JP2014061285A - Base material and device for suppressing organ inflammation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a base material and a device for suppressing organ inflammation effective for suppressing organ inflammation caused by a disease that occurs or worsens through the intervention of cytokines/chemokines represented by SIRS.SOLUTION: The invention is a base material 120 for suppressing organ inflammation that includes a water-insoluble carrier and an ethylene-vinyl alcohol copolymer coating at least part of the surface of the water-insoluble carrier, and is disposed in a blood extracorporeal circulation circuit so as to contact the blood to be treated, and a device 100 provided with this base material.

Description

  The present invention relates to a substrate and device for suppressing organ inflammation.

  Systemic inflammatory response syndrome (SIRS) is still a disease with a high fatality rate in the intensive care region, but there are few effective treatments.

  1991 American Society of Thoracic Diseases (ACCO) / American Intensive Care Society (SCCM) Joint Conference Infection, trauma, burns, pancreatitis, and other biological invasions triggered various excess cytokines and systemic inflammation The state of increased response was defined as systemic inflammatory response syndrome (SIRS), and SIRS associated with infection was defined as sepsis. It has been clarified that these conditions further involve various humor mediators centering on cytokines, resulting in organ failure from a severely injured pathological condition after invasion. The process of organ damage after invasion is described by Ogawa in a second attack theory. In the case of excessive invasiveness that does not directly lead to organ damage, neutrophils are primed and accumulated in important organs, and second attack such as infection causes organ damage, resulting in multiple organ failure. Therefore, unlike the case of multiple organ failure in which a plurality of organs are directly and simultaneously impaired, the possibility of preventing or treating the transition to multiple organ failure due to a second attack has been pointed out.

Attempts to start treatment early are being made to prevent multiple organ failure, but not all of SIRS falls into multiple organ failure. Therefore, several severity determinations and diagnostic criteria have been proposed. The ACCO / SCCM joint conference provides diagnostic criteria for SIRS and sepsis, but does not provide diagnostic criteria for multiple organ dysfunction syndrome (MODS). In the multiple organ dysfunction score (MOD score), the measurement items are respiratory (PaO ₂ / FiO ₂ ratio), kidney (creatinine value [μmol / L]), liver (bilirubin value [μmol / L]), cardiovascular ( There are Par: pressure adjusted heart rate), blood (platelet count [number / mL × 10 ⁻³ ]), and nerve (Glasgow Coma Scale), and the score and mortality are set to correlate. In addition, a SOFA (Sepsis-related Organ Failure Assessment) score created by the European Intensive Care Society (ESICM) in 1994 includes, as measurement items, respiratory function (PaO ₂ / FiO ₂ [torr]), coagulation function (platelet count [ × 10 ³ / mm ² ]), liver function (bilirubin value [mg / dL]), circulatory function (blood pressure lowering), central nervous function (Glasgow Coma Scale), renal function (creatinine value [mg / dL]) Although it was made focusing on MODS associated with sepsis, it was not limited to sepsis, so SOFA is now an abbreviation for Sequential Organ Failure Assessment and is widely used.

  SIRS with infection, that is, sepsis, and various humal mediators, mainly cytokines, are involved, resulting in multiple organ failure from severe injuries after invasion. The main points of multidisciplinary treatment for this are as follows: Treatments such as maintenance of tissue oxygen metabolism by surgery, antibiotic administration, etc., treatment of cell damage by removing various humor mediators using blood purification methods, enhancement of self-defense function by nutrition management, etc. have been considered.

  In patients with multiple organ failure, functions such as metabolism and excretion in the kidney and liver are significantly reduced, and pathogenic substances and metabolites accumulate in the body, and it is difficult to secure an infusion space. In recent years, a blood purification method has attracted attention as one of effective treatment methods in order to overcome such adverse conditions.

  For example, Non-Patent Document 1 discloses continuous blood filtration (CHF) / continuous hemofiltration dialysis (CHDF), intermittent plasma exchange (CHF) as main blood purification methods used for multiple organ failure. Examples include IPE (intermittent plasma exchange) / continuous plasma exchange (CPE), endotoxin adsorption therapy (PMX).

  The greatest advantage of CHF / CHDF for multi-organ failure is ease of management of water / electrolyte / acid-base balance in addition to the original significance of substance removal in the medium molecular weight region and part of the low molecular weight protein region. . The more severe the pathological condition, the greater the number of drugs to be administered and the need for sufficient nutritional administration, but the introduction of CHF / CHDF enables safe water management. The effect of CHF / CHDF on the removal of humal mediators has not yet been confirmed, but the theory that effective removal of cytokines cannot be performed is being supported. However, it cannot be denied that currently unmeasured substances are removed by CHF / CHDF and contribute to the improvement of circulatory dynamics and oxygenation.

