JP7228882B2 - Artificial blood vessel and method for manufacturing artificial blood vessel - Google Patents

Description

TECHNICAL FIELD The present invention relates to an artificial blood vessel and a method for manufacturing an artificial blood vessel.

In surgery using an artificial indwelling object such as an artificial blood vessel, if postoperative infection occurs, it is likely to be intractable, and often the infection cannot be cured unless the artificial object is removed. In particular, postoperative infection has been a problem in valve replacement surgery in the field of cardiovascular surgery and aortic surgery using artificial blood vessels (Non-Patent Document 1).

To remedy this situation, an endoprosthesis characterized by an active agent coating consisting of at least one antibiotic, such as an aminoglycoside antibiotic, and a pantothenic acid derivative, said endoprosthesis An invention relating to an endoprosthesis is known (Patent Document 1). However, since this method is intended for coating artificial organs, which are inorganic substances, it is difficult to apply this method to artificial blood vessels using chemical polymer resins, such as the need for a drying process in a drying oven for four hours.

Further, the controlled drug release member for indwelling in vivo comprises a base material composed of at least one of a metal material, a ceramic material and a polymer material, and a multilayer film covering the surface of the base material and releasing the drug. , wherein the multilayer film contains a drug such as an antithrombotic agent and an antibiotic, and includes a polymer film that slowly releases the drug, and a protective film that covers the polymer film and limits the sustained release of the drug. A controlled drug release member containing such is also known (Patent Document 2). However, the member was made for a stent, and the compatibility of such a member with the surface of a blood-infused vascular prosthesis has never been verified.

Advances in artificial blood vessel technology have led to the development of heparin-containing artificial blood vessels that prevent thrombosis, but no antibiotic-containing heparin-containing artificial blood vessels have been described (Non-Patent Document 2).
Artificial blood vessels have the problem of bleeding and plasma leakage in the body relatively early after surgery, and gelatin-coated artificial blood vessels are known as artificial blood vessels that do not cause bleeding (Patent Document 3). ).

A vascular prosthesis containing rifampicin in a gelatin-coated layer is known for use in aortic surgery using a vascular prosthesis (Non-Patent Document 3). However, rifampicin has a side effect of liver dysfunction, and resistant bacteria are likely to emerge. In addition, the leaching method of rifampicin is complicated, and it is difficult to incorporate other drugs at the same time. Vancomycin-soaked artificial blood vessels are also known to be effective against MRSA (Non-Patent Document 4), but vancomycin is not effective against Pseudomonas aeruginosa.

Patent No. 5964867 WO2010/029753 Patent No. 2815752

Edited by Akira Tsuji, Seminar on Hospital Infection Control Q&A, 74 pages, 2006, published by the Japanese Association for Infectious Diseases Takeshi Baba, Yuji Kanaoka, Takao Oki, "Current status and recent progress of artificial blood vessels", Artificial organs, Vol.44, No.3, 2015, pp.146-151 Satoshi Hirata, Tadahiro Sasajima, Kazutomo Go, Susumu Koshiko, Eiji Yayayanagi, Hirosuke Yamazaki, Yoshihiko Kubo, Kaoru Chiba, "Rifampicin-bonded Gelatin-sealed Dacron Synthetic Aortic Resection for T4 Lung Cancer", Japanese Journal of Respiratory Surgery, Vol. 12, No. 7, 1998, pp. 42-46 Satoshi Michimoto, Toshihisa Asakura, Hiroyuki Koike, Kozo Morita, Dr. Niinami, Yuji Suda, "A case of surgical trial for ruptured thoracoabdominal aortic aneurysm with active stage infection complicated by empyema", Journal of the Japanese Society of Vascular Surgery, Vol.22, 2013 year, pages 1013-1016

There is a demand for a simple method for producing a vascular graft containing an antibacterial agent that has few side effects and a wide antibacterial spectrum, and an antibacterial agent-containing artificial vascular graft that can be mixed with various drugs.

