JP2011241174A - Solution composition containing amyloid formation inhibitor - Google Patents

Abstract

A solution composition that can reduce cell damage caused by a solution containing GDP (glucose degradation product) or a solution capable of generating GDP.
A solution composition containing an electrolyte salt, glucose or a glucose polymer, and an amyloid formation inhibitor. According to this solution composition, a solution composition capable of reducing damage to cells by GDP can be provided, and preferably a peritoneal dialysis solution capable of reducing damage to peritoneal mesothelial cells by GDP can be provided.
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Description

  The present invention relates to a solution composition containing an amyloid formation inhibitor, and particularly to a peritoneal dialysate preparation containing an amyloid formation inhibitor.

  Conventionally, hemodialysis therapy, peritoneal dialysis therapy, and the like have been performed as treatments for patients with renal failure. Peritoneal dialysis (CAPD) is a method of placing a catheter in the abdominal cavity of a patient with renal failure. It is a method of repeating the operation of discharging outside the body several times a day. The peritoneal dialysate generally contains glucose in addition to the electrolyte salt, and glucose plays a role of an osmotic agent in the peritoneal dialysate. That is, glucose is used for the peritoneal dialysis solution in order to remove excess water in the blood by utilizing the difference in osmotic pressure.

  Problems in performing peritoneal dialysis therapy include deterioration of peritoneal function and hardening / thickening of the peritoneum due to peritoneal dialysis over a long period of time. Deterioration of peritoneal function and hardening / thickening of the peritoneum may cause problems such as the development of encapsulating peritoneal sclerosis (EPS), which deteriorates the ability to remove solutes and remove water. As one of the causes, glucose degradation products (GDPs) generated by degrading glucose contained in the peritoneal dialysis fluid as an osmotic agent, and final glycation products (AGEs) generated by the reaction between sugar and protein are introduced into the peritoneum. Negative effects have been pointed out.

  GDP includes methylglyoxal (MGO), glyoxal (GO), formaldehyde, acetaldehyde, furfural, 5-hydroxymethylfurfural (5-HMF), 3-deoxyglucosone (3-DG), 3,4-dideoxyglucosone -3-ene (3,4-DGE). These substances are generated when glucose is decomposed by a heat load when the peritoneal dialysate is heat sterilized. It is known that these substances have cytotoxicity, and it has been considered that degradation of peritoneal function and hardening / thickening of the peritoneum are caused by the cytotoxicity of GDPs contained in peritoneal dialysis fluid.

  When heat is applied to the glucose solution, the closer the hydrogen ion concentration (pH) of the solution is to 3, the more the generation of GDPs due to heat load can be reduced. However, since the peritoneal dialysis solution is injected into the body, the pH is preferably neutral close to that of the living body. In order to solve this conflicting problem, a peritoneal dialysis solution as described in Patent Document 1 has been developed. According to this technology, a solution containing glucose and a solution not containing glucose are divided in a bag, and the solution-side chamber containing glucose is heated and sterilized with a configuration in which glucose is easily stabilized. Generation of GDPs at the time can be suppressed. In addition, by preparing the liquid in each chamber so that the pH of the dialysate after mixing the liquid in each chamber is close to that of the living body, damage to the living body due to the pH can be prevented.

  There is also an invention described in Patent Document 2. Patent Document 2 discloses a substance that suppresses the generation of AGEs and the configuration of a peritoneal dialysis fluid using the knowledge that AGEs are involved in peritoneal sclerosis. However, it has not been elucidated how GDPs and AGEs act on the peritoneum and by what mechanism the peritoneum is cured and thickened.

Japanese Patent No. 4284737 WO2005 / 018649

  The present invention provides a solution composition capable of reducing cell damage caused by a GDP-containing solution or a solution capable of generating GDP.

  In one aspect, the present invention relates to a solution composition containing an electrolyte salt, glucose or a glucose polymer, and an amyloid formation inhibitor.

  In another aspect, the present invention relates to a peritoneal dialysis fluid preparation containing glucose or a glucose polymer and an amyloid formation inhibitor. Moreover, this invention is related with the prefilled syringe in which the amyloid formation inhibitory substance accommodated in the peritoneal dialysate or a peritoneal dialysate bag in another aspect. Furthermore, this invention relates to the intraperitoneal washing | cleaning liquid containing an amyloid formation inhibitor in another aspect. Furthermore, in another aspect, the present invention relates to a method for preparing a peritoneal dialysis fluid, which comprises co-injecting an amyloid formation inhibitor into a peritoneal dialysis fluid preparation containing glucose or a glucose polymer.

  ADVANTAGE OF THE INVENTION According to this invention, the solution composition which can reduce the damage to the cell by GDP can be provided, Preferably, the peritoneal dialysate which can reduce the damage | damage to the peritoneal mesothelial cell by GDP can be provided.

