the organoids within the animal (e.g., non-patent documents 1-3).

[0004] It should be noted that Non-Patent Document 3 is a document published after the priority date of this international patent application.

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent document 1: Genes and Development, Vol. 28, 2014, pp. 1752-1757

[0008] Non-patent literature 2: Cell Stem Cell, 2018, Vol. 22, pp. 1-6

[0009] Non-patent literature 3: Nature, 2021, Vol. 592, 1 April, 99-104 Summary of the Invention

[0010] Problems to be solved by the invention

[0011] However, in the methods described in the aforementioned non-patent documents, EDTA leaks into the bloodstream and enters the systemic circulation during the exfoliation of the large intestinal epithelium, potentially causing serious side effects such as hypocalcemia. Therefore, minimizing risks such as mucosal damage and blood transfusion of chelating agents when exfoliating normal mucosal epithelium by allowing chelating agents such as EDTA to act on the epithelium has become a challenge.

[0012] The present invention was completed in view of the processing and repair of biological tissue in regenerative medicine. The purpose of the present invention is to provide a material that has both the effect as a biological tissue embrittlement agent and safety to the living body, and to provide a safe method for exfoliating the surface of biological tissue.

[0013] Means for solving problems

[0014] The present inventors have conducted intensive studies to solve the above-mentioned problems and have consequently completed the present invention.

[0015] That is, the present invention relates to a compound (Z) represented by the general formula (1) or the general formula (2); a composition containing the compound (Z); a living tissue embrittlement agent containing the compound (Z); a method for exfoliating a living tissue surface, comprising: a step of bringing the compound (Z) into contact with living tissue to embrittle the surface of the living tissue; and a step of exfoliating cell tissue from the embrittled surface portion of the living tissue; and a living tissue exfoliation kit comprising the living tissue embrittlement agent.

[0016] [Chemistry 1]

[0017]

[0018] [Chemistry 2]

[0019]

[0020] [In general formula (1) and general formula (2), X is each independently a carboxyl group, a carboxylate group or a monovalent group represented by general formula (3);

[0021] At least one of the multiple Xs in the general formula (1) and the general formula (2) is a carboxyl group or a carboxylate group;

[0022] At least one of the plurality of Xs in the general formula (1) and the general formula (2) is a monovalent group represented by the general formula (3).]

[0023] -C(O)-Y-(AO) n -R(3)

[0024] [In general formula (3), -Y- is -O-, -NH-, or -S-; A is an alkylene group having 2 to 4 carbon atoms; R is a hydrogen atom or a monovalent hydrocarbon group having 1 to 15 carbon atoms in which the hydrogen atom may be substituted by an alkoxy group having 1 to 10 carbon atoms; n is an integer from 4 to 1000; at least one of the n A's present is an ethylene group; when there are multiple monovalent groups represented by general formula (3) in general formula (1) and general formula (2), -Y-, A, R, and n may be the same or different.]

[0025] Effects of the Invention

[0026] According to the present invention, a material that achieves both an effect as a biological tissue embrittlement agent and safety to a living body can be provided, and a safe method for exfoliating a biological tissue surface can be provided.

[0027] Furthermore, it is possible to provide a tissue embrittlement agent useful for tissue processing and repair in regenerative medicine, enabling simple and safe processing and modification of tissue to be removed. This is particularly useful for removing mucosal epithelium and transplanting new organoids into the removed area. DETAILED DESCRIPTION

[0028] The present invention relates to a compound (Z) represented by the general formula (1) or (2).

[0029] [Chemistry 3]

[0030]

[0031] [Chemistry 4]

[0032]

[0033] In the general formula (1) and the general formula (2), X is each independently a carboxyl group, a carboxylate group, or a monovalent group represented by the general formula (3).

[0034] In general formulae (1) and (2), at least one of the plurality of Xs is a carboxyl group or a carboxylate group. From the perspective of the tissue embrittlement effect, preferably at least two of the Xs are carboxyl groups or carboxylate groups, and more preferably at least three of the Xs are carboxyl groups or carboxylate groups.

[0035] In general formulae (1) and (2), at least one of the plurality of Xs is a monovalent group represented by general formula (3). From the viewpoint of safety to the organism, it is preferred that at least two of the Xs are monovalent groups represented by general formula (3).

[0036] Aminopolycarboxylic acid compounds have long been used as chelating agents in industrial applications. DTPA (corresponding to the skeleton of general formula (2)) and EDTA (corresponding to the skeleton of general formula (1)) have almost equivalent chelate-forming abilities. The stability constant (log K) for calcium ions is 10.9 for EDTA and 10.7 for DTPA. Furthermore, regarding safety, it is known that there is no significant difference in the efficacy of Ca-EDTA and Ca-DTPA when administered intravenously to beagle dogs for one month.

[0037] Therefore, it is considered that the compound represented by the general formula (1) or (2) exhibits the same effect from the viewpoint of chelating ability and safety.

[0038] When X in general formula (1) and general formula (2) is a carboxylate group, examples of the counter ion of the carboxylate group include alkali metal ions (such as sodium ions and potassium ions) and alkaline earth metal ions (such as calcium ions), and sodium ions are preferred.

[0039] -C(O)-Y-(AO) n -R(3)

[0040] In the general formula (3), -Y- is -O-, -NH- or -S-. -Y- is preferably -O-.

[0041] Even if -Y- is -NH- or -S-, since they have a heteroatom with unshared lone electron pairs, they exhibit the same chelating effect as when Y is -O-. In addition, even if -Y- is -NH- or -S-, it is equally safe as when -Y- is -O-.

[0042] In the general formula (3), A is an alkylene group having 2 to 4 carbon atoms (such as ethylene, 1,2-propylene, 1,3-propylene, 1,2-butylene, 1,3-butylene, 1,4-butylene, or 2,3-butylene).

[0043] In general formula (3), R is a hydrogen atom or a monovalent hydrocarbon group having 1 to 15 carbon atoms (such as methyl, ethyl, isopropyl, tert-butyl, neopentyl, and tetradecyl) in which the hydrogen atom may be substituted by an alkoxy group having 1 to 10 carbon atoms.

[0044] In general formula (3), n is an integer of 4 to 1000. From the viewpoint of safety to the living body, n is preferably 10 to 500. From the viewpoint of tissue embrittlement, n is more preferably 4 to 100, and even more preferably 4 to 50.

[0045] In general formula (3), at least one of the n A's present is an ethylene group. From the viewpoint of formulation stability and safety to living organisms, it is preferred that 85% or more of the n A's present are ethylene groups.

[0046] It should be noted that the chelating effect is similarly exhibited regardless of whether the number of carbon atoms in the alkylene group of A is 2 to 4. Furthermore, the safety is similar regardless of whether the number of carbon atoms in the alkylene group of A is 2 to 4.

[0047] In general formula (1) and general formula (2), when there are a plurality of monovalent groups represented by general formula (3), Y, A, R, and n may be the same or different.

[0048] From the viewpoint of the biological tissue embrittlement effect, the total molar number of the carboxyl group and the carboxylate group in the compound (Z) is preferably 0.04 to 14 mmol / g, more preferably 0.04 to 5 mmol / g, based on the weight of the compound (Z).

[0049] From the viewpoint of safety to a living body, the weight average molecular weight of the compound (Z) is preferably 500 to 20,000, more preferably 500 to 10,000, and even more preferably 2,000 to 10,000.

[0050] In the case of a composition containing a plurality of compounds (Z) represented by the general formula (1) or (2), the weight average molecular weight of all compounds corresponding to the compound (Z) is preferably within the above range.

[0051] In the present invention, the weight average molecular weight can be measured by the following method.

