Artificial blood vessels

Abstract

This invention suppresses the occurrence of stenosis due to intimal thickening within the lumen of porous tubes used as artificial blood vessels, thereby improving the lifespan of the artificial blood vessels. [Solution] The artificial blood vessel according to this disclosure is composed of a porous tube coated with a chymase inhibitor on its surface.

Description

[Technical Field]

[0001] The present invention relates to artificial blood vessels, and more particularly to artificial blood vessels coated with a chymase inhibitor on their surface. [Background technology]

[0002] In patients with chronic renal failure, hemodialysis is essential for maintaining life. Typically, prior to hemodialysis, an arteriovenous anastomosis (arteriovenous fistula) is performed to enable blood flow. Furthermore, in patients where multiple surgeries have resulted in the loss of suitable blood vessels for anastomosis, an artificial blood vessel is used as a hemodialysis shunt.

[0003] In recent years, porous tubes made of polytetrafluoroethylene (PTFE), which has excellent flexibility and biocompatibility, have been commonly used as artificial blood vessels (see, for example, Patent Document 1). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2005-152178 [Overview of the project] [Problems that the invention aims to solve]

[0005] While porous PTFE tubes offer the advantages mentioned above, they are not permanently usable in a patient's body, and stenosis of the artificial blood vessel may occur approximately two years after surgery. One reason for this is the thickening of the intimal layer within the lumen of the artificial blood vessel, which leads to stenosis. As a result, the patient may require re-operation to implant a new artificial blood vessel, which is burdensome for the patient.

[0006] This invention has been made in view of the above-mentioned problems, and its purpose is to suppress the occurrence of stenosis due to intimal thickening in the lumen of porous tubes used as artificial blood vessels, and to improve the lifespan of the artificial blood vessel. [Means for solving the problem]

[0007] To achieve the above objective, the inventors, through diligent research, discovered that by coating the surface of a porous tube with a chymase inhibitor, the migration of fibroblasts and other cells from the periphery of an artificial blood vessel into the lumen, which occurs when the porous tube is used as an artificial blood vessel, can be suppressed. As a result, the present invention was completed.

[0008] An artificial blood vessel according to one embodiment of the present invention is characterized in that a chymase inhibitor is coated on the surface of a porous tube.

[0009] According to an artificial blood vessel of one embodiment, since the surface of the porous tube is coated with a chymase inhibitor, the chymase inhibitor can suppress the migration of fibroblasts and other cells from the periphery of the artificial blood vessel into the lumen, and as a result, intimal thickening of the artificial blood vessel can be suppressed. This can suppress stenosis within the lumen of the artificial blood vessel, thereby improving the lifespan of the artificial blood vessel.

[0010] In an artificial blood vessel according to one embodiment of the present invention, the porous tube is preferably a tube made of PTFE.

[0011] PTFE is suitable as a material for artificial blood vessels because of its excellent flexibility and biocompatibility.

[0012] In an artificial blood vessel according to one embodiment of the present invention, the chymase inhibitor preferably comprises a peptide having a chymase inhibitory region and an anchor region for binding to polytetrafluoroethylene.

[0013] In this case, the peptide is (PhO)2-Phe, which is represented by the following chemical formula. P-Pro-Val-Suc-(Gly)3-(Lys-Tyr)3-NH2 is preferred.

[0014]

Chemical formula

[0015] By doing so, the above peptide as a chymase inhibitor can be suitably bound to the artificial blood vessel and can function as a chymase inhibitor in the long term. Therefore, the occurrence of stenosis in the lumen of the artificial blood vessel can be suppressed in the long term, and as a result, the lifespan of the artificial blood vessel can be further improved.

Advantages of the Invention

[0016] According to the artificial blood vessel of the present invention, the occurrence of stenosis due to intimal hyperplasia in the lumen of the porous tube used as the artificial blood vessel can be suppressed, and the lifespan of the artificial blood vessel can be improved.

Brief Description of the Drawings

[0017] [Figure 1] FIG. 1 is a photograph of a section of a paraffin block of an artificial blood vessel composed of a PTFE porous tube prepared in an embodiment, stained with mast cells, chymase, angiotensin II (AngII), and transforming growth factor β (TGF-β). [Figure 2] FIG. 2 is a photograph of a section of a paraffin block of an artificial blood vessel composed of a PTFE porous tube prepared in an embodiment, stained with vimentin 4 weeks and 8 weeks after transplantation. [Figure 3] FIG. 3 is a graph comparing the intimal hyperplasia areas of an artificial blood vessel composed of a PTFE porous tube and an artificial blood vessel composed of a tube with a non-porous layer in an embodiment. [Figure 4] FIG. 4 is a graph comparing the intimal hyperplasia areas of an artificial blood vessel coated with a chymase inhibitor and an artificial blood vessel not coated in an embodiment.