FIG. 1 shows the chemical formula of 3,4-DGE.

  The present inventor pays particular attention to 3,4-DGE among the above-mentioned GDPs, and 3,4-DGE causes protein degeneration, and the denatured protein has an amyloid structure, thereby causing cell death of peritoneal mesothelial cells. Found to cause. Moreover, it discovered that the cell death by 3, 4-DGE could be suppressed by suppressing the amyloid formation of the denatured protein. The present invention has been made based on these findings.

  That is, the present invention relates to a solution composition (hereinafter, also referred to as “the solution composition of the present invention”) containing an electrolyte salt, glucose or a glucose polymer, and an amyloid formation inhibitor. According to the solution composition of the present invention, the cytotoxicity of a glucose degradation product (GDP) contained in or contained in the solution composition can be reduced. Therefore, the solution composition of the present invention can be used as a peritoneal dialysate preparation that can reduce damage to peritoneal mesothelial cells.

  The mechanism by which the cytotoxicity of GDP can be reduced by the solution composition of the present invention is considered as follows. That is, the cause of cell death by GDP is apoptosis induced by amyloidation of final glycation products (AGEs) by GDP, and apoptosis is suppressed by the amyloid formation inhibitor contained in the solution composition of the present invention. Conceivable. However, the present invention is not limited to this mechanism.

[Electrolyte salt]
From the viewpoint of using the solution composition of the present invention as a peritoneal dialysis solution, the electrolyte salt in the solution composition of the present invention is preferably a composition and concentration used in a conventional peritoneal dialysis solution. Examples of the electrolyte salt include sodium chloride, magnesium chloride, calcium chloride, sodium lactate, sodium bicarbonate, hydrates thereof, combinations thereof, and the like, and those close to the composition and concentration in serum are preferable.

  In one non-limiting embodiment of the solution composition of the present invention, the combination of electrolyte salt contained and its concentration include 0.4 to 0.6 w / v sodium chloride, 0.01 to 0.02 w. / V% calcium chloride hydrate, 0.005-0.02 w / v% magnesium chloride, and 0.3-0.5 w / v% sodium lactate.

[Glucose and glucose polymer]
The solution composition of the present invention contains glucose and / or glucose polymer. From the viewpoint of using the solution composition of the present invention as a peritoneal dialysis solution, the glucose and glucose polymer in the solution composition of the present invention are preferably in the composition and concentration used in the conventional peritoneal dialysis solution. Glucose and glucose polymers serve to increase osmotic pressure when used as peritoneal dialysis fluid. In general, the osmotic pressure ratio of the peritoneal dialysis solution is preferably 1.1 to 1.6, more preferably 1.2 to 1.5. Accordingly, the content of glucose and / or glucose polymer in the solution composition of the present invention is preferably included as a peritoneal dialysis solution in a necessary amount to form these osmotic pressure ratios, and is 1.4 to 2.5 w / v. % Is preferable, and 1.5 to 2.4 w / v% is more preferable.

  In the present specification, the glucose polymer refers to a polymer containing glucose as a structural unit or a polymer in which the structural unit is composed only of glucose. The glucose polymer is not particularly limited as long as it can form an osmotic pressure difference when used as a peritoneal dialysis solution, but a glucose polymer whose structural unit is α-glucose is preferable from the viewpoint of enabling use as a peritoneal dialysis solution. Specific examples of the glucose polymer in the present specification include dextran, starch, glycogen, cellulose, pullulan, curdlan, schizophyllan, lentinan, and pestarothian.

[PH]
The pH of the solution composition of the present invention is preferably 6.4 to 7.6, more preferably 6.5 to 7.5, from the viewpoint that it can be used as a peritoneal dialysis solution.

[GDP and 3,4-DGE]
The dialysate needs to be sterilized under high temperature and high pressure conditions. The peritoneal dialysis solution containing glucose contains glucose degradation product (GDP) by receiving the sterilization treatment. As described above, the GDP in the peritoneal dialysis fluid includes methylglyoxal (MGO), glyoxal (GO), formaldehyde, acetaldehyde, furfural, 5-hydroxymethylfurfural (5-HMF), 3-deoxyglucosone (3-DG). ), 3,4-DGE has been reported. Therefore, the solution composition of the present invention contains at least one kind of GDP as one aspect thereof. All of GDP is considered to damage peritoneal mesothelial cells in the peritoneal dialysate, and among them, the cytotoxicity of 3,4-DGE tends to be remarkably high. According to the solution composition of the present invention, even when 3,4-DGE is contained, the effect of reducing the damage to peritoneal mesothelial cells can be exhibited. In the present specification, “3,4-DGE” refers to 3,4-dideoxyglucosone-3-ene shown in FIG.