The present invention
[1] An artificial blood vessel coated with gelatin containing colistin.
[2] The artificial blood vessel according to [1], which contains vancomycin.
[3] The method for producing an artificial blood vessel according to [1] or [2], wherein the gelatin-coated artificial blood vessel is immersed in colistin-containing physiological saline for 5 to 10 minutes.
[4] The production method of [3], wherein vancomycin is added to colistin-containing physiological saline.
[5] The production method of [3] or [4], wherein the concentration of colistin or vancomycin in physiological saline is 400 to 800 µg/ml.
[6] The production method according to any one of [3] to [5], wherein the gelatin-coated artificial blood vessel is immersed in colistin-containing physiological saline for 5 minutes.
[7] The production method of any one of [3] to [6], wherein the concentration of colistin or vancomycin in physiological saline is 600 μg/ml.

INDUSTRIAL APPLICABILITY According to the present invention, an artificial blood vessel free from infection after surgery and a simple manufacturing method thereof are provided.

FIG. 1 shows the amount of bacteria inside and outside a gelatin-coated artificial blood vessel (CTL-ABV) containing no antibacterial agent as a control for Pseudomonas aeruginosa PA01 (Pseudomonas aeruginosa PA01: hereinafter referred to as Pseudomonas aeruginosa). is the result of the change over time of FIG. 2 shows the time-dependent change in bacterial load of Pseudomonas aeruginosa inside and outside gelatin-coated vascular grafts containing colistin (COL-ABV). FIG. 3 shows the time course of the amount of Pseudomonas aeruginosa inside and outside gelatin-coated vascular grafts (COL+REP-ABV) containing rifampicin and colistin. FIG. 4 shows the time-course results of bacterial load of Pseudomonas aeruginosa inside and outside gelatin-coated vascular grafts (REP-ABV) containing rifampicin. FIG. 5 shows the amount of bacteria inside and outside a gelatin-coated artificial blood vessel (CTL-ABV) containing no antibacterial agent as a control for Staphylococcus aureus N315 (Staphylococcus aureus N315: hereinafter referred to as MRSA). This is the result of aging. FIG. 6 shows the time-course results of bacterial load on the inside and outside of vancomycin-containing gelatin-coated vascular grafts (VCM-ABV) for MRSA. FIG. 7 shows the time-course results of bacterial load on the inside and outside of rifampicin-containing gelatin-coated vascular grafts (REP-ABV) for MRSA. FIG. 8 shows the correlation between the immersion time of colistin (a) or vancomycin (b) and the amount of binding to the artificial blood vessel.

The gelatin-coated artificial blood vessel in the present invention refers to an artificial blood vessel coated with gelatin that does not require pre-clotting. Artificial blood vessels made of synthetic polymer materials, artificial blood vessels derived from biomaterials processed from animal blood vessels, hybrid artificial blood vessels in which artificial materials and animal materials are mixed, etc., but polyester artificial blood vessels and Teflon artificial blood vessels are preferred. is mentioned.
In the gelatin-coated artificial blood vessel containing colistin of the present invention, the inclusion of colistin means that in surgery using the artificial blood vessel, colistin is added even after surgery in order to prevent infection with bacteria such as Pseudomonas aeruginosa after surgery. It shows a state in which colistin is contained in the gelatin layer so that even in the gelatin-coated artificial blood vessel, colistin is retained in the gelatin layer and exhibits an antibacterial effect.

The necessary amount of colistin contained in the gelatin-coated artificial blood vessel is considered to be the condition under which the artificial blood vessel exhibits an antibacterial effect after transplantation. Even after immersion, it is preferable that the material is maintained so as to exhibit an antibacterial effect against Pseudomonas aeruginosa.

The specific amount of colistin contained in the gelatin-coated artificial blood vessel containing colistin of the present invention is preferably 50 μg or more, preferably 100 μg, of colistin per 3 cm of the artificial blood vessel.

In the present invention, "containing vancomycin" means that the artificial blood vessel coated with gelatin containing colistin further contains vancomycin. The combination of colistin and vancomycin is effective in providing the artificial blood vessel with a wide range of infectious disease prevention effects, since vancomycin is also effective against MRSA, which is not inhibited by colistin or other antibiotics.

The amount of vancomycin contained in the artificial blood vessel of the present invention is determined after immersing the artificial blood vessel coated with colistin-containing gelatin in physiological saline for 24 hours. Even so, it is preferable that it is maintained so as to exhibit an antibacterial effect against MRSA. A specific amount of vancomycin is 50 μg or more, preferably 100 μg per 3 cm of artificial blood vessel.