Best Mode for Carrying Out the Invention

[0018] Hereinafter, embodiments for carrying out the present invention will be described. The following description of the preferred embodiments is merely illustrative in nature and is not intended to limit the present invention, its application method, or its uses.

[0019] One embodiment of the present invention is an artificial blood vessel characterized in that a chymase inhibitor is coated on the surface of a porous tube.

[0020] In this embodiment, as the porous tube constituting the artificial blood vessel, commercially available products generally used as artificial blood vessels can be used. For example, a porous artificial blood vessel formed by stretching PTFE can be used. Since PTFE is excellent in flexibility, biocompatibility, etc., it is suitably used as a material for artificial blood vessels.

[0021] In this embodiment, the chymase inhibitor inhibits the action of chymase. Chymase is known as an enzyme having the ability to produce angiotensin II in addition to angiotensin-converting enzyme (ACE) in human vascular tissue, and is a type of serine protease particularly expressed in mast cells. Chymase present in normal vascular tissue is stored in a state without enzymatic activity in mast cell granules, that is, in a state without the ability to produce angiotensin II. That is, chymase is considered to exhibit an enzymatic function only in pathological tissues. Also, chymase has been reported to be related to tissue fibrosis in renal failure, liver cirrhosis, heart failure, etc. For example, in balloon catheter injury or grafted vascular tissue, chymase is released from mast cells and functions as an enzyme that produces angiotensin II. Also, the relationship between mast cells and fibroblasts has been focused on, and it has been clarified that chymase secreted from mast cells directly proliferates fibroblasts and is involved in fibrosis in the heart, kidney, etc.

[0022] The chymase inhibitor is not particularly limited as long as it contains a substance that inhibits the action of chymase as described above. For example, well-known serpines (serine protease inhibitors), antibodies such as anti-chymase antibodies, and other peptide-based chymase inhibitors may be used. The chymase inhibitor may contain other substances as long as it contains such chymase inhibitors. However, in this embodiment, since the chymase inhibitor needs to be coated on the surface of the porous tube, it is preferable that it contains a substance or portion that can be maintained on the surface of the porous tube. In this specification, "coating on the surface of the porous tube" means that the chymase inhibitor is present on and maintained on the surface of the porous tube, and its form is not limited. For example, the chymase inhibitor may exist on the surface of the porous tube in any form, such as bound, fixed, or adsorbed.

[0023] Therefore, in this embodiment, the chymase inhibitor preferably comprises a peptide having a chymase inhibitory region and an anchor region for binding to a porous tube. When the porous tube is made of PTFE, the anchor region is composed of a peptide that can bind to PTFE.

[0024] In this embodiment, the peptide constituting the above-mentioned chymase inhibitor is (PhO)2-Phe, represented by the following chemical formula. P -Pro-Val-Suc-(Gly)3-(Lys-Tyr)3-NH2 can be used.

[0025] [ka]

[0026] In the above peptide, (PhO)2-Phe P The -Pro-Val-Suc portion acts as a chymase inhibitory region, and the (Gly)3-(Lys-Tyr)3-NH2 portion acts as an anchor region. Such peptides can be synthesized using techniques well known to those skilled in the art, for example, by using the Fmoc solid-phase synthesis method.

[0027] Regarding the method of coating the surface of a porous tube with a chymase inhibitor, for example, methods known in the art can be used. Such methods include applying a chymase inhibitor solution to the surface of the porous tube, or immersing the porous tube in a chymase inhibitor solution and then drying it to achieve coating. For example, when coating the surface of a porous tube made of PTFE with the peptide, the tube can be immersed in a solution containing the peptide, subjected to reduced pressure, then an oxidizing agent is added to the solution to react, and finally dried to achieve coating. This method is preferable because it allows the peptide to be present not only on the outer surface of the porous tube, but also on the inner surface and within the porous parts of the tube's walls, thereby improving the chymase inhibitory effect. [Examples]

[0028] The following are examples illustrating the artificial blood vessel of the present invention in detail.