  Although it has been reported that plasma exchange that has been performed for acute liver failure is useful for the removal of endotoxin and cytokines, the clearance is low, so humoral has a large distribution volume in short-time plasma exchange (IPE) There is no great expectation for removing the mediator. Devices such as Continuous Albumin Purification System (CAPS) in which albumin is added to the dialysate for the purpose of extending the plasma exchange time (CPE) and removing toxins bound to albumin have been devised.

  However, the above CHF / CHDF and IPE / CPE are concepts that attempt to remove all mediators involved in a complex cytokine network, at least in quantitative terms. The effectiveness of different mechanisms is also considered.

  On the other hand, PMX is covered by insurance for septic shock caused by Gram-negative bacilli, and it is reported that it is useful for improvement after treatment for infected lesions, particularly early in the onset (at the time of hyperdynamic state). Endotoxin also has an effect of enhancing the toxicity (TSST-1) of Gram-positive bacteria, and thus is expected to have an effect on Gram-positive bacteria infection cases. However, the endotoxin measurement method has not been established, There are many unclear points such as an early pressor effect that cannot be explained by the mechanism of endotoxin removal.

  The findings regarding endotoxin shock include that the LPS / LBP complex binds to CD14 / TLR4 and a signal is transmitted into the cell to release the cytokine, but the early mediator released at the cell membrane before this cytokine release. Endogenous cannabis (two types of macrophages from anandamide (ANA) and platelets from 2-arachidonyl glyceride (2-AG)) and cytokine release, high mobility group-- a late mediator released from the nucleus of macrophages Two mediators such as 1 (HMGB-1) protein were discovered. ANA exhibits hypotension and neuropsychiatric symptoms via CB1 and CB2 receptors. On the other hand, it has been reported that HNGB-1 protein is lethal and that the serum HMGB-1 protein concentration is significantly higher in the death group than in the sepsis survival group. Cannabinoid is a metabolite produced from phosphophatic acid and arachidonic acid, and has a molecular weight of only a few hundred daltons, but HMGB-1 protein has a molecular weight of 30 kD. It has been reported that ANA is adsorbed to PMX via polymixin-B.

  In Non-Patent Document 2, as a blood purification method after cardiovascular surgery, CHDF, CPE, and PMX treatments that consider humor mediators for acute renal failure (ARF), acute liver failure, and septic shock are also available. Has been done. The results of treatment with CHDF for ARF are as follows: low cardiac output syndrome (LOS) mortality rate is 75%, sepsis mortality rate is 83%, and acute liver failure is the target. When the combination therapy of CHDF and IPE or CHDF and CPE is compared, the prognosis of life is improved, but the prognosis of acute liver failure associated with sepsis is still poor. In addition, we examined the relationship between septic shock and PMX treatment for the group of chest infections caused by pneumonia and mediastinal sinusitis that cause sepsis after cardiovascular surgery. Yes.

  Moreover, in nonpatent literatures 3-6, the trial by CHF and CHDF by various membrane materials is performed with respect to sepsis and septic multiple organ failure.

  In Non-Patent Document 3, the authors conducted ILF, IL-8, which are inflammatory cytokines on the filtrate side and the blood side, respectively, by performing CHF using a polyacrylonitrile (PAN) membrane in 10 cases of septic multi-organ failure. The clearance of IL-10 which is an anti-inflammatory cytokine is calculated and examined. As a result, clearances obtained from filtrates of IL-6, IL-8, and IL-10 were 11.9 ± 0.6, 8.1 ± 1.9, respectively, at a filtration amount of 800 to 1000 [mL / hour]. It is 2.2 ± 0.5 [mL / min], and it is said that it is so small that it cannot be compared with the intrinsic clearance. In addition, the polymethyl methacrylate (PMMA) membrane has higher adsorption capacity than other membranes, and it is said that removal of cytokines with CHDF using the PMMA membrane is effective. When examined in the examples, it is reported that there was no significant difference in the clearance by adsorption of about 10 [mL / min] at most.

  Non-Patent Document 4 describes that CHDF using a PMMA membrane is effective against high humor mediatoremia such as severe sepsis, septic shock and ARDS.

  Non-Patent Document 5 describes the enforcement of CHDF / CHD in the perioperative period.

  Non-Patent Document 6 describes the removal of various humor mediators when CHDF is performed using a PMMA film.

  Non-patent documents 7 to 9 discuss cytokines and chemokines in blood in septic patients and SIRS model rats.

Non-Patent Document 7 describes 25 cases of septic surgery diagnosed as peritonitis. Heparinized whole blood was collected before and after surgery, and the centrifuged plasma was stored frozen at −80 ° C. Various cytokines and chemokines are measured in 30 μl of plasma using Cytokine 17-plex panel ^™ (BIO-RAD Laboratories, Hercules, Calif., USA), which is a system. Specifically, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, G -The items of CSF, CM-CSF, IFN-γ, TNF-α, MCP-1, and MIP-1β are measured.