[0052] Device body: HLC-8320GPC (manufactured by Tosoh Corporation)

[0053] Column: TSK gel Super AW made by Tosoh Corporation

[0054] Detector: RI (refractive index)

[0055] Eluent: 0.01M LiBr·DMF

[0056] Eluent flow rate: 0.6 ml / min

[0057] Column temperature: 40°C

[0058] Sample concentration: 0.125 wt%

[0059] Injection volume: 20 μl

[0060] Standard material: TSK standard polyethylene oxide manufactured by Tosoh Corporation

[0061] From the viewpoint of safety to a living body, the number average molecular weight of the compound (Z) is preferably 500 to 20,000, more preferably 500 to 10,000, and even more preferably 2,000 to 10,000.

[0062] In the case of a composition containing a plurality of compounds (Z) represented by the general formula (1) or (2), the number average molecular weight of all compounds corresponding to the compound (Z) is preferably within the above range.

[0063] In the present invention, the number average molecular weight can be measured by the following method.

[0064] The number average molecular weight can be used 1For example, when compound (Z) is a mixture of a compound having one monovalent group represented by general formula (3) in general formula (1) and a compound having two monovalent groups represented by general formula (3) in general formula (1), and A is an alkylene group having 2 carbon atoms, the following method can be used for calculation.

[0065] The molar ratio (α:β) of the compound having one monovalent group represented by the general formula (3) in the general formula (1) to the compound having two monovalent groups represented by the general formula (3) in the general formula (1) can be obtained. 1 The integral value (γ) of the signal derived from the methylene group of A in H-NMR is calculated as follows. 1 In the case of the integrated value of H-NMR, the total integrated value of the signals derived from the two methylene groups sandwiched between the two nitrogen atoms in the general formula (1) was set to 4.

[0066] n sum =γ / {[4×α+8×β] / (α+β)}

[0067] (number average molecular weight) = 44 × n sum +292

[0068] < 1 H-NMR evaluation conditions>

[0069] Solvent: Deuterated DMSO

[0070] Device: AVANCE III HD400 (manufactured by Bruker Japan Co., Ltd.)

[0071] Frequency: 400MHz

[0072] The above-mentioned compound (Z) can be produced by the following method.

[0073] EDTA dianhydride and water (preferably 1 molar equivalent relative to EDTA dianhydride) are mixed in an organic solvent (DMF or the like) at 70 to 80° C. (preferably for 2 to 4 hours) to obtain EDTA monoanhydride.

[0074] Next, a basic compound (such as triethylamine and diisopropylethylamine) and a compound represented by the general formula (4) are added and mixed at 25 to 70°C (preferably for 2 to 24 hours), thereby producing compound (Z) [In this case, compound (Z) having one monovalent group represented by the general formula (3) is obtained as the main product. -Y- in the general formula (3) is -O-.].

[0075] HO-(AO) n -R(4)

[0076] It should be noted that A, R and n in the general formula (4) are the same as A, R and n in the general formula (3).

[0077] In addition, the following compounds may be used instead of the compound represented by the general formula (4).

[0078] H2N-(AO) n -R(5)

[0079] HS-(AO) n -R(6)

[0080] When the compound represented by the general formula (5) is used, -Y- in the general formula (3) is -NH-, and when the compound represented by the general formula (6) is used, -Y- in the general formula (3) is -S-.

[0081] It should be noted that A, R and n in the general formulae (5) and (6) are the same as A, R and n in the general formula (3).

[0082] Furthermore, even if it is a method other than the above, it can be produced by, for example, the following method.

[0083] EDTA dianhydride and a compound in which a group represented by the general formula (3) is bonded to a hydroxyl group are mixed in an organic solvent (DMF, etc.) in the presence of the above-mentioned basic compound at 25 to 70° C. (preferably for 2 to 24 hours), thereby producing compound (Z) [in this case, compound (Z) having two monovalent groups represented by the general formula (3) is obtained as the main product].

[0084] The composition of the present invention contains the above-mentioned compound (Z).

[0085] The above-mentioned compound (Z) may be used alone or in combination of two or more.

[0086] The composition of the present invention may be in the form of any of powder, tablet, gel, sol and solution. In the case of a solution, it preferably contains water in addition to the compound (Z).

[0087] Furthermore, the composition of the present invention may contain salt (sodium chloride, etc.) and the like in addition to the above-mentioned compound (Z) and water, within a range that does not impair the effects of the invention.

[0088] When the composition is a solution, the weight ratio of the compound (Z) is preferably 1 to 500 mM, more preferably 2 to 250 mM, based on the weight of the composition, from the viewpoint of the biological tissue embrittlement effect.

[0089] The composition of the present invention can be used as a biological tissue embrittlement agent described later and is useful.

[0090] The biological tissue embrittlement agent of the present invention contains the above-mentioned compound (Z). Preferred embodiments of the biological tissue embrittlement agent of the present invention are the same as those of the composition of the present invention.

[0091] The biological tissue embrittlement agent may be in the form of a powder, a tablet, a gel, a sol, or a solution. In the case of a solution, it preferably contains water in addition to the compound (Z).

[0092] Furthermore, the biological tissue embrittlement agent of the present invention may contain salt (sodium chloride, etc.) and the like in addition to the compound (Z) and water as long as the effects of the invention are not impaired.

[0093] When the biological tissue embrittlement agent is a solution, the weight ratio of the compound (Z) is preferably 1 to 500 mM, more preferably 2 to 250 mM, based on the weight of the biological tissue embrittlement agent, from the viewpoint of the biological tissue embrittlement effect.

[0094] Examples of biological tissues suitable for biological tissue embrittlement agents include digestive tract epithelium, mucous membranes, skin, and connective tissue. Biological tissue embrittlement is primarily used in regenerative medicine as a pretreatment to remove unwanted tissue from the epithelium and mucous membranes of the transplanted site when transplanting new cell structures, or to remove damaged or necrotic tissue to promote tissue regeneration. In this case, to minimize damage to the underlying tissue, it is necessary to minimize invasiveness and efficiently remove the target tissue. Based on this perspective, the present inventors have successfully developed a highly functional complex based on EDTA, which is registered in the Japanese Pharmacopoeia and can also be used in the medical field, in order to maintain its chelating ability while improving the safety of its biological effects.

[0095] When the digestive tract is targeted, the large and small intestinal epithelium are preferred from the perspective of tissue fragility. It is conceivable to remove the large intestinal epithelium and transplant various organoids (such as large and small intestinal organoids) into this area.

[0096] The tissue embrittlement agent of the present invention chelates calcium ions required for cell adhesion, weakening intercellular adhesion and enabling cell-freeing. Therefore, when applied to living tissue, any method, such as fixation, coating, or retention, is acceptable, as long as the agent is brought into contact. Furthermore, tissue removal after embrittlement can be performed by any method, such as peeling, friction, or washing, to free cells and connective tissue. Furthermore, there are no particular limitations on the procedure for transplanting cells, tissue organoids, or the like into the area where tissue embrittlement and removal has been performed using this agent. Furthermore, the transplanted cell or tissue structure can be in any shape, such as a sheet, block, or organoid.

[0097] For example, the present invention's method for exfoliating the surface of living tissues can be used to exfoliate the large intestinal epithelium and transplant other tissues, thereby imparting properties corresponding to the transplanted tissue. When applying this technology to regenerative medicine for short bowel syndrome, small intestinal organoids (described in Non-Patent Documents 1 and 2, etc.) are preferred.