[0029] (Mechanism of intimal thickening in artificial blood vessels) First, we conducted experiments to clarify the mechanism of intimal thickening within the lumen of artificial blood vessels. Specifically, male beagle dogs were anesthetized, an inguinal incision was made, the femoral artery and vein were dissected, and after blood flow occlusion, an artificial blood vessel (Getting Group Japan Co., Ltd. (Tokyo), Advanta PTFE Artificial Blood Vessel VXT, product number: 21007), consisting of a PTFE porous tube with an inner diameter of 4 mm and a length of approximately 10 cm, was implanted in a loop between the carotid artery and vein. Eight weeks after transplantation, the artificial blood vessel was harvested, fixed with Carnoy's fixation to analyze the intimal thickening of the artificial blood vessel, and paraffin blocks were prepared at a later date. Subsequently, toluidine blue staining was performed on sections of the paraffin blocks for mast cells using a standard method, and immunostaining was performed for angiotensin II, chymase, and TGF-β using antibodies that recognize each respective. The results are shown in Figure 1.

[0030] As shown in Figure 1, mast cells are present on the outside of the artificial blood vessel 8 weeks after transplantation, and in particular, chymase-producing cells are observed to be concentrated. Angiotensin II (AngII) and transforming growth factor β (TGF-β), which are activated by chymase production, were also observed.

[0031] Next, the following experiment was conducted to investigate cell migration in the artificial blood vessel wall. Specifically, artificial blood vessels were transplanted into beagle dogs in the same manner as described above, and the artificial blood vessels were harvested at 4 and 8 weeks later. Carnoy's fixation was performed to analyze the intimal thickening of the artificial blood vessels, and paraffin blocks were prepared at a later date. Subsequently, immunostaining using anti-vimentin antibodies was performed on sections of the paraffin blocks using a standard method to stain fibroblasts. The results are shown in Figure 2.

[0032] As shown in Figure 2, four weeks after artificial blood vessel transplantation, some cells were observed in the wall of the artificial blood vessel, but no cells were found in the lumen of the artificial blood vessel. On the other hand, eight weeks after artificial blood vessel transplantation, fibroblasts were observed throughout the wall of the artificial blood vessel, and furthermore, fibroblasts had accumulated in the wall of the lumen of the artificial blood vessel, forming a thickened intima. From these results, it is thought that after artificial blood vessel transplantation, fibroblasts surrounding the artificial blood vessel pass through the pores of PTFE to reach the lumen of the artificial blood vessel, where they accumulate on its inner wall and form a thickened intima.

[0033] Furthermore, to investigate this phenomenon, we compared the behavior of cells in artificial blood vessels made of PTFE porous tubing with those made of non-porous material in the intermediate layer of the wall (Terumo Corporation, Gracil®). Specifically, the two types of artificial blood vessels were implanted into beagle dogs (5 of each) in the same manner as above. After 16 weeks, the artificial blood vessels were harvested, fixed with Carnoy's fixation to analyze the intimal thickening of the artificial blood vessels, and paraffin blocks were prepared at a later date. Subsequently, sections of the paraffin blocks were stained with Mallory azocarmine G stain and aniline (Muto Chemical Co., Ltd., Japan) using standard Azan staining. The paraffin blocks were prepared by dividing the artificial blood vessel into three equal parts: arterial side, venous side, and intermediate part, and sections from the central part of each were observed under a microscope. The intimal thickening area of ​​the lumen of the artificial blood vessel was measured and calculated as the intimal area of ​​the lumen per unit length of the artificial blood vessel. Specifically, the area of ​​intimal thickening observed within the section and the length of the artificial blood vessel within the section were measured using the software WinROOF2021 (Mitani Corporation), and the intimal area per unit artificial blood vessel length (mm²) was calculated by dividing the measured intimal thickening area by the length of the artificial blood vessel. 2 The value ( / mm) was calculated. The results are shown in Figure 3. Note that the graph on the right in Figure 3 shows the results for the arterial side, venous side, and intermediate section separately, while the graph on the left shows the average calculated by adding them together.

[0034] As shown in Figure 3, in PTFE artificial blood vessels, intimal thickening of the inner wall increased over time, both 8 weeks and 16 weeks after transplantation. On the other hand, while intimal thickening also occurred in Gracil artificial blood vessels, it was suppressed compared to PTFE. Furthermore, this suppression was particularly pronounced in the middle portion of the artificial blood vessel. Therefore, it is suggested that intimal thickening of artificial blood vessels after transplantation, except near the ends of the artificial blood vessel, such as the arterial and venous sides, i.e., outside the anastomosis site, is closely related to the migration of fibroblasts from around the artificial blood vessel into the lumen.