  In addition, cytokines in the serum of four healthy individuals have been measured, and as a result, TNF-α, IL-1β, IL-4, IL-5, IL-7, IL-10, and IL-12 are measured. (The numerical value that can be read from the graph is at most 10 [pg / mL] or less). On the other hand, IL-6, monocytochemical chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1β) are high, and each max value read from the graph is about 100 [pg / mL]. , About 300 [pg / mL], about 100 [pg / mL].

Non-Patent Document 8 describes that 17 types of cytokines and chemokines were given to Cytokine 17-plex panel ^™ (USA, USA) for 29 patients who had severe sepsis or septic shock within 24 hours after entering ICU. There is a report that it measured using BIO-RAD Laboratories, Hercules, CA). Specifically, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, G -The items of CSF, CM-CSF, IFN-γ, TNF-α, MCP-1, and MIP-1β are measured. As a result, the IL-6, IL-8, IL-10 and MCP-1 values of the deceased patients are higher than those of the surviving patients, and in particular, the IL-10 values of the deceased patients are It was described that it was extremely high compared to the value of the patients who had received it.

  In Non-patent Document 9, when LPS was administered 6 hours after the administration of LPS, a model rat with systemic inflammation was subjected to a blood purification method using CHF / CHDF using a small membrane area dialyzer made of polysulfone membrane. It has been reported that the survival rate is improved in the order of CHDF group> CHF group> control group as compared with the control group that did nothing after administration.

  Patent Documents 1 to 4 describe an ethylene vinyl alcohol copolymer (hereinafter sometimes referred to as “EVAL”). An ethylene vinyl alcohol copolymer is known as a polymer compound characterized by hydrophilicity and blood compatibility. Patent Document 1 discloses a method for producing a hollow fiber, and Patent Document 2 discloses production of a nonwoven fabric. A method has been proposed. Further, because of its excellent hydrophilicity, Patent Document 3 discloses a method for producing a polysulfone hollow fiber hydrophilized with an ethylene vinyl alcohol copolymer. Further, Patent Document 4 discloses ethylene vinyl alcohol. Hydrophobic materials made hydrophilic by dehydration condensation of hydrophilic polymers such as copolymers and methods thereof have been proposed.

Japanese Patent No. 3203047 Japanese Patent Publication No. 6-72350 Japanese Patent No. 3305778 JP 2004-332138 A

Rara Nara, Hitoshi Imaizumi, Yasufumi Asai, “Blood Purification of Multiple Organ Failure”, Kidney and Dialysis, Vol. 48, no. 5 (2000), p. 653-659 Hitoshi Imaizumi, Masamitsu Kaneko, “Apheresis for post-cardiovascular complications”, Journal of Japanese Society of Apheresis, Vol. 18, No. 1 (1999), p. 62-66 Toshio Naka, Masahiro Shinozaki, Toshihiko Morinaga, Hidenori Nasu, Seizo Inui, Tomitsu Abe, “Removal of cytokines by continuous hemofiltration (CHF) / continuous hemofiltration dialysis (CHDF): its mechanisms and effects”, Japan Journal of Apheresis Society, Vol. 18, No. 1 (1999), p. 48-52 Kenichi Matsuda, Hiroyuki Hirasawa, Adult Oda, Hidetoshi Shiga, Kazuya Nakanishi, Masataka Nakamura, “Non-Relual Indication of Continuous Hemofiltration Dialysis (CHDF)”, Medical Engineering Treatment, Vol. 14, no. 2 (2002), p. 107-114 Kenichi Matsuda, Hiroyuki Hirasawa, Hidetoshi Shiga, Masataka Nakamura, “Perioperative CHDF”, intensive care, vol. 10, no. 11 (1998), p. 1217-1227 Kenichi Matsuda, Hiroyuki Hirasawa, Hidetoshi Shiga, Masataka Nakamura, “Removal of human mediator with CHDF”, intensive care, vol. 10, no. 12 (1998), p. 1341-1355 Masanori Hakozaki, Nobuhiro Sato, Masahiro Oga, Masaaki Ogawa, Manabu Takahashi, Yasushi Suzuki, Chihaya Maezawa, Tsuyoshi Wakabayashi, "Simultaneous Quantification of Blood Cytokines and Chemokines in Sepsis-Examination Using Suspension Array System", Iwate Journal, Vol. 59, No. 2 (June 2007), p. 127-138 David Andauluz-Ojeda, Felipe Bobillo, Veronica Iglesias, Raquel Almansa, Lucia Rico, Francis Gandia, Savador Resino, Edurado Tamulaj. Bermejo-Martin, “A combined score of pro- and anti-inflammatory interleukins improvise vitality prediction in seven steps”, Cytokine 57 (20). 332-336 Satoshi Hagiwara, Hideo Iwasaka, Akira Hasegawa, Seigo Hidaka, Aya Uno, Kaori Ueno, Tomohisa Uchida, and Takayuki Noguchi, "Continuous Hemodiafiltration Therapy ameliorates LPS-induced Systemic Inflammation in a Rat Model", Journal of Surgical Research, Vol. 171. No. 2, p. 791-796 (2011)

  In Japan, the Japan Society for Lifesaving Medicine in 1990 defined broad organ failure (MOF) and narrow MOF for 7 organs and systems including blood coagulation system, and since 1998, discussions for scoring Has been made. However, in any score, various humor mediators centering on cytokines that cause SIRS are not mentioned. Therefore, it is not clear what treatment should be performed when and how many cytokines in the blood are present.