[0098] Furthermore, the tissue embrittlement agent of the present invention can also be used for debridement of skin tissue that has ulcers, injuries, or necrosis, and can also be applied to the regeneration of healthy tissue. It can be used for cleaning wounds at the site of bedsores, cleaning anal fistulas or fistulas after surgery, and exfoliating and cleaning inflamed mucous membranes. Furthermore, if the area is subcutaneous tissue or within a body cavity, the agent can also be applied as an exfoliation aid to areas where organs or tissues are in poor condition due to adhesions, to promote adhesion repair. During surgical procedures, adhesions of tissues and organs can sometimes make the procedure difficult, and the technology of the present invention also provides an effective solution in such cases.

[0099] In the field of skin care, this agent is used as an auxiliary agent in chemical peels to remove old stratum corneum, effectively removing the stratum corneum while minimizing skin irritation.

[0100] Furthermore, it can be widely used for the exfoliation and separation of mucosal tissue during clinical examinations. Even when tissue slices are conventionally obtained through excision, scraping, or wiping, the biological tissue embrittlement agent of the present invention can be exfoliated from the target tissue surface to obtain tissue slices while preserving the cell biochemical structure. Therefore, it can be effectively used as a tissue collection method suitable for histopathological diagnosis in clinical examinations and three-dimensional observation in cytodiagnosis. For example, it is useful in cervical cancer screening.

[0101] Specifically, the present invention will be described using the method for exfoliating the surface of biological tissue, described later. However, as long as the biological tissue embrittlement agent can be in contact with the biological tissue surface of the target site for a certain period of time, the form of the biological tissue embrittlement agent of the present invention can be any of a powder, tablet, gel, sol, cream, patch, patch, or solution. For example, one preferred method is to contact the gel with the mucosal epithelium and, after a certain period of time, remove the tissue at the contact site by washing or friction exfoliation. Alternatively, contacting the agent with the target site can be achieved using a probe, catheter, injection, spray, endoscope, laparoscope, thoracoscope, patch, brush, or the like. When removing the biological tissue embrittlement agent of the present invention after a certain period of contact, the contact site can be removed by performing operations such as washing, suction, and exfoliation.

[0102] The method for exfoliating the surface of a living tissue of the present invention comprises the following steps: a step of bringing the compound (Z) into contact with the living tissue to embrittle the surface of the living tissue (hereinafter sometimes referred to as the "embrittlement step"); and a step of exfoliating cell tissue from the embrittled surface portion of the living tissue (hereinafter sometimes referred to as the "exfoliation step").

[0103] Here, in the present invention, the surface of a biological tissue is not limited to the aforementioned skin, gastrointestinal epithelium, external mucosa, etc. as long as cells are bonded in a surface layer, and is a concept that also includes surfaces existing inside organs.

[0104] Specific aspects of the embrittlement step include the following.

[0105] Examples of methods for contacting the compound (Z) contained in the biological tissue embrittlement agent include methods such as exposing the biological tissue to be embrittled (contact site) by surgical means (methods such as those described in Non-Patent Documents 1 and 2 can be used), and administering the biological tissue embrittlement agent using a probe, a catheter, injection, or spray.

[0106] For example, when the living tissue to be embrittled is the large intestinal epithelium, a method can be used in which the large intestine is exposed to the outside of the abdominal cavity by a surgical method and a living tissue embrittlement agent is administered into the intestine using a catheter.

[0107] The biological tissue embrittlement agent is preferably heated to 20 to 50° C. before administration to the biological tissue to be embrittled (contact site).

[0108] Specific aspects of the peeling step include the following.

[0109] Methods for peeling off cell tissue from the brittle surface of biological tissue include methods such as washing peeling (peeling using the pressure of washing with saline or the like) and friction peeling (peeling using friction with a brush (such as an interdental brush)) to remove cell tissue from the contact area.

[0110] The peeling step is preferably performed immediately after the compound (Z) is brought into contact with the living tissue in the embrittlement step.

[0111] The method for exfoliating a living tissue surface of the present invention may include, after the exfoliation step, a removal step of removing the living tissue embrittlement agent from the living body.

[0112] Examples of methods for removing the biological tissue embrittlement agent include methods of removing the biological tissue embrittlement agent by washing, suction, etc. It should be noted that the above-mentioned removal step can be performed simultaneously with the washing and peeling and friction peeling in the peeling step.

[0113] The living tissue exfoliation kit of the present invention is a kit comprising the living tissue embrittlement agent of the present invention as an essential component.

[0114] The above-mentioned biological tissue exfoliation kit preferably includes, in addition to the biological tissue embrittlement agent of the present invention, an instrument for injecting the biological tissue embrittlement agent (probe, catheter, injection, spray, etc.) and materials used in the exfoliation step (physiological saline solution, brush, etc.). It can be used for medical purposes or skin care applications, etc.

[0115] Example

[0116] Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

[0117] It should be noted that parts hereinafter represent parts by weight.

[0118] <Production Example 1: Production of EDTA Monoanhydride>

[0119] 148.5 parts of EDTA dianhydride (manufactured by Tokyo Chemical Industry Co., Ltd.) were dissolved in 701 parts of DMF (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) at 90°C. After cooling to 75°C, a mixture of 140.1 parts of DMF and 10.4 parts of water (1 molar equivalent relative to EDTA monoanhydride) was added dropwise over 2 hours. After the dropwise addition, the mixture was mixed at 75°C for 2 hours, and the precipitated solid was suction filtered to obtain Compound (PX-1) (EDTA monoanhydride) with a yield of 62% and a purity of 93% by weight.

[0120] <Production Example 2: Method for producing the compound represented by general formula (4)>

[0121] 275 parts of methoxypolyethylene glycol [a compound represented by the general formula (4) (main component: n=12), number average molecular weight 550, "UNIOX M-550" manufactured by NOF Corporation] and 0.40 parts of an aqueous potassium hydroxide solution ["SuperCali" manufactured by Toagosei Co., Ltd.] were placed in a stainless steel autoclave equipped with stirring and temperature control functions. After replacing the atmosphere with argon, the pressure was reduced and the temperature was raised to 130°C.

[0122] After dehydration at 2.7 kPa and 130°C for 2 hours, the temperature was raised to 150°C, and 725 parts of ethylene oxide was slowly added dropwise over 5 hours at a temperature of 145-155°C so that the internal pressure of the autoclave did not exceed 0.4 MPa. After the completion of the dropwise addition, the autoclave was aged at 145-155°C for 1 hour until the internal pressure of the autoclave reached the same pressure as at the start of the dropwise addition, thereby obtaining 1000 parts of methoxypolyethylene glycol, a compound represented by the general formula (4) (the compound having n=45 as the main component).

[0123] <Production Example 3: Method for producing the compound represented by general formula (4)>

[0124] 137 parts of methoxypolyethylene glycol [a compound represented by the general formula (4) (main component: n=12), number average molecular weight 550, "UNIOX M-550" manufactured by NOF Corporation] and 0.4 parts of an aqueous potassium hydroxide solution ["SuperCali" manufactured by Toagosei Co., Ltd.] were placed in a stainless steel autoclave equipped with stirring and temperature control functions, the atmosphere was replaced with argon, the pressure was reduced, and the temperature was raised to 130°C.

[0125] After dehydration at 2.7 kPa and 130°C for 2 hours, the temperature was adjusted to 120°C, and 863 parts of ethylene oxide was slowly added dropwise at 115-125°C over 10 hours so that the internal pressure of the autoclave did not exceed 0.4 MPa. After the completion of the dropwise addition, the autoclave was aged at 115-125°C for 1 hour until the internal pressure of the autoclave reached the same pressure as at the start of the dropwise addition, thereby obtaining 1000 parts of methoxypolyethylene glycol, a compound represented by the general formula (4) (the compound having n=90 as the main component).