In the present invention, colistin may contain rifampicin, which exhibits an antibacterial effect against MRSA, and vancomycin may also be contained.
The amount of rifampicin contained in the artificial blood vessel of the present invention is determined after immersing the artificial blood vessel coated with gelatin containing rifampicin in physiological saline for 24 hours. Even so, it is preferable that it is maintained so as to exhibit an antibacterial effect against MRSA.

In the present invention, the method for producing an artificial blood vessel coated with gelatin containing colistin comprises:
Any method for producing an artificial blood vessel containing colistin and gelatin and having the antibacterial function may be used. A method of adding colistin, a method of processing the polymer on the surface of the artificial blood vessel to form a hydroxyl group, a carboxyl group, an epoxy group, an amino group, etc., and a method of adding colistin to the surface of which gelatin is bound may also be used. . The means for forming hydroxyl groups, carboxyl groups, epoxy groups, or amino groups on the entire surface of a tube made of a porous synthetic polymer such as polyester or Teflon is graft polymerization if the synthetic polymer is Teflon or polyester. employ graft polymerization or partial hydrolysis. The vascular prosthesis of the present invention also includes one in which gelatin is covalently bonded to the thus formed functional group and colistin is further added. The present invention can also be produced by adding colistin to commercially available gelatin-coated artificial blood vessels.

In the present invention, the method of adding colistin to the artificial blood vessel coated with gelatin includes a method of applying colistin dissolved in a solution to the gelatin layer of the artificial blood vessel by spraying, and a colistin solution in which colistin is dissolved in a solution. Examples include a method of immersing an artificial blood vessel coated with gelatin and a method of inkjet printing colistin on the gelatin layer of the artificial blood vessel. However, it is preferable to use a method of impregnating the gelatin layer with colistin by an immersion method.

In the present invention, the method of immersing an artificial blood vessel coated with gelatin in a colistin solution in which colistin is dissolved in a solution includes 400 μg of colistin dissolved in a solution such as physiological saline, sterile water for injection, and phosphate buffer. /ml to 800 μg/ml, preferably 500 μg/ml to 700 μg/ml, most preferably 600 μg/ml of colistin aqueous solution is prepared, and a gelatin-coated artificial blood vessel is added thereto at 40 degrees without affecting the gelatin layer. It is a method of immersing for 4 to 15 minutes, preferably 5 to 10 minutes, particularly preferably 5 minutes at the following temperature, preferably room temperature of 30°C to 1°C, preferably normal temperature of 25°C to 15°C.

A gelatin-coated vascular prosthesis containing colistin and vancomycin is produced by preparing a mixed solution of colistin and vancomycin, since the physicochemical properties of colistin and vancomycin are similar. can be done.

A method for producing a gelatin-coated artificial blood vessel containing colistin and vancomycin includes adding colistin and vancomycin to the gelatin-coated artificial blood vessel. Methods such as spray coating on the gelatin layer of blood vessels, immersing a gelatin-coated artificial blood vessel in a mixed solution of colistin and vancomycin dissolved in a solution, inkjet printing colistin and vancomycin on the gelatin layer of the artificial blood vessel, etc. However, it is preferable to use a method of impregnating the gelatin layer with colistin and vancomycin by an immersion method.

In the case of manufacturing by the immersion method, vancomycin is dissolved together with colistin in a solution such as physiological saline, sterilized water for injection, phosphate buffer, etc., at 400 μg/ml to 800 μg/ml, preferably 500 μg/ml to 700 μg/ml. , most preferably 600 μg/ml vancomycin in water. An artificial blood vessel coated with gelatin is placed in the solution at a temperature of 40 degrees or less that does not affect the gelatin layer, preferably a room temperature of 30 degrees to 1 degree, preferably a room temperature of 25 degrees to 15 degrees, for 4 minutes to 15 minutes. , preferably 5 to 10 minutes, particularly preferably 5 minutes.