  The solution composition of the present invention preferably contains no glucose degradation product (GDP). However, for example, when glucose or a glucose polymer-containing liquid is sterilized, GDP is generated, and therefore it contains GDP. Generally, the GDP that can be generated when glucose and / or glucose polymer is sterilized under neutral conditions is 50 μM or less, and the GDP that can be generated when sterilized under acidic conditions is 300 to 400 μM or less. is there.

[Amyloid formation inhibitor]
GDP that can be included in the solution composition of the present invention is thought to cause cell injury via amyloidation. Therefore, a known amyloid formation inhibitor can be used as the amyloid formation inhibitor in the solution composition of the present invention. The fact that amyloidation is involved in the cytotoxicity of GDP in peritoneal dialysis fluid and that the cytotoxicity of GDP can be reduced by an amyloid formation-inhibiting substance are new findings that have not been conventionally known.

  Preferable examples of the amyloid formation inhibitor in the solution composition of the present invention include congo red, thioflavine, curcumin, and derivatives thereof. These substances may be in the form of salts. Among these amyloid formation inhibitors, curcumin and curcumin derivatives are preferred from the viewpoint of reducing the cytotoxicity of GDP. As the curcumin derivative, those having a structure similar to curcumin and having an amyloid formation inhibitory effect are preferable.

  The content of the amyloid formation inhibitor in the solution composition of the present invention is, for example, 0.1 μM to 100 mM from the viewpoint of reducing the cytotoxicity of GDP.

[Peritoneal dialysate preparation]
In another aspect, the present invention relates to a peritoneal dialysate preparation (hereinafter also referred to as “the peritoneal dialysate preparation of the present invention”), and relates to a peritoneal dialysate preparation containing glucose or a glucose polymer and an amyloid formation inhibitor. . That is, the peritoneal dialysate preparation of the present invention is obtained by adding the aforementioned amyloid formation inhibitor to a conventional glucose-containing peritoneal dialysate. Therefore, the above-mentioned GDP is included after the heat sterilization process. With the peritoneal dialysate preparation of the present invention, it is possible to reduce the damage to peritoneal mesothelial cells caused by GDP that is difficult to exclude from the peritoneal dialysate using glucose. The kind and concentration of the amyloid formation inhibitor in the peritoneal dialysate preparation of the present invention can be the same as described above.

  The peritoneal dialysate preparation of the present invention, as one embodiment, separates and accommodates a first solution in which an electrolyte salt is dissolved and glucose and glucose polymer are not contained, and a second solution in which glucose or glucose polymer is dissolved. The peritoneal dialysate preparation, wherein at least one of the first liquid and the second liquid contains an amyloid formation inhibitor. According to this embodiment, an amyloid formation inhibitor can be added at the time of manufacture, and there exists an advantage which can save the effort at the time of use. Whether the amyloid formation inhibitor is included in either the first liquid or the second liquid can be appropriately determined from the pH stability of the amyloid formation inhibitor. This embodiment can be implemented using, for example, a conventional two-liquid type dialysate bag.

  As another embodiment, the peritoneal dialysate preparation of the present invention separates and accommodates the first solution in which the electrolyte salt is dissolved and does not contain glucose and glucose polymer, and the second solution in which glucose or glucose polymer is dissolved. The present invention relates to a peritoneal dialysate preparation that further contains a third liquid containing an amyloid formation inhibitor and that is separated and accommodated. According to this embodiment, there is a merit that contact between the amyloid formation inhibitor and the first liquid or the second liquid can be avoided until use. This embodiment can be implemented using, for example, a conventional three-liquid type dialysate bag.

  The peritoneal dialysis fluid preparation of the present invention relates to a peritoneal dialysis preparation in combination with a conventional 1-3 liquid type peritoneal dialysis fluid and an amyloid formation inhibitor for co-infusion into the peritoneal dialysis fluid as another embodiment. The amyloid formation inhibitor is preferably a prefilled syringe containing the amyloid formation inhibitor from the viewpoint of ease of operation. Therefore, this invention relates to the prefilled syringe which accommodated the amyloid formation inhibitory substance for co-injecting into a peritoneal dialysate as another aspect. The prefilled syringe of the present invention can employ a conventionally known prefilled syringe except that the chemical solution is an amyloid formation inhibitor or a solution thereof. In addition, the prefilled syringe of the present invention may be accompanied by an instruction manual for use as a peritoneal dialysis solution.

[Intraperitoneal washing solution]
In still another embodiment, the present invention relates to an intraperitoneal washing solution containing an amyloid formation inhibitor. In the present specification, the intraperitoneal washing liquid is a liquid that is introduced into the abdominal cavity in order to wash the abdominal cavity. For example, when peritoneal dialysis is stopped, it is injected into the abdominal cavity instead of the dialysate and washed. Refers to liquid. Since there is a possibility that GDP remains in the peritoneal cavity even after the dialysate is drained, peritoneal mesothelial cells can be further protected by using an intraperitoneal washing solution containing an amyloid formation inhibitor. The content of the amyloid formation inhibitor in the intraperitoneal washing solution of the present invention can be the same as described above. Ingredients other than the amyloid formation inhibitor may be the same as in the past, for example, physiological saline.