[0126] <Example 1: Production of Compound (Z-1)>

[0127] 22 parts of methoxypolyethylene glycol [a compound represented by the general formula (4) (the compound with n=12 as the main component), number average molecular weight 550, "UNIOX M-550" manufactured by NOF Corporation] were dissolved in 107 parts of DMF. 5 parts of diisopropylethylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) and 11 parts of the compound (PX-1) produced in Production Example 1 were added in this order at 60°C. The mixture was mixed at 60°C for 5 hours. 855 parts of tert-butyl methyl ether (manufactured by Nacalai Tesque) was added, and the mixture was filtered off with suction. Thus, a mixture of compound (Z-1) [compound (Z-1-1) having one monovalent group represented by the general formula (3) in the general formula (1) (82% by weight), compound (Z-1-2) having two monovalent groups represented by the general formula (3) in the general formula (1) (15% by weight)] and methoxypolyethylene glycol (3% by weight) as an impurity was obtained. The purity of the compound (Z-1) contained in 100% by weight of the mixture was 97% by weight, and the yield of the mixture was 45%.

[0128] The number average molecular weights of the main component (Z-1-1) and the subcomponent (Z-1-2) of the compound (Z-1) are 850 and 1400, respectively. The total molar number of carboxyl groups possessed by the main component (Z-1-1) of the compound (Z-1) is 1.2 mmol / g, based on the weight of the main component (Z-1-1), and the total molar number of carboxyl groups possessed by the subcomponent (Z-1-2) is 1.4 mmol / g, based on the weight of the subcomponent (Z-1-2).

[0129] In addition, the number average molecular weight of the components corresponding to compound (Z) [main component (Z-1-1) and subcomponent (Z-1-2)] was 935, and the total molar number of carboxyl groups and carboxylate groups possessed by the components corresponding to compound (Z) was 1.2 mmol / g.

[0130] It should be noted that the compound (Z-1) 1 The H-NMR spectrum is shown below.

[0131] 1 H-NMR(D2O,400MHz): δ3.25(t,2H,-NCH2CH2N-),3.36(s,3H,-OMe),3.44(t,2H,-NCH2CH2N-),3.58-3.80(m,PEG),3.90(s,4H,-NCH2COOH and-NCH2COO-),3.93(s,4H,-NCH2COOH×2),4.33(m,2H,-COOCH2CH2O-)

[0132] <Example 2: Production of Compound (Z-2)>

[0133] 46 parts of methoxypolyethylene glycol [a compound represented by the general formula (4) (the compound with n=12 as the main component), number average molecular weight 550, "UNIOX M-550" manufactured by NOF Corporation] were dissolved in 104 parts of DMF. 10.5 parts of diisopropylethylamine and 10.5 parts of EDTA dianhydride were added in this order at 25°C, and the mixture was mixed at 60°C for 5 hours. 829 parts of tert-butyl methyl ether was added, and the mixture was filtered with suction to obtain a mixture of compound (Z-2) [compound (Z-2-2) having one monovalent group represented by the general formula (3) in the general formula (1) (2% by weight), compound (Z-2-1) having two monovalent groups represented by the general formula (3) in the general formula (1) (95% by weight)] and methoxypolyethylene glycol (3% by weight) as an impurity. The purity of compound (Z-2) contained in 100% by weight of the mixture was 97% by weight. The yield of the mixture was 86%.

[0134] The number average molecular weights of the main component (Z-2-1) and the subcomponent (Z-2-2) of compound (Z-2) are 1400 and 850, respectively. The total molar number of carboxyl groups possessed by the main component (Z-2-1) of compound (Z-2) is 1.4 mmol / g based on the weight of (Z-2-1), and the total molar number of carboxyl groups possessed by the subcomponent (Z-2-2) is 1.2 mmol / g based on the weight of the subcomponent (Z-2-2).

[0135] The number average molecular weight of the components corresponding to compound (Z) [main component (Z-2-1) and subcomponent (Z-2-2)] was 1390, and the total molar number of carboxyl groups and carboxylate groups possessed by the components corresponding to compound (Z) was 1.4 mmol / g.

[0136] It should be noted that the compound (Z-2) 1 The H-NMR spectrum is shown below.

[0137] 1 H-NMR (D2O, 400MHz): δ3.30(s,4H,-NCH2CH2N-), 3.31(s,6H,-OMe×2), 3.50-3.86(m,PEG), 4.02(m,4H,-NCH2COOH×2), 4.35(m,4H,-COOCH2CH2O-×2)

[0138] <Example 3: Production of Compound (Z-3)>

[0139] 23.5 parts of methoxypolyethylene glycol produced in Production Example 2 was dissolved in 110.5 parts of DMF. 0.5 parts of diisopropylethylamine and 3.5 parts of compound (PX-1) produced in Production Example 1 were added in this order at 60°C, and the mixture was mixed at 60°C for 5 hours. 862 parts of tert-butyl methyl ether was added, and the mixture was filtered with suction. This gave a mixture of compound (Z-3) [compound (Z-3-1) (83% by weight) having one monovalent group represented by general formula (3) in general formula (1) and compound (Z-3-2) (11% by weight) having two monovalent groups represented by general formula (3) in general formula (1)] and methoxypolyethylene glycol (6% by weight) as an impurity. The purity of compound (Z-3) contained in 100% by weight of the mixture was 94% by weight. The yield of the mixture was 64%.

[0140] The number average molecular weights of the main component (Z-3-1) and the subcomponent (Z-3-2) of the compound (Z-3) are 2300 and 4300, respectively. The total molar number of carboxyl groups possessed by the main component (Z-3-1) of the compound (Z-3) is 0.4 mmol / g based on the weight of the main component (Z-3-1), and the total molar number of carboxyl groups possessed by the subcomponent (Z-3-2) is 0.5 mmol / g based on the weight of the subcomponent (Z-3-2).

[0141] The number average molecular weight of the components corresponding to compound (Z) [main component (Z-3-1) and subcomponent (Z-3-2)] was 2530, and the total molar number of carboxyl groups and carboxylate groups possessed by the components corresponding to compound (Z) was 0.4 mmol / g.

[0142] It should be noted that the compound (Z-3) 1 The H-NMR spectrum is shown below.

[0143] 1 H-NMR(D2O,400MHz): δ3.25(t,2H,-NCH2CH2N-),3.42(s,3H,-OMe),3.44(t,2H,-NCH2CH2N-),3.59-3.79(m,PEG),3.90(s,4H,-NCH2COOH and-NCH2COO-),3.98(s,4H,-NCH2COOH×2),4.35(m,2H,-COOCH2CH2O-)

[0144] <Example 4: Production of Compound (Z-4)>

[0145] 27 parts of methoxypolyethylene glycol produced in Production Example 2 was dissolved in 126 parts of DMF. 2 parts of diisopropylethylamine and 2 parts of EDTA dianhydride were added in this order at 25°C, and the mixture was mixed at 60°C for 5 hours. 843 parts of tert-butyl methyl ether was added, and the mixture was filtered with suction to obtain a mixture of compound (Z-4) [compound (Z-4-2) having one monovalent group represented by general formula (3) in general formula (1) (1% by weight), compound (Z-4-1) having two monovalent groups represented by general formula (3) in general formula (1) (96% by weight)] and methoxypolyethylene glycol (3% by weight) as an impurity. The purity of compound (Z-4) contained in 100% by weight of the mixture was 97% by weight. The yield of the mixture was 85%.

[0146] The number average molecular weights of the main component (Z-4-1) and the subcomponent (Z-4-2) of the compound (Z-4) are 4300 and 2300, respectively. The total molar number of carboxyl groups possessed by the main component (Z-4-1) of the compound (Z-4) is 0.5 mmol / g based on the weight of the main component (Z-4-1), and the total molar number of carboxyl groups possessed by the subcomponent (Z-4-2) is 0.4 mmol / g based on the weight of the subcomponent (Z-4-2).