[0035] (Effects of chymase inhibitor-coated artificial blood vessels) Based on the above studies, we investigated the usefulness of artificial blood vessels coated with chymase inhibitors in preventing intimal thickening. The methods and results are described below. First, we will explain the chymase inhibitors used in this study. In this study, we used peptide-type chymase inhibitors, particularly (PhO)2-Phe, which is represented by the following chemical formula. P -Pro-Val-Suc-(Gly)3-(Lys-Tyr)3-NH2 was used. This peptide was synthesized by the conventional Fmoc solid-phase synthesis method.

[0036] [ka]

[0037] The coating of the above peptide as a chymase inhibitor onto the surface of a PTFE porous tube used as an artificial blood vessel is described below. First, a PTFE porous tube with an inner diameter of 6 mm and a length of approximately 6 cm was ultrasonically cleaned in methanol for 30 minutes. Then, it was ultrasonically cleaned in ultrapure water for 30 minutes, and again in methanol for 30 minutes. Subsequently, 5 mL of a solution containing the above peptide (4.0 × 10⁻³) was placed in a 5 mL tube. -4 A PTFE porous tube was immersed in the peptide solution (M, solvent is sterile ultrapure water) and subjected to reduced pressure treatment for 5 minutes. After that, an oxidizing agent (5.3 × 10⁻¹⁰) was added to the peptide solution. -3 M CuCl2 aqueous solution, 4.8 × 10 -5 A aqueous solution of H2O2 (M) was added and shaken at 50°C for 24 hours. After that, the PTFE porous tube was washed with ultrapure water, placed in a sterile pack, and dried under reduced pressure. In this way, the peptide described above, acting as a chymase inhibitor, was coated onto the surface of the PTFE porous tube used as an artificial blood vessel.

[0038] The above-coated artificial blood vessels and the uncoated PTFE porous tubes used as artificial blood vessels were each transplanted into beagle dogs (5 each) in the same manner as above. Six months later, the artificial blood vessels were collected, fixed with Carnoy's solution for analyzing intimal hyperplasia of the artificial blood vessels, and paraffin blocks were prepared at a later date. Thereafter, ordinary Azan staining using Mallory's azocarmine G staining solution and aniline (Muto Chemical Co., Ltd., Japan) was performed on sections of the paraffin blocks. The paraffin blocks were prepared by dividing the artificial blood vessels into three equal parts on the arterial side, venous side, and middle part, and the sections at the center of each part were observed under a microscope. Then, the intimal hyperplasia area of the artificial blood vessel lumen was measured and calculated as the luminal intimal area per unit artificial blood vessel length. Specifically, in the same manner as the above experiment, the area of intimal hyperplasia observed in the section and the length of the artificial blood vessel in the section were measured by software WinROOF2021 (Mitani Shoji Co., Ltd.), and the measured intimal hyperplasia area was divided by the artificial blood vessel length to calculate the luminal intimal area (mm 2 / mm) per unit artificial blood vessel length. Also, at that time, the measurements and calculations were performed separately for the arterial side, venous side, and middle part of the artificial blood vessel, and finally, they were totaled to calculate the average. The results are shown in FIG. 4.

[0039] As shown in FIG. 4, when an artificial blood vessel not coated with the above peptide was used, intimal hyperplasia occurred in the lumen of the artificial blood vessel. On the other hand, when an artificial blood vessel coated with the above peptide was used, the occurrence of such intimal hyperplasia was significantly suppressed. Thus, it was revealed by this test that by coating a surface of an artificial blood vessel composed of a porous tube with a chymase inhibitor, the occurrence of intimal hyperplasia occurring in the lumen of the artificial blood vessel can be suppressed.

[0040] From the above, according to the artificial blood vessel of the present invention, the occurrence of stenosis due to intimal hyperplasia in the lumen of the porous tube used as the artificial blood vessel can be suppressed, and the lifespan as an artificial blood vessel can be improved.

Claims

[Claim 1] An artificial blood vessel composed of a porous tube coated with a chymase inhibitor on its surface, The porous tube is a tube made of polytetrafluoroethylene, The chymase inhibitor comprises a chymase inhibitory region and a region that binds to polytetrafluoroethylene. It includes a peptide having an anchor region for, The aforementioned peptide is an artificial blood vessel represented by the following chemical formula: (PhO) 2-Phe P-Pro-Val-Suc-(Gly) 3-(Lys-Tyr) 3-NH 2. 【Chemistry 1】

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