[Peritoneal dialysis method]
In still another aspect, the present invention relates to a peritoneal dialysis method using the solution composition of the present invention or the peritoneal dialysate preparation of the present invention as a peritoneal dialysate. A specific method can be performed in the same manner as the known CAPD therapy.

[Method of inhibiting apoptosis]
In still another aspect, the present invention relates to a method for inhibiting apoptosis of cells in contact with a glucose degradation product solution, the method comprising containing an amyloid formation inhibitor in the solution. The cell may be a cell in the body or an in vitro cell.

[Preparation of cell culture medium containing 3,4-DGE]
Congo red (Sigma, final concentration 100 μM), which is an amyloid formation inhibitor, and 3,4-DGE (final concentration 100 μM) isolated and purified in-house from the dialysis solution are prepared in M199 medium (10% FBS, 100 U / ml penicillin). And 100 μg / ml streptomycin, and the same applies hereinafter) to obtain a culture solution 1. Culture solution 2 is the same as culture solution 1 except that Congo red is replaced with curcumin (Sigma, final concentration 10 μM). Congo red is the same as Culture solution 1 except that Congo red is replaced with Thioflavin T (Wako Pure Chemicals, final concentration 100 μM). A culture solution 3 was prepared. Moreover, the M199 culture medium which did not contain the amyloid formation inhibitory substance of the culture solutions 1-3, and added only 3, 4-DGE (final density | concentration of 100 micromol) was used as the comparison culture solution 1. The comparative culture solution 2 is a normal M199 medium that does not contain an amyloid formation inhibitor and 3,4-DGE.

  Using the culture solutions 1 to 3 and the comparison culture solutions 1 and 2 prepared as described above, the cell death inhibitory effect of the amyloid formation inhibitor was confirmed under the following measurement conditions.

[Confirmation test of cell death suppression effect]
Culture medium 1-3 and comparative culture medium 1-2 (500 μl) were added to 8 Well culture slides in which rat peritoneal mesothelial cells were cultured. After further incubation for 4 hours, nuclear staining using Hoechst 33342 and anti-Vimentin antibody were used. Immunostaining was performed. Cell morphology was confirmed by staining the cytoskeleton with Vimentin immunostaining, and constriction of the nucleus observed characteristically in apoptotic cells was confirmed with Hoechst 33342 with a confocal laser microscope. Based on the observation results, the total number of cells and the number of dead cells were counted, and the ratio (%) of dead cells was calculated. The results are shown in Table 1 below.

  As shown in Table 1 above, in the peritoneal mesothelial cells cultured in the culture solutions 1 to 3 containing the amyloid formation inhibitory substance, the cytotoxicity induced by 3,4-DGE as compared with the case of culturing in the comparative culture solution 1 Was reduced.

  In addition, when the cell after culture | cultivation with the comparison culture solutions 1-2 was dye | stained with Congo red, the cell was dye | stained notably with the comparison culture solution 1 compared with the comparison culture solution 2 (Data not showing). In addition, it was confirmed by YO-PRO-1 staining and Hoechst 33258 staining that cell death induced in peritoneal mesothelial cells cultured in Comparative Culture Medium 1 was apoptotic (Data not shown). From these results, it is considered that as a result of amyloidization being promoted by 3,4-DGE, apoptosis is induced and cell death occurs.

  The present invention is useful, for example, in the field of peritoneal dialysis.

Claims (8)

A solution composition containing an electrolyte salt, glucose or glucose polymer, and an amyloid formation inhibitor. Furthermore, the solution composition of Claim 1 containing a glucose degradation product. A peritoneal dialysis fluid preparation containing glucose or a glucose polymer and an amyloid formation inhibitor. A peritoneal dialysis fluid formulation that separates and accommodates a first liquid in which an electrolyte salt is dissolved and that does not contain glucose and glucose polymer, and a second liquid in which glucose or glucose polymer is dissolved. The peritoneal dialysate preparation according to claim 3, wherein at least one of the two liquids contains an amyloid formation inhibitor, or a third liquid containing an amyloid formation inhibitor is further separated and accommodated. Furthermore, the peritoneal dialysate preparation of Claim 3 or 4 containing a glucose degradation product. A prefilled syringe containing an amyloid formation-inhibiting substance to be mixed with peritoneal dialysis fluid. An intraperitoneal washing solution containing an amyloid formation inhibitor. A method for preparing a peritoneal dialysis fluid, comprising mixing an amyloid formation inhibitor with a peritoneal dialysis fluid preparation containing glucose or a glucose polymer.

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