[0147] The number average molecular weight of the components corresponding to compound (Z) [main component (Z-4-1) and subcomponent (Z-4-2)] was 4280, and the total molar number of carboxyl groups and carboxylate groups possessed by the components corresponding to compound (Z) was 0.5 mmol / g.

[0148] It should be noted that the compound (Z-4) 1 The H-NMR spectrum is shown below.

[0149] 1 H-NMR (D2O, 400MHz): δ3.30(s,4H,-NCH2CH2N-), 3.36(s,6H,-OMe×2), 3.49-3.85(m,PEG), 4.03(m,4H,-NCH2COOH×2), 4.36(m,4H,-COOCH2CH2O-×2)

[0150] <Example 5: Production of Compound (Z-5)>

[0151] 32.3 parts of methoxypolyethylene glycol produced in Production Example 3 was dissolved in 110.9 parts of DMF. 1.4 parts of diisopropylethylamine and 3.4 parts of compound (PX-1) produced in Production Example 1 were added in this order at 60°C, and the mixture was mixed at 60°C for 12 hours. 852 parts of tert-butyl methyl ether was added, and the mixture was suction filtered. This gave a mixture of compound (Z-5) [compound (Z-5-1) having one monovalent group represented by general formula (3) in general formula (1) (82% by weight), compound (Z-5-2) having two monovalent groups represented by general formula (3) in general formula (1) (15% by weight)] and methoxypolyethylene glycol (3% by weight) as an impurity. The purity of compound (Z-5) contained in 100% by weight of the mixture was 97% by weight. The yield of the mixture was 91%.

[0152] The number average molecular weights of the main component (Z-5-1) and the subcomponent (Z-5-2) of the compound (Z-5) are 4300 and 8300, respectively. The total molar number of carboxyl groups possessed by the main component (Z-5-1) of the compound (Z-5) is 0.23 mmol / g based on the weight of the main component (Z-5-1), and the total molar number of carboxyl groups possessed by the subcomponent (Z-5-2) is 0.24 mmol / g based on the weight of the subcomponent (Z-5-2).

[0153] The number average molecular weight of the components corresponding to compound (Z) [main component (Z-5-1) and subcomponent (Z-5-2)] was 4920, and the total number of moles of carboxyl groups in the components corresponding to compound (Z) was 0.2 mmol / g.

[0154] It should be noted that the compound (Z-5) 1 The H-NMR spectrum is shown below.

[0155] 1 H-NMR (D2O, 400MHz): δ3.20(t,2H,-NCH2CH2N-),3.32(s,3H,-OMe),3.37(t,2H,-NCH2CH2N-),3.55-3.75(m,PEG),3.88(s,4H,-NCH2COOH and-NCH2COO-),3.98(s,4H,-NCH2COOH×2),4.30(m,2H,-COOCH2CH2O-)

[0156] The compounds (Z-1) to (Z-5) synthesized in Examples 1 to 5 were evaluated for stability and chelating ability as follows.

[0157] <Evaluation of Stability of Compound (Z) (pH: 7)>

[0158] Compound (Z) was dissolved in deuterated water at a concentration of 1% by weight to obtain a test solution (pH of the solution was 7).

[0159] Then, the mixture was allowed to stand at a temperature of 25°C, 40°C, or 60°C for 3 hours.

[0160] Afterwards, use 1 Each solution was analyzed by H-NMR (conditions are as follows), and the following α was calculated.

[0161] α=100×[“integrated value of the signal of the methylene group adjacent to the oxygen atom originating from the ester bond in the general formula (3) at around 4.3 ppm” / 2] / [“integrated value of the signal of the R (methyl group) in the general formula (3) at around 3.35 ppm” / 3]

[0162] Next, the retention rate of α before and after temperature adjustment was calculated [100×(α after standing for 3 hours) / (α before standing for 3 hours)]. The results of evaluation based on the following criteria are shown in Table 1.

[0163] The retention rate of the ester bond derived from the general formula (3) is 95% or more...

[0164] The retention rate of the ester bond derived from the general formula (3) is 90% or more and less than 95%...○

[0165] The retention rate of the ester bond derived from the general formula (3) is 80% or more and less than 90%...△

[0166] The retention rate of the ester bond derived from the general formula (3) is less than 80%···×

[0167] < 1 H-NMR evaluation conditions>

[0168] Solvent: heavy water

[0169] Device: AVANCE III HD400 (manufactured by Bruker Japan Co., Ltd.)

[0170] Frequency: 400MHz

[0171] <Evaluation of Stability of Compound (Z) (pH: 8)>

[0172] Compound (Z) was dissolved in heavy water, and the pH was adjusted to 8 with sodium acetate, and the final weight ratio of Compound (Z) was adjusted to 1% by weight to obtain a solution for testing.

[0173] The test was conducted in the same manner as in <Evaluation of Stability of Compound (Z) (pH: 7)>, except that the test solution was changed to the test solution obtained above. The retention rate of α was calculated. The results are shown in Table 1.

[0174] <Evaluation of Stability of Compound (Z) (pH: 4)>

[0175] Compound (Z) was dissolved in heavy water, and the pH was adjusted to 4 with acetic acid, and the final weight ratio of Compound (Z) was adjusted to 1% by weight to obtain a solution for testing.

[0176] The test was conducted in the same manner as in the "Evaluation of Stability of Compound (Z) (pH: 7)" above, except that the test solution was replaced with the test solution obtained above. The retention rate of α was calculated. The results are shown in Table 1.

[0177] The higher the retention rate of α, the more excellent the stability of the compound (Z), and the more capable it is of inhibiting the formation of EDTA, which causes serious side effects such as hypocalcemia due to the formation of the compound (Z). Here, as described above, even when the pH of the compound (Z) of the present invention is changed between 4 and 8, the formation of EDTA can be inhibited. Therefore, it is implied that the compound (Z) of the present invention can also inhibit the formation of EDTA in the pH environment in vivo.

[0178] <Evaluation of Chelation Ability of PEG-EDTA>

[0179] The compound (Z) was mixed with calcium chloride at a molar ratio of 1:1 and diluted with ion-exchanged water to a concentration of 10 ppm. For this sample, molecular weight measurement was performed using LC / TOF-MS (conditions are as described below) to confirm whether a signal from the chelate of the compound (Z) and calcium ions could be observed.

[0180] The case where a signal from the chelate of the compound (Z) and calcium ions could be confirmed was evaluated as ○, and the case where a signal from the chelate could not be confirmed was evaluated as ×. The results are shown in Table 1.

[0181] <Evaluation Conditions Using LC / TOF-MS>

[0182] Apparatus main body: Manufactured by Waters

[0183] LC: Aquity UFLC, semi-micro ultra-high speed LC

[0184] MS: SYNAPT HDMS, Q-TOF hybrid MS

[0185] Sample concentration: 10 ppm

[0186] Injection volume: 0.2 μl or 1.0 μl

[0187] [Table 1]

[0188]

[0189] <Examples 6 to 11: Manufacture of Compositions Containing Compound (Z)>

[0190] To the compound (Z-1), water at room temperature was added little by little, and it was stirred and shaken using a vortex mixer (Vortex-Genie 2 manufactured by KENIS Corporation) until it was completely dissolved. It was dissolved in about 5 minutes, and then adjusted to pH 8 using 1M NaOH. Then, it was diluted to the molar concentrations shown in Table 2 using Hanks' balanced salt solution (without Ca and Mg, containing phenol red) (Nacalai Tesque Cat#17460-15) to obtain compositions (C-1) to (C-6).

[0191] <Comparative Examples 1 to 7: Production of Comparative Compositions Containing Comparative Compound (Z')>

[0192] As a comparative compound (Z'-1), a composition containing Na2EDTA (disodium ethylenediaminetetraacetate) was prepared by the following method.