  In addition, the blood purification methods described in Non-Patent Documents 1 and 2 are occupying an extremely important position as a multidisciplinary treatment method for SIRS including multiple organ failure, but unfortunately still a complete treatment method. The current situation is not enough. The reason for this is that (i) in the blood purification method, blood is once taken out of the body and subjected to blood filtration, filtration dialysis and adsorption, so that the blood (blood cells and plasma) components are not in contact with the device material which is a foreign substance. It is difficult to avoid and is not a suitable device made of a more biocompatible material (suitable for SIRS treatment), and (ii) the patient's blood condition (cytokine concentration) is treated in a condition suitable for treatment. This is probably because the therapeutic treatment in the appropriate concentration region of cytokines and chemokines in the blood has not been performed.

  By the way, cytokines bind to cell surface receptors of immunocompetent cells in the inflamed region, and once they enter the blood, they are inactivated as immune complexes by binding to α2 macroglobulin and soluble receptors. The half-life of cytokines in the blood is as extremely short as 5 to 10 minutes. For example, it is said that IL-6 is 3 to 7 minutes and TNF-α is 5 to 10 minutes. Cytokines that enter the blood decrease exponentially, but the reason why cytokines continue to show high levels in SIRS is thought to be because they are continuously produced in inflammatory areas. For example, when the circulating blood volume of an adult weighing 60 kg is about 4600 mL, if the cytokine is halved in 5 to 10 minutes, half of 4600 mL, that is, 2300 mL of cytokine is removed in 5 to 10 minutes. The endogenous clearance of the cytokine is 230 to 460 [mL / min]. Therefore, since the clearance by CHF / CHDF is 10 [mL / min] at most as described above, most of the clearance of the cytokine is removed by the in vivo elimination mechanism. That is, it can be seen that treatment of SIRS and multiple organ failure is difficult only by the effect of removal of cytokines by simple CHF / CHDF.

  As described above, in Non-Patent Documents 3 to 6, trials using various membrane materials for CHF and CHDF are made for sepsis and septic multiple organ failure.

  However, in Non-Patent Document 3, only IL-6, IL-8, and IL-10 are measured as cytokines. The time since the patient was infected, the timing of treatment, and the cytokines at the time of treatment. No mention is made of blood conditions such as the concentration of chemokines.

  Non-Patent Document 4 shows the result of clearance of IL-6 in the blood, but it is about 20 [mL / min] at most, and as described above, only the removal of IL-6 by the PMMA membrane, There is no good explanation for the effectiveness of treatment. Furthermore, for high-value cases in which the concentration of IL-6 in the blood is 100 [pg / mL] or higher, CHDF by PMMA film, CTA (cellulose triacetate) film, PEPA film, EVAL film, PAN film, and PS film The result that the concentration of IL-6 is lowered by performing for 3 days is disclosed. However, the exact treatment timing and the extent of their therapeutic effect, such as the blood concentration of cytokines and chemokines other than IL-6 in cases where CHDF was actually performed by these membranes, etc. There was no mention of whether it was.

  Non-Patent Document 5 discloses an implementation example using a PMMA membrane or an EVAL membrane, and includes an example in which EVAL-CHD was implemented in a case of septic shock that falls into the category of SIRS. However, the timing of the operation and the level of blood levels of cytokines and chemokines in the blood at the time of the operation are not described, and the outcome of the case is fatal.

  Non-Patent Document 6 discloses data on blood concentrations of TNF, IL-6, IL-8, C3a, lipid peroxide, type II PLA2, granulocyte elastase and their clearance. However, no mention is made of cytokines or chemokines other than the primary disease of the subject patient and the above-mentioned humal mediator. In addition, there is no mention of the therapeutic effect of CHDF on the PMMA film. Non-Patent Document 6 also discloses data showing the relationship between the blood concentration and clearance regarding the removal of TNF, IL-6, and IL-8 when CHDF is performed by a PAN membrane. However, this also does not mention cytokines / chemokines other than TNF, IL-6, and IL-8, and does not mention any therapeutic effect of the PAN membrane by CHDF. In addition, Non-Patent Document 6 also discloses data showing the relationship between the blood concentration and clearance for the removal of TNF, IL-6, and IL-8 at the time of enforcement by CHD of the EVAL membrane. Cytokines and chemokines other than TNF, IL-6, and IL-8 are not mentioned, and the therapeutic effect of CHD on the EVAL membrane is not mentioned at all.