In addition, when rifampicin is used, the solubility of rifampicin is considerably worse than that of colistin or vancomycin, so it is preferable to prepare an aqueous rifampicin solution in advance and add colistin or vancomycin. Rifampicin is dissolved and sodium chloride or the like is added to prepare an aqueous rifampicin solution of 400 μg/ml to 800 μg/ml, preferably 500 μg/ml to 700 μg/ml, most preferably 600 μg/ml. The rifampicin solution is added with colistin prepared as described above and, if necessary, vancomycin, and the gelatin-coated artificial blood vessel is placed at a temperature of 40°C or less, preferably at room temperature of 30°C to 1°C so as not to affect the gelatin layer. , preferably at room temperature of 25°C to 15°C, for 4 minutes to 15 minutes, preferably 5 minutes to 10 minutes, particularly preferably 5 minutes.

[Example 1]
Colistin (colistin sulfate, manufactured by Sigma) was dissolved in physiological saline and adjusted to a concentration of 600 µg/ml. A colistin-containing vascular prosthesis (hereinafter referred to as COL-ABV) was prepared by immersing Techselweave (manufactured by Terumo Corporation) at room temperature for 5 minutes.

[Example 2]
25 mL of the physiological saline injection was put into a syringe, and 150 mg of rifampicin (Rifampicin Capsules, manufactured by Sandoz Co.) was put into another syringe. These two syringes are connected with a three-way stopcock and mixed, mixed left and right 20 times, moved to one side, filtered by connecting two types of filters of 0.8 μm and 0.22 μm, and transferred to another syringe. An aqueous solution adjusted to a rifampicin concentration of 600 μg/ml was prepared by inserting, locking the syringe, and expelling. To 30 mL of this rifampicin aqueous solution, 18 mg of colistin (colistin sulfate, manufactured by Sigma) was added to prepare a colistin and rifampicin mixed solution so that each of colistin and rifampicin was 600 μg/ml. A gelatin-coated artificial blood vessel with an inner diameter of 8 mm and a length of 6 cm (trade name: Vasque Tech Zellweave, manufactured by Terumo Corporation) was immersed in 30 ml of the mixed solution at room temperature for 5 minutes to form a colistin- and rifampicin-containing artificial blood vessel (hereinafter referred to as COL + RFP-ABV). ) was produced.

[Example 3]
A gelatin-coated artificial blood vessel with an inner diameter of 8 mm and a length of 6 cm (product A vancomycin-containing artificial blood vessel (hereinafter referred to as VCM-ABV) was prepared by immersing a Vasque Tech Zellweave (manufactured by Terumo Corporation) at room temperature for 5 minutes.

[Example 4]
The colistin-containing gelatin-coated artificial blood vessel (COL-ABV) prepared in Example 1 above, the colistin- and rifampicin-containing gelatin-coated artificial blood vessel (COL+RFP-ABV) prepared in Example 2 above, and the vancomycin-containing gelatin-coated artificial blood vessel prepared in Example 3. An artificial blood vessel (VCM-ABV), a gelatin-coated artificial blood vessel (CTL-ABV) containing no control antibacterial agent prepared in Reference Example 1, and an artificial blood vessel containing lymphampicin prepared in Reference Example 2 (RFP- ABV) was immersed in physiological saline at room temperature for 24 hours in order to obtain conditions that match the conditions for using artificial blood vessels after surgery.

In the Pseudomonas aeruginosa test group, after the 24-hour immersion operation, control gelatin-coated artificial blood vessels (CTL-ABV), colistin-containing gelatin-coated artificial blood vessels (COL-ABV), rifampicin-containing gelatin-coated artificial blood vessels (RFP) -ABV), colistin and rifampicin-containing gelatin-coated artificial blood vessels (COL + RFP-ABV) are folded and placed in a 6-well plate, and Pseudomonas aeruginosa PA01 is placed on the outside of the artificial blood vessel. Physiological saline solution was injected into the inside of the fungus solution (amount of bacteria, 10 ⁶ CFU/ml) and cultured under conditions of 37° C. reaction temperature and 5% CO ₂ . At 0 hours, 6 hours, 12 hours, 18 hours, and 24 hours after the start of culture for each artificial blood vessel during culture, the aqueous solution inside and outside the artificial blood vessel were collected, and the amount of Pseudomonas aeruginosa was measured (n = 3, the results are calculated as the mean value). Fig. 1 shows changes over time in the amount of Pseudomonas aeruginosa outside and inside the control gelatin-coated artificial blood vessel (CTL-ABV), and time-course changes in the amount of Pseudomonas aeruginosa outside and inside the colistin-containing gelatin-coated artificial blood vessel (COL-ABV). Figure 2 shows changes over time in the amount of Pseudomonas aeruginosa outside and inside gelatin-coated artificial blood vessels containing colistin and rifampicin (COL + RFP-ABV). FIG. 4 shows the change over time in the amount of Pseudomonas aeruginosa inside.