[0193] Na2EDTA (0.5 mol / l EDTA solution pH 8.0, Nacalai Tesque Cat#14347-21) was diluted to the molar concentrations shown in Table 2 using Hanks' balanced salt solution (Ca-free, Mg-free, containing phenol red) (Nacalai Tesque Cat#17460-15) to obtain comparative compositions (C'-1) to (C'-7).

[0194] The performance was evaluated by the following methods using the compositions (C-1) to (C-6) obtained in the Examples and the comparative compositions (C'-1) to (C'-7).

[0195] <Evaluation of Crypt Isolation Effect in Vitro 1>

[0196] Mice (C57B6 / j 8-week-old male Japanese CLEA) were sacrificed by cervical dislocation, and the entire large intestine was removed and washed with Hanks' balanced salt solution (Ca-free, Mg-free, phenol red-containing) (Nacalai Tesque Cat#17460-15). The intestine was unfolded along the longitudinal axis to prepare 5mm long tissue pieces, which were placed one by one in a 1.5ml microtube containing 1ml of Hanks' balanced salt solution. The tissue pieces were washed by stirring for 10 minutes using a microtube mixer (MT-400 manufactured by TOMY SEIKO CO., LTD.). After washing, the tissue pieces were transferred to a 1.5ml microtube containing 1ml of new Hanks' balanced salt solution and the same washing operation was performed. The tissue pieces were transferred to a 1.5ml microtube containing 1ml of composition (C-1) to (C-6) or comparative composition (C'-1) to (C'-7) and allowed to stand at 37°C for 30 minutes.

[0197] After 30 minutes, the tissue pieces were transferred to a 1.5 ml microtube containing 1 ml of Hanks' balanced salt solution and stirred for 5 minutes using a microtube mixer to free the crypts. After 5 minutes, 0.5 ml of 4% paraformaldehyde-phosphate buffer (Nacalai Tesque Cat#09154-85) was added to the microtube containing the tissue to fix the tissue. 50 μl was collected and added dropwise to a dish, and the number of crypts contained was measured using an optical microscope. The measurement of the number of crypts was repeated 3 times for each concentration, and the average value was calculated. The results are shown in Table 2.

[0198] It should be noted that as long as the number of crypts is confirmed to be 1 or more, it can be considered that a sufficient embrittlement effect is obtained.

[0199]

[0200] <Examples 12 to 19: Production of Compositions Containing Compound (Z)>

[0201] Room temperature water was added gradually to the compound (Z) listed in Table 3 in small amounts, and the mixture was stirred and shaken using a vortex mixer (Vortex-Genie 2, manufactured by KENIS Corporation). Water was then added until the mixture was completely dissolved. Dissolution took approximately 5 minutes, and the pH was then adjusted to 8 using 1M NaOH. Subsequently, the mixture was diluted to the molar concentrations shown in Table 3 using Hanks' balanced salt solution (Ca- and Mg-free, containing phenol red) (Nacalai Tesque Cat#17460-15), yielding compositions (C-7) to (C-14).

[0202] <Comparative Examples 8-9: Preparation of Comparative Compositions Containing Comparative Compound (Z')>

[0203] As a comparative compound (Z'-1), a composition containing Na2EDTA (disodium ethylenediaminetetraacetate) was prepared by the following method.

[0204] Na2EDTA (0.5 mol / l EDTA solution, pH 8.0, Nacalai Tesque Cat#14347-21) was diluted with Hanks' balanced salt solution (Ca-free, Mg-free, containing phenol red) (Nacalai Tesque Cat#17460-15) to the molar concentrations shown in Table 3 to obtain comparative compositions (C'-8) to (C'-9).

[0205] The properties of the compositions (C-7) to (C-14) obtained in the Examples and the comparative compositions (C'-8) to (C'-9) were evaluated by the following methods.

[0206] <Evaluation of Crypt Isolation Effect in Vitro 2>

[0207] Mice (C57B6 / j 8-week-old male Japanese CLEA) were killed by cervical dislocation, and 4 cm of the distal large intestine was removed and cleaned with Hanks' balanced salt solution (Ca, Mg-free, containing phenol red) (Nacalai Tesque Cat#17460-15). The intestine was unfolded along the longitudinal axis and minced meat was made using a razor (Feather Cat#FAS-10). 40 ml of minced meat tissue was suspended in Hanks' balanced salt solution, and 4 ml was divided into 15 ml tubes. Centrifuge at 1500 rpm for 1 minute to precipitate the minced meat tissue and discard the supernatant. Add 1 ml of Hanks' balanced salt solution to suspend the minced meat tissue and transfer the minced meat tissue together with the solution to a 1.5 ml microtube. Stir for 10 minutes using a microtube mixer (MT-400 manufactured by TOMY SEIKOCO., LTD.) to clean the minced meat tissue. Centrifuge the tube at 15000 rpm for 1 minute to precipitate the minced meat tissue and discard the supernatant. Add 1 ml of Hanks' balanced salt solution and repeat the same washing procedure. Centrifuge the tube at 15,000 rpm for 1 minute to pellet the minced meat tissue. Discard the supernatant and add 1 ml of compositions (C-7) to (C-14) or comparative compositions (C'-8) to (C'-9). Incubate at 37°C for 30 minutes.

[0208] After 30 minutes, the tube was centrifuged at 15000rpm for 1 minute to precipitate the minced meat tissue and the supernatant was discarded. 1ml of Hanks' balanced salt solution was added and stirred for 5 minutes using a microtube mixer to free the crypts. After 5 minutes, 0.5ml of 4% paraformaldehyde·phosphate buffer (Nacalai Tesque Cat#09154-85) was added to the microtube containing the tissue to fix the tissue. 50μl was collected from it and added dropwise to the dish, and the number of crypts contained was measured using an optical microscope. Regarding the measurement of the number of crypts, one concentration was repeated 3 times and the average value was calculated. The experiment was performed 6 times each and the average value was calculated. The results are shown in Table 3.

[0209] It should be noted that as long as the number of crypts is confirmed to be 1 or more, it can be considered that a sufficient embrittlement effect is obtained.

[0210]

[0211] <Examples 20-24: Production of Living Tissue Embrittling Agent>

[0212] Room temperature water was added gradually to the compound (Z) listed in Table 4. The mixture was stirred and shaken using a vortex mixer (Vortex-Genie 2, manufactured by KENIS Corporation). Water was then added until the mixture was completely dissolved. Dissolution took approximately 5 minutes, and the pH was then adjusted to 8 using 1M NaOH to obtain tissue embrittlement agents (M-1) to (M-5). The concentrations of compound (Z) in the tissue embrittlement agent (M) are listed in Table 4.

[0213] <Comparative Examples 10-12: Production of Comparative Tissue Embrittling Agents>

[0214] As a comparative compound (Z'-1), a biological tissue embrittlement agent containing Na2EDTA (disodium ethylenediaminetetraacetate) was produced.

[0215] Na2EDTA (0.5 mol / l EDTA solution, pH 8.0, Nacalai Tesque Cat#14347-21) was diluted with water to the molar concentrations shown in Table 4 to obtain comparative tissue embrittlement agents (M'-1) to (M'-3).

[0216] The biological tissue embrittlement agents (M-1) to (M-5) obtained in the Examples and the biological tissue embrittlement agents (M'-1) to (M'-3) for comparison were used to evaluate their performance by the following method.