  On the other hand, regarding the changes in blood concentrations of TNF, IL-6, IL-8, C3a, lipid peroxide, type II PLA2, and granulocyte elastase when CHDF by PMMA membrane was performed for MOF patients for 3 days, It is examined in the high value group and the low value group. As a result, for TNF, IL-6, and the like, in the high value group, it was reported that the data after 3 days from the implementation were significantly lower when compared with before CHFD and 3 days after the implementation. The group says there was little change. Further, as other membrane materials, the above-described cytokine data before and 3 days after the EVAL membrane group and the PAN membrane have been studied. In the PMMA film, TNF, IL-6, and IL-8 were decreased, but the data that the EVAL film and the PAN film did not change are described. However, in any case, the therapeutic effect using these membranes is not described, and further, TNF, IL-6, IL-8, C3a, lipid peroxide, type II PLA2, granulocyte elastase Other cytokines and chemokines have not been examined, and the exact timing of CHDF administration is not described at all.

  Further, as described above, Non-Patent Documents 7 to 9 discuss cytokines and chemokines in blood in septic patients and SIRS model rats.

  However, in Non-Patent Document 7, although the above-described cytokines are measured before the operation, blood such as CHF / CHDF is once taken out of the body and brought into contact with the blood treatment device. There is no blood purification method.

  Also, in Non-Patent Document 8, what kind of treatment has been done on the patient, and blood purification methods such as CHF / CHDF etc., where blood is once taken out of the body and brought into contact with the blood treatment device. It is not described whether or not.

  Also, Non-Patent Document 9 does not describe cytokines other than IL-6 and TNF-α.

  Thus, when blood purification therapy for treating SIRS is performed, there has been no knowledge regarding the types and concentrations of effective cytokines and chemokines in blood for estimating the treatment timing. Moreover, until now, there has been no base material or device optimal for the blood purification method.

  In view of such a current situation, the present invention provides a substrate and a device for suppressing organ inflammation that are effective in suppressing organ inflammation caused by a disease that develops or deteriorates through cytokines and chemokines typified by SIRS. For the purpose. Another object of the present invention is to identify cytokines and chemokines for determining the treatment timing of organ inflammation caused by the above-mentioned diseases, and to clarify blood concentrations suitable for treatment.

  As a result of intensive studies to solve the above problems, the present inventors measured cytokines and chemokines in blood of rats (systemic inflammatory response syndrome model rats) that showed SIRS status after LPS administration, When a specific chemokine is subjected to blood purification by extracorporeal blood circulation to a rat with a specific concentration or higher using a specific substrate, it is used in conventional dialysis membranes and continuous slow blood filters. As a result, the present inventors have found that the survival rate is increased as compared with the case where the blood purification method is performed using the polysulfone membrane.

  As described above, it is known that the ethylene vinyl alcohol copolymer has blood compatibility. However, the substrate for organ inflammation suppression according to the present invention can be used in the blood extracorporeal circuit without directly contacting the organ. It is quite surprising that the effect of suppressing the inflammation of the organ can be obtained by contacting with.

  The present invention comprises a water-insoluble carrier and an ethylene vinyl alcohol copolymer coated on the surface of the water-insoluble carrier, and is arranged (connected to the blood extracorporeal circuit so as to come into contact with the blood to be treated. ) Provide a substrate for organ inflammation suppression. The base material for organ inflammation suppression may be formed of an ethylene vinyl alcohol copolymer.

  By performing a blood purification method by extracorporeal blood circulation using such a base material, organ inflammation caused by a disease that develops or worsens through cytokines and chemokines typified by SIRS can be suppressed. That is, the above-mentioned base material is suitable for organ inflammation suppression. Further, the ethylene vinyl alcohol copolymer is suitable as an organ inflammation inhibitor or an organ inflammation inhibitor for use outside the body in a blood purification method by extracorporeal blood circulation.

  The water-insoluble carrier may be a hollow fiber, a particle, or a nonwoven fabric. Or when said base material itself is formed from the ethylene vinyl alcohol copolymer, the shape of a hollow fiber, particle | grains, or a nonwoven fabric may be sufficient as an ethylene vinyl alcohol copolymer. By using such a substrate for organ inflammation suppression, a blood purification method can be performed efficiently.

  The treatment target preferably has a GRO concentration in blood of 3500 [pg / mL] or higher or a MIP-1α concentration of 5100 [pg / mL] or higher. Further, the concentration of GRO in blood is more preferably 100,000 [pg / mL] or less, and the concentration of MIP-1α is more preferably 100,000 [pg / mL] or less. That is, the above-mentioned base material is for a treatment object whose concentration of GRO in blood is in the above range.

  The blood purification method using the above-described base material has a high therapeutic effect for a treatment target in which the concentrations of GRO and MIP-1α are in the above range.

  The present invention also provides a device comprising a container having an inlet port through which blood flows in and an outlet port through which blood flows out, and the above-mentioned substrate filled in the container.

  Such a device can be suitably used for a blood purification method that suppresses organ inflammation caused by a disease that develops or worsens through the intervention of cytokines and chemokines typified by SIRS.