In the control vascular prosthesis (CTL-ABV) and the rifampicin-containing gelatin-coated vascular prosthesis (RFP-ABV) of the known art, the amount of bacteria inside and outside the vascular prosthesis increased with time (Fig. 1, Fig. 3). In the colistin-containing gelatin-coated artificial blood vessel (COL-ABV) of the present invention, the amount of bacteria on the outside of the artificial blood vessel decreased significantly until 12 hours after the start of the experiment, and then increased slightly, but did not grow on the inside (Fig. 2). In the artificial blood vessel containing colistin and rifampicin (COL+RFP-ABV), Pseudomonas aeruginosa disappeared 6 hours after the start of the experiment, and did not grow inside (Fig. 4).

In the MRSA test group, after the 24-hour immersion operation, control gelatin-coated artificial blood vessels (CTL-ABV), vancomycin-containing gelatin-coated artificial blood vessels (VCM-ABV), and rifampicin-containing gelatin-coated artificial blood vessels (RFP-ABV) were used. ) is folded and placed in a 6-well plate, and a bacterial solution of Staphylococcus aureus N315 (Staphylococcus aureus N315: hereinafter referred to as MRSA) (bacterial amount, 10 ⁶ CFU/ml) is placed outside the artificial blood vessel. , saline was injected inside, and culture was carried out under the conditions of a reaction temperature of 37°C and 5% CO ₂ . At 0 hour, 6 hours, 12 hours, 18 hours, and 24 hours after the start of culture for each artificial blood vessel during culture, the aqueous solution inside and outside the artificial blood vessel were collected, and the amount of MRSA bacteria was measured (n = 3). , calculated as the mean value). Figure 5 shows the time-dependent changes in the amount of MRSA bacteria outside and inside the control artificial blood vessel (CTL-ABV), and Figure 6 shows the time-dependent changes in the amount of MRSA bacteria outside and inside the vancomycin-containing gelatin-coated artificial blood vessel (VCM-ABV). In addition, FIG. 7 shows changes over time in the amount of MRSA bacteria outside and inside the rifampicin-containing gelatin-coated artificial blood vessel (RFP-ABV).

In the control gelatin-coated artificial blood vessel (CTL-ABV), the amount of bacteria increased on the outside and rapidly increased on the inside after 6 hours (Fig. 5). In vancomycin-containing gelatin-coated artificial blood vessels (VCM-ABV) and rifampicin-containing gelatin-coated artificial blood vessels (RFP-ABV), the amount of bacteria decreased on the outside and did not grow on the inside (Figs. 6 and 7).

The above results show that colistin-containing artificial blood vessels have a strong effect against Pseudomonas aeruginosa to completely inhibit the growth of Pseudomonas aeruginosa inside blood vessels after surgery, compared to the use of known rifampicin-containing artificial blood vessels. It was suggested that combined use with vancomycin and/or colistin would provide a vascular prosthesis with broad antibacterial effects against Pseudomonas aeruginosa and MRSA.

[Example 5]
The ability of the drug to bind to the vascular prosthesis of the present invention was examined. Specifically, the binding property to artificial blood vessels was measured over time by the immersion method of colistin or vancomycin, and the binding property was examined.

30 mL of an aqueous solution prepared by dissolving colistin (colistin sulfate, manufactured by Sigma) or vancomycin (hydrochloride of vancomycin, manufactured by Wako Pure Chemical Industries, Ltd.) in physiological saline (drug concentration: 600 μg/ml) was placed in a culture tube. A gelatin-coated artificial blood vessel with a length of 3 cm was inserted into the colistin or vancomycin aqueous solution in the culture tube and immersed at room temperature. After immersion for 5 minutes, 10 minutes, and 30 minutes, the artificial blood vessel was removed, and the colistin concentration or vancomycin concentration of the solution remaining in the culture tube was measured as follows.