[0217] <In vivo evaluation of epithelial exfoliation effect 1>

[0218] Mice (C57B6 / j 8-week-old male Japanese CLEA) were fixed in the supine position under isoflurane anesthesia (FUJIFILM Cat#099-06571). A 1 cm longitudinal incision was made in the middle of the abdomen to expose the proximal large intestine to the outside of the abdominal cavity. A small incision was made on the anal side of the proximal large intestine 1 cm away from the cecum, and a catheter was inserted toward the anus. The inner cavity was cleaned with Dulbecco's phosphate buffered saline (Ca, Mg-free) (Nacalai Tesque Cat#14249-24). A small incision was made on the anal side 1 cm away from the catheter insertion site, and a catheter was inserted toward the mouth. In this way, 1 cm of the intestine can be perfused through two catheters. The catheters were tied and fixed from the outside of the intestine with a thread, thereby blocking the blood flow of the 1 cm long intestine clamped by the two catheters. In this state, 8 ml of the biological tissue embrittlement agent (M-1) or the biological tissue embrittlement agents (M'-1) to (M'-3) heated to 50°C were injected from the catheter over 2 minutes.

[0219] Immediately after the injection of the biological tissue embrittlement agent, 25 ml of Dulbecco's phosphate buffered saline (without Ca and Mg) was injected three times continuously over 45 seconds to cause the crypts to fall off. 20 ml of high-glucose Dulbecco's modified Eagle's medium (Sigma-Aldrich Cat#D5796) was injected to antagonize the biological tissue embrittlement agent. The proximal large intestine that had been operated on was cut out and the tissue was fixed using 4% paraformaldehyde·phosphate buffer. The large intestine was cut open along the long axis and unfolded into a plane, and photographed using a stereo fluorescence microscope (Leica M165FC) and an integrated system (All-in-One) fluorescence microscope (KEYENCE BZ-X800). The images were analyzed using ImageJ and the peeling area was measured. The results are shown in Table 4.

[0220] <In vivo evaluation of epithelial exfoliation effect 2>

[0221] To evaluate whether extending the duration of PEG-EDTA administration could enhance the exfoliation effect, the exfoliation area was measured in the same manner as in <In vivo Evaluation of Epithelial Exfoliation Effect 1>, except that 8 ml of tissue embrittlement agents (M-1) to (M-5) heated to 50°C were injected through the catheter over 2 minutes four times rather than just once. The results are shown in Table 4.

[0222] It should be noted that as long as the peeling of the epithelium can be confirmed, it can be considered that a sufficient embrittlement effect has been obtained (in the case of this evaluation, it is preferably possible to confirm 1mm 2 above epithelial detachment).

[0223]

[0224] <Evaluation Example and Comparative Evaluation Example: Toxicity Evaluation by Oral Administration>

[0225] Mice (8-week-old male C57B6 / j Japanese CLEA) were fasted from 8:00 PM on the day before dosing (with access to drinking water). Dosing began 12 hours later at 8:00 AM on the day of examination. Body weight was measured immediately before dosing, and the LD50 equivalent of Na2EDTA (reference: Initial Risk Assessment of Chemical Substances, Provisional Version No. 14, Law on the Control and Management of Chemical Substance Releases, Government Decree No. 1-47, CAS Registry No. 60-00-4), half of this amount (0.5 times the LD50), and 1.5 times this amount (1.5 times the LD50) were calculated.

[0226] 1 ml of aqueous solution containing Na2EDTA (0.5 mol / l EDTA solution pH 8.0 Nacalai Tesque Cat#14347-21) equivalent to the LD50 or half of it (0.5 times the LD50) was prepared and used as toxicity evaluation reagents (P'-1) to (P'-2).

[0227] Separately, 1 ml of an aqueous solution containing the same amount of Na2EDTA as that contained in the toxicity evaluation reagent (P'-1) was prepared and used as the toxicity evaluation reagent (P-1). Separately, reagents using (Z-3), (Z-5), or (Z-4) instead of (Z-1) were designated as the toxicity evaluation reagents (P-3), (P-4), and (P-5), respectively.

[0228] 1 ml of an aqueous solution containing the compound (Z-1) in the same amount of substance as that of Na2EDTA contained in the toxicity evaluation reagent (P'-2) was prepared and used as the toxicity evaluation reagent (P-2).

[0229] 1 ml of an aqueous solution containing compound (Z-3) in an amount 1.5 times the amount of Na2EDTA contained in the toxicity evaluation reagent (P'-1) (1.5 times the LD50) was prepared as a toxicity evaluation reagent (P-6).

[0230] Next, the toxicity evaluation reagents (P-1) to (P-6) or the toxicity evaluation reagents (P'-1) or (P'-2) were administered to the mice.

[0231] Toxicity evaluation reagents were administered orally under isoflurane anesthesia using a disposable oral probe (FUCHIGAMI Cat#4200). For each solution, the LD50 dose was administered to three animals, 0.5 times the LD50 dose was administered to five animals, and 1.5 times the LD50 dose was administered to five animals. Evaluation was performed according to the following criteria. The results are shown in Tables 5 to 7.

[0232] ◎: Patients confirmed alive more than 1 week after full dose administration

[0233] ○: Survival confirmed more than 15 minutes after full dose administration

[0234] ×: Death confirmed within 15 minutes after full dose administration

[0235] [Table 5]

[0236]

[0237] [Table 6]

[0238]

[0239] [Table 7]

[0240]

[0241] <Evaluation Examples 7 to 25 and Comparative Evaluation Examples 3 to 5: Toxicity Evaluation by Intraperitoneal Administration>

[0242] Mice (C57B6 / j 8-week-old male Japanese CLEA) were administered as follows. Immediately before administration, body weight was measured for the administered solution, and the LD50 equivalent of Na2EDTA (reference: Provisional Version No. 14 of the Initial Risk Assessment of Chemical Substances, Law on the Control and Management of Chemical Substance Releases, Government Decree No. 1-47, CAS Registry No. 60-00-4), half of it (0.5 times the LD50), 1.5 times of it (1.5 times the LD50), 2.0 times of it (2.0 times the LD50), 3.0 times of it (3.0 times the LD50), 4.0 times of it (4.0 times the LD50), and 8.0 times of it (8.0 times the LD50) were calculated.

[0243] 0.5 ml of aqueous solutions containing Na2EDTA (0.5 mol / l EDTA solution, pH 8.0, Nacalai Tesque Cat#14347-21) at an amount equivalent to the LD50, half of its amount (0.5 times the LD50), or 1.5 times its amount (1.5 times the LD50) were prepared and used as reagents for toxicity evaluation (P'-3) to (P'-5).

[0244] Separately, 0.5 ml of aqueous solutions containing the compound (Z-1) having the same amount of Na2EDTA as that contained in the toxicity evaluation reagents (P'-3) to (P'-5) were prepared and used as toxicity evaluation reagents (P-7) to (P-9).

[0245] A 0.5 ml aqueous solution containing 1.5 times the amount of Na2EDTA contained in the toxicity evaluation reagent (P'-3) (1.5 times the LD50) of compound (Z-3) was prepared and used as the toxicity evaluation reagent (P-10). Separately, reagents using (Z-5) or (Z-4) instead of (Z-3) were designated as toxicity evaluation reagents (P-11) and (P-12), respectively.

[0246] A 0.5 ml aqueous solution containing 2.0 times the amount of Na2EDTA contained in the toxicity evaluation reagent (P'-3) was prepared as a toxicity evaluation reagent (P-13). Separately, reagents using (Z-3), (Z-5), or (Z-4) instead of (Z-1) were designated as toxicity evaluation reagents (P-14), (P-15), and (P-16), respectively.

[0247] A 0.5 ml aqueous solution containing 3.0 times the amount of Na2EDTA contained in the toxicity evaluation reagent (P'-3) was prepared and used as the toxicity evaluation reagent (P-17). Separately, reagents using (Z-5) or (Z-4) instead of (Z-3) were designated as toxicity evaluation reagents (P-18) and (P-19), respectively.