  The present invention also includes a substrate comprising a water-insoluble carrier and an ethylene vinyl alcohol copolymer coated on the surface of the water-insoluble carrier, and disposed in a blood extracorporeal circuit so as to come into contact with blood to be treated. A method for treating organ inflammation comprising the step of contacting with blood to be treated is provided.

  The present invention also includes a step of contacting a substrate made of ethylene vinyl alcohol copolymer, which is disposed in contact with blood to be treated in an extracorporeal blood circuit, with the blood to be treated. A method of treatment is provided.

  The present invention also provides a substrate comprising a water-insoluble carrier and an ethylene vinyl alcohol copolymer coated on the surface of the water-insoluble carrier, and disposed in a blood extracorporeal circuit so as to come into contact with blood to be treated. Provided for use as an organ inflammation inhibitor.

  The present invention also provides the use of a substrate made of an ethylene vinyl alcohol copolymer and disposed in a blood extracorporeal circuit so as to come into contact with blood to be treated as an organ inflammation inhibitor.

  The present invention also includes a substrate comprising a water-insoluble carrier and an ethylene vinyl alcohol copolymer coated on the surface of the water-insoluble carrier, and disposed in a blood extracorporeal circuit so as to come into contact with blood to be treated. An organ inflammation inhibitor is provided.

  The present invention also provides an organ inflammation inhibitor comprising a base material which is made of an ethylene vinyl alcohol copolymer and is disposed in a blood extracorporeal circuit so as to come into contact with blood to be treated.

  INDUSTRIAL APPLICABILITY According to the present invention, it is effective in treating diseases that develop or worsen through cytokines and chemokines typified by SIRS, particularly septic shock, acute organ damage and organ failure, and organ inflammation caused by ischemia reperfusion. Bases and devices for organ inflammation suppression are provided.

It is a graph which shows the blood level of cytokine chemokine. 4 is a photograph showing the results of lung tissue observation of Example 2. It is a graph which shows the result of the tissue observation evaluation of the lungs of Example 3, Comparative Example 2, and Comparative Example 4. It is a graph which shows the measurement result of the density | concentration of MDA in the lung tissue of Example 3 and Comparative Example 2. 6 is a photograph showing the results of lung tissue observation in Comparative Example 1. 6 is a photograph showing the results of lung tissue observation in Comparative Example 3. 10 is a photograph showing the result of lung tissue observation in Comparative Example 5. 1 is a cross-sectional view illustrating one embodiment of a device. FIG. 6 is a cross-sectional view illustrating another embodiment of a device.

  The present invention is for organ inflammation suppression comprising a water-insoluble carrier and an ethylene vinyl alcohol copolymer coated on the surface of the water-insoluble carrier, and disposed in a blood extracorporeal circuit so as to come into contact with blood to be treated. A substrate is provided. The present invention also provides a base material for organ inflammation suppression which is made of an ethylene vinyl alcohol copolymer and is disposed in a blood extracorporeal circuit so as to come into contact with blood to be treated.

(Ethylene vinyl alcohol copolymer)
An ethylene vinyl alcohol copolymer is a copolymer of ethylene and vinyl alcohol, but generally produced by saponifying an ethylene-vinyl acetate copolymer obtained by copolymerizing a vinyl acetate monomer and ethylene. Is done. As a typical polymer resin, EVAL ^TM resin manufactured by Kuraray Co., Ltd. may be mentioned.

  The ethylene vinyl alcohol copolymer may be any type of copolymer such as random, block, or graft. Moreover, it is preferable that the ethylene content of an ethylene vinyl alcohol copolymer is 20-70 mol% or less. When the ethylene content having a hydrophobic property is less than 20 mol%, the adhesiveness to the water-insoluble carrier tends to be low when the water-insoluble carrier is coated (coating). The coating layer may peel off. Further, when the ethylene content of the ethylene vinyl alcohol copolymer exceeds 70 mol%, the hydrophilicity of the coating layer of the ethylene vinyl alcohol copolymer tends to be lost, and unfavorable properties such as increased non-selective adsorption are exhibited. May show. From the balance between adhesiveness and hydrophilicity, the ethylene content of the ethylene vinyl alcohol copolymer is more preferably 25 to 50 mol%, and still more preferably 29 to 44 mol%. As a monomer component which forms an ethylene vinyl alcohol copolymer, you may include components other than ethylene and vinyl alcohol within the range which does not inhibit the effect of this invention.

  The weight average molecular weight (Mw) of the ethylene vinyl alcohol copolymer is preferably 10,000 to 3,000,000. When the weight average molecular weight of the ethylene vinyl alcohol copolymer is less than 10,000, the molecular weight of the polymer may decrease when the sterilization treatment, particularly the radiation sterilization treatment is performed, and the elution amount with respect to water may increase. Moreover, when the weight average molecular weight of an ethylene vinyl alcohol copolymer exceeds 3 million, the solubility to the solvent at the time of coating to a water-insoluble support | carrier may fall. Further, when the ethylene vinyl alcohol copolymer is polymerized, it may not be produced stably. The weight average molecular weight of the ethylene vinyl alcohol copolymer is more preferably 20,000 to 2,000,000. The weight average molecular weight of the ethylene vinyl alcohol copolymer is determined by various known methods. In this specification, the measurement is performed by gel permeation chromatography (hereinafter referred to as “GPC”) using polyethylene oxide as a standard. Is adopted.