Colistin was analyzed by high-performance liquid chromatography of the acetone-extracted solution after fluorescent derivatization of colistin in a solid-phase extraction cartridge using an oral derivatization reagent, 9-fluorenylmethylchloroformate, to determine its concentration. It was measured.

In addition, vancomycin is measured by the chemiluminescence immunoassay method by allowing vancomycin to react with antibody-bound magnetic particles, then secondary-reacting an antibody labeled with a chemiluminescence substance with vancomycin, and measuring the luminescence intensity of the chemiluminescence substance. The concentration of vancomycin was determined by

By comparing the concentration of colistin or vancomycin in the aqueous solution in the culture tube before the start of immersion and the concentration of colistin or vancomycin in the remaining solution after immersion in the gelatin-coated artificial blood vessel, the amount of colistin or vancomycin bound to the gelatin-coated artificial blood vessel was determined. It was measured.

The results of the above binding test over time are shown in FIG. 8. Both colistin and vancomycin were found to bind to artificial blood vessels, and the amount of binding reached a plateau in about 10 minutes.
The above tests confirmed that colistin or vancomycin binds to gelatin-coated artificial blood vessels not only by functional analysis but also by chemical analysis.

As described above, it was shown that colistin-containing artificial blood vessels have a stronger effect of suppressing Pseudomonas aeruginosa infection than conventional rifampicin-containing artificial blood vessels, and vancomycin-containing artificial blood vessels, which have a strong effect on MRSA, are manufactured in the same manner as colistin-containing artificial blood vessels. Since it was confirmed that they can be obtained by the method, it is expected that the artificial blood vessel containing colistin and vancomycin will have a strong inhibitory effect on both Pseudomonas aeruginosa and MRSA.

Reference example 1
For comparison with the present invention, a gelatin-coated artificial blood vessel with an inner diameter of 8 mm and a length of 6 cm (trade name: Vasque Tech Zellweave, manufactured by Terumo Corporation) was immersed in 30 ml of physiological saline for 5 minutes. hereinafter referred to as CTL-ABV).

Reference example 2
Take 25 mL of physiological saline injection into a syringe, put 150 mg of rifampicin (Rifampicin Capsules, manufactured by Sandoz) into another syringe, connect these two syringes with a three-way stopcock, mix, and mix 20 times to the left and right. 2 types of filters of 0.8 μm and 0.22 μm are connected and filtered, put into another syringe, locked with a syringe, and dispensed so that the concentration of rifampicin becomes 600 μg/ml. A prepared aqueous solution was prepared. A gelatin-coated artificial blood vessel with an inner diameter of 8 mm and a length of 6 cm (trade name: Vasquetec Zellweave, manufactured by Terumo Corporation) was immersed in 30 ml of this rifampicin aqueous solution for 5 minutes to obtain a rifampicin-containing artificial blood vessel (hereinafter referred to as RFP-ABV). ) was made.

INDUSTRIAL APPLICABILITY The present invention provides an artificial blood vessel that does not cause postoperative infections and a method for producing the same.

Control: control group COL: group of artificial blood vessels containing colistin RFP: group of artificial blood vessels containing rifampicin RFP+COL: group of artificial blood vessels containing rifampicin and colistin VCM: group of artificial blood vessels containing vancomycin

Claims (5)

A gelatin-coated vascular prosthesis characterized by containing 50 μg to 200 μg of colistin per 3 cm of the vascular graft to inhibit growth of Pseudomonas aeruginosa inside the vessel . 2. The vascular prosthesis of Claim 1, further comprising vancomycin to inhibit MRSA growth within the vessel . An aqueous colistin solution of 400 μg/ml to 800 μg/ml is prepared by dissolving colistin in a solution such as physiological saline, sterilized water for injection, or phosphate buffer, and an artificial blood vessel coated with gelatin is placed on the gelatin layer . 2. The method for producing a colistin-containing artificial blood vessel according to claim 1, wherein the immersion is performed for 4 to 15 minutes at a temperature of 40° C. or lower, which does not affect . 4. The method according to claim 3, wherein vancomycin is added to the colistin aqueous solution . 5. The production method according to claim 3, wherein the concentration of vancomycin in the aqueous solution is 400-800 μg/ml.

Images