[0248] A 0.5 ml aqueous solution containing 4.0 times the amount of Na2EDTA contained in the toxicity evaluation reagent (P'-3) (4.0 times the LD50) of compound (Z-3) was prepared and used as the toxicity evaluation reagent (P-20). Separately, reagents using (Z-5) or (Z-4) instead of (Z-3) were designated as toxicity evaluation reagents (P-21) and (P-22), respectively.

[0249] A 1.5 ml aqueous solution containing 8.0 times the amount of Na2EDTA contained in the toxicity evaluation reagent (P'-3) (8.0 times the LD50) of compound (Z-3) was prepared and used as the toxicity evaluation reagent (P-23). Separately, reagents using (Z-5) or (Z-4) instead of (Z-3) were designated as toxicity evaluation reagents (P-24) and (P-25), respectively.

[0250] Next, the toxicity evaluation reagents (P-7) to (P-25) or the toxicity evaluation reagents (P'-3) or (P'-5) were administered to the mice.

[0251] Toxicity evaluation reagents were administered intraperitoneally under isoflurane anesthesia using a syringe equipped with a needle [0.5 ml of an aqueous solution containing 0.5 to 4.0 times the mass of the compound using a 1 ml syringe with a 29G needle (Terumo Cat#SS-10M2913A). Alternatively, 1.5 ml of an aqueous solution containing 0.5 to 4.0 times the mass of the compound using a 2.5 ml syringe (Terumo Cat#SS-02SZ) with a 27G needle (Terumo Cat#NN-2719S)]. For each solution, three animals were used each at the LD50, 0.5 times the LD50, 1.5 times the LD50, 2.0 times the LD50, and 3.0 times the LD50, and two animals were used each at 4.0 times the LD50 and 8.0 times the LD50. Evaluation was performed according to the following criteria. The results are shown in Tables 8 to 14.

[0252] ◎: Patients confirmed alive more than 1 week after full dose administration

[0253] ○: Survival confirmed after 60 minutes or more after full dose administration

[0254] △: Survival confirmed more than 15 minutes after full dose administration

[0255] ×: Death confirmed within 15 minutes after full dose administration

[0256] [Table 8]

[0257]

[0258] [Table 9]

[0259]

[0260] [Table 10]

[0261]

[0262] [Table 11]

[0263]

[0264] [Table 12]

[0265]

[0266] [Table 13]

[0267]

[0268] [Table 14]

[0269]

[0270] <Evaluation Examples 26 to 29 and Comparative Evaluation Examples 6 to 7: Evaluation of Ca Concentration after Intraperitoneal Administration>

[0271] As in "Toxicity evaluation by intraperitoneal administration", 0.5 ml of an aqueous solution containing 2.0 times the LD50 equivalent of Na2EDTA (0.5 mol / l EDTA solution, pH 8.0, Nacalai Tesque Cat#14347-21) was prepared and used as a toxicity evaluation reagent (P'-6).

[0272] 0.5 ml of an aqueous solution containing the compound (Z-1) in the same amount of Na2EDTA as that contained in the toxicity evaluation reagent (P'-6) was prepared and used as the toxicity evaluation reagent (P-26).

[0273] In addition, reagents using (Z-3), (Z-5), or (Z-4) instead of (Z-1) were designated as reagents for toxicity evaluation (P-27), (P-28), and (P-29), respectively.

[0274] The aqueous solution was intraperitoneally administered to the mice using the same method as in "Toxicity Evaluation by Intraperitoneal Administration," and blood was collected 2 minutes later. The serum obtained from the collected blood was used to determine the corrected Ca concentration (mg / dl) [Ca concentration excluding Ca bound to blood albumin from Ca present in the blood] according to the following formula.

[0275] Corrected Ca concentration (mg / dL) = Measured Ca concentration (mg / dL) + 4-Alb (g / dL)

[0276] In the above formula, "measured Ca concentration (mg / dL)" and "Alb (g / dL)" are values obtained by the following measurement.

[0277] Measured Ca concentration (mg / dL): The Ca concentration (mg / dL) of serum obtained from the collected blood was measured using the Arsenazo III method (contracted to BML Co., Ltd.) [measured Ca concentration].

[0278] Alb (g / dL): The albumin concentration (g / dL) of serum obtained from the collected blood was measured using Albumin IIHA-Testwako (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (the measurement was commissioned by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0279] In addition, water was administered instead of the above-mentioned toxicity evaluation reagent, and the corrected Ca concentration (mg / dl) was measured by the same method as above. The toxicity evaluation reagent using water was designated as (P'-7).

[0280] Toxicity evaluation was performed using three mice per reagent, and the average corrected Ca concentration (mg / dl) was determined. The results are shown in Table 15.

[0281] Note that from the perspective of biological safety, the corrected Ca concentration is preferably 7.0 mg / dl or higher. If the corrected Ca concentration is less than 7.0 mg / dl, neurological symptoms such as tetany and generalized spasms, as well as severe arrhythmias such as QT prolongation and bradycardia, may occur.

[0282] [Table 15]

[0283]

[0284] Industrial Applicability

[0285] When a composition containing the compound of the present invention is used as a biological tissue embrittlement agent, it can achieve the same tissue embrittlement effect as conventional EDTA, while exhibiting a less toxic effect to the living body. Therefore, the composition is useful as a biological tissue embrittlement agent.

Claims

1. Use of a compound (Z) represented by the general formula (1) or (2) in the manufacture of a composition for embrittlement of living tissues, [Chemistry 1] [Chemistry 2] In general formula (1) and general formula (2), X is each independently a carboxyl group, a carboxylate group, or a monovalent group represented by general formula (3); At least one of the multiple Xs in the general formula (1) and the general formula (2) is a carboxyl group or a carboxylate group; At least one of the plurality of Xs in the general formula (1) and the general formula (2) is a monovalent group represented by the general formula (3), -C(O)-Y-(AO) n -R(3) In the general formula (3), -Y- is -O-, -NH- or -S-; A is an alkylene group having 2 to 4 carbon atoms; R is a hydrogen atom or a monovalent hydrocarbon group having 1 to 15 carbon atoms in which the hydrogen atom may be substituted by an alkoxy group having 1 to 10 carbon atoms; n is an integer from 4 to 1000; at least one of the n A's present is an ethylene group; in the case where the general formulas (1) and (2) contain multiple monovalent groups represented by the general formula (3), -Y-, A, R and n may be the same or different.

2. A biological tissue embrittlement agent comprising a compound (Z) represented by the general formula (1) or (2), [Chemistry 1] [Chemistry 2] In general formula (1) and general formula (2), X is each independently a carboxyl group, a carboxylate group, or a monovalent group represented by general formula (3); At least one of the multiple Xs in the general formula (1) and the general formula (2) is a carboxyl group or a carboxylate group; At least one of the plurality of Xs in the general formula (1) and the general formula (2) is a monovalent group represented by the general formula (3), -C(O)-Y-(AO) n -R(3) In the general formula (3), -Y- is -O-, -NH- or -S-; A is an alkylene group having 2 to 4 carbon atoms; R is a hydrogen atom or a monovalent hydrocarbon group having 1 to 15 carbon atoms in which the hydrogen atom may be substituted by an alkoxy group having 1 to 10 carbon atoms; n is an integer from 4 to 1000; at least one of the n A's present is an ethylene group; in the case where the general formulas (1) and (2) contain multiple monovalent groups represented by the general formula (3), -Y-, A, R and n may be the same or different.

3. The biological tissue embrittlement agent according to claim 2, wherein The biological tissue is digestive tract epithelium, mucosa, skin or connective tissue. A biological tissue peeling kit comprising the biological tissue embrittlement agent according to claim 2 .

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