  Further, the base material for organ inflammation suppression may be any base material as long as at least a part, preferably all, of the surface thereof is an ethylene vinyl alcohol copolymer. For example, the surface of the water-insoluble carrier may be coated with an ethylene vinyl alcohol copolymer, or the substrate itself may be formed from an ethylene vinyl alcohol copolymer. The ethylene content and the weight average molecular weight of the ethylene vinyl alcohol copolymer when the substrate itself is formed from the ethylene vinyl alcohol copolymer are the same as those described above when the surface of the water-insoluble carrier is coated. Same as coalescence.

  When the organ inflammation suppression base material itself is formed from an ethylene vinyl alcohol copolymer, the base material is preferably molded into a hollow fiber, non-woven fabric, particle, or the like shape. More preferably.

  As a method of coating the water-insoluble carrier with the ethylene vinyl alcohol copolymer, a method that can uniformly coat the water-insoluble carrier without significantly blocking the pores of the water-insoluble carrier and without significantly exposing the surface of the water-insoluble carrier is particularly preferable. There are no restrictions and various methods can be used. For example, a method in which a solution in which an ethylene vinyl alcohol copolymer is dissolved is impregnated with a water-insoluble carrier, a method in which a solution in which an ethylene vinyl alcohol copolymer is dissolved is sprayed onto a water-insoluble carrier, and a monomer that constitutes the ethylene vinyl alcohol copolymer. Examples thereof include a graft polymerization method on the surface of a water-insoluble carrier. Among these, the method of impregnating a solution in which an ethylene vinyl alcohol copolymer is dissolved with a water-insoluble carrier is more preferable because the coating layer can be made uniform and the cost is low. Specifically, a water-insoluble carrier is immersed in a water-miscible organic solvent alone or a solution of an ethylene vinyl alcohol copolymer dissolved in a mixed solvent of a water-miscible organic solvent and water. After removing the excess solution, it may be dried.

The organic solvent in which the ethylene vinyl alcohol copolymer is dissolved is a water-miscible organic solvent, the solubility in water is 20% by mass or more at a temperature below the boiling point of the solvent, and the Hildebrand solubility parameter is 9.5 [ An organic solvent of (cal · cm ⁻³ ) ^1/2 ] or more is preferable. Here, the solubility parameter of Hildebrand is defined as “J. H. Hildebrand, R.A. L. The δ value described in Scott “The Solubility of Nonelectrolytes, 3rd Ed.” (Dover Pub., New York) is used, and can be calculated by the equation (1).
σ = (E / V) ^1/2 (1)
Here, E is the cohesive energy [cal mol ⁻¹ ], and V is the molar volume [cm ³ mol ⁻¹ ].

  Examples of preferable organic solvents include alcohols such as methanol, ethanol, n-propanol, isopropanol, sec-butanol, t-butanol and cyclohexanol, polyhydric alcohols such as ethylene glycol, propylene glycol and glycerin, tetrahydrofuran and dioxane. Dimethylformamide, dimethylsulfoxide, dimethylacetamide, formamide, ethylene chlorohydrin and the like. Among these, n-propanol, ethanol, and dimethyl sulfoxide are particularly preferable because the ethylene vinyl alcohol copolymer has good solubility and low toxicity.

  These organic solvents can be used alone, but can also be used in a mixed solvent system, and a mixed solvent system with water is particularly preferably used. The ethylene vinyl alcohol copolymer is composed of a non-polar and hydrophobic ethylene moiety and a polar and hydrophilic vinyl alcohol moiety. When dissolved in a strongly polar solvent system, for example, when coated on a non-polar and hydrophobic filter carrier, the non-polar ethylene moiety is localized on the filter carrier side and the polar vinyl alcohol moiety is likely to be localized on the surface side. it is conceivable that. This phenomenon is a preferable phenomenon because the adhesion between the coating layer and the filter carrier is improved and the hydrophilicity of the coating layer surface is improved. It is preferable to add water to the above organic solvent to form a mixed solvent system because the polarity of the solvent is further increased and the above phenomenon is promoted. The ratio of water to be added is preferably large as long as the solubility of the ethylene vinyl alcohol copolymer is not inhibited. The ratio varies depending on the ethylene content of the ethylene vinyl alcohol copolymer, the temperature of the solution, etc. 60 mass% is mentioned as a preferable range.

  Although the density | concentration of the ethylene vinyl alcohol copolymer solution used for coating can select arbitrary density | concentrations suitable for coating, For example, the density | concentration of about 0.005-10 mass% is suitable. When the solution concentration is less than 0.005% by mass, the polymer coated on the surface may be reduced. On the other hand, when it exceeds 10 mass%, a solution viscosity may become high and a handleability may fall. From the above viewpoint, the concentration of the ethylene vinyl alcohol copolymer solution is more preferably 0.02% by mass or more and less than 5% by mass.

  The temperature of the ethylene vinyl alcohol copolymer solution used for coating is not particularly limited, but generally a higher temperature is preferable because the solubility of the ethylene vinyl alcohol copolymer is better and the viscosity of the solution is reduced. To 100 ° C. is preferred.

(Carrier)
A water-insoluble carrier means a carrier insoluble in water. By being insoluble in water, it is possible to prevent elution into blood when the substrate comes into contact with blood.

  As the water-insoluble carrier, any shape such as a hollow fiber shape, a particle shape, a nonwoven fabric shape, a fiber shape, a sheet shape, and a sponge shape can be used, and a hollow fiber shape is preferable.

  The hollow fiber is a hollow fiber that has pores having a size according to the purpose on a straw-like fiber wall surface, and can exchange substances inside and outside the hollow fiber. The material of the hollow fiber membrane is not particularly limited, and examples thereof include polysulfone, polysulfone and polyvinylpyrrolidone, ethylene vinyl alcohol, polyacrylonitrile, polyethylene, regenerated cellulose, cellulose diacetate, cellulose triacetate, polyethersulfone, Examples include polymethyl methacrylate.

  When a hollow fiber-like carrier is used as the water-insoluble carrier, the hollow fiber preferably has an inner diameter of 100 to 300 μm from the viewpoint of flowing blood inside the hollow fiber and mass exchange efficiency. The performance of the hollow fiber is not particularly limited. On October 1, 2011, the hollow fibers of type I to V described in the “Dializer Function Classification Implementation Manual” published by the Japan Medical Equipment Manufacturers Association were used. It can be used suitably.

  When a particulate carrier is used as the water-insoluble carrier, a particulate carrier having an average particle diameter of 25 to 2500 μm can be used. The particles may be spherical. Here, the particle diameter means the length of the longest straight line among straight lines connecting any two points on the outline of the carrier when observed with a microscope. The average particle diameter means an arithmetic average value of the particle diameters of 100 particulate carriers selected at random.

  The particulate carrier preferably has an average particle size of 50 to 1500 μm in view of its specific surface area and fluidity of body fluid, and more preferably 100 to 1000 μm in consideration of allowing whole blood to pass through.

  As the particulate carrier, organic or inorganic porous materials such as cellulose gel, dextran gel, agarose gel, polyacrylamide gel, porous glass, vinyl polymer gel can be used, and they are used for ordinary affinity chromatography. Any material for the carrier can be used.

Specific particulate carriers include Asahi Kasei Microcarrier (manufactured by Asahi Kasei Corporation), CM-Cellulofine (registered trademark) CH (trade name, exclusion limit protein molecular weight: about 3 × 10 ⁶ , Seikagaku Corporation ) Sales) and the like, a completely porous activated gel described in JP-B-1-44725, CM-Toyopearl (registered trademark) 650C (trade name, exclusion limit protein molecular weight: 5 × 10 ⁶ , Tosoh ( Etc.), etc., such as polyvinyl alcohol carrier, CM-Trisacryl M (CM-Trisacryl M, trade name, exclusion limit protein molecular weight: 1 × 10 ⁷ , manufactured by Pharmacia-LKB, Sweden), etc. Polyacrylamide carrier, Sepharose CL-4B (Sepharose CL-4B, trade name, exclusion limit protein) Molecular weight: 2 × 10 ⁷ , organic carrier such as agarose carrier such as Pharmacia-LKB (Pharmacia-LKB), Sweden, and CPG-10-1000 (trade name, exclusion limit protein molecular weight: 1 × 10 ⁸ And an inorganic carrier such as porous glass such as an average pore diameter of 100 nm, manufactured by Electro-nucleonics, Inc., and a carrier made of an ethylene vinyl alcohol copolymer.

  The composition of the particulate carrier may be a known polymer that can take a porous structure, such as a polyamide-based, polyester-based, polyurethane-based, or vinyl compound polymer.

  Non-woven fabric refers to fabric made by bonding or intertwining fibers by thermal, mechanical or chemical action. There is no restriction | limiting in particular in the thickness and fiber diameter of the nonwoven fabric, If it is a form which can pass the blood, it can be used. The composition of the nonwoven carrier is not particularly limited, and examples thereof include polyester, polypropylene, regenerated cellulose, and ethylene vinyl alcohol copolymer.

(Measurement method of surface area of carrier)
When the carrier is a hollow fiber membrane, the surface area of the carrier can be calculated and calculated by hollow fiber membrane inner diameter × 3.14 × hollow fiber length × number of hollow fibers. The hollow fiber membrane has an inner diameter of a certain number of hollow fibers embedded in a paraffin block, sliced to a desired thickness using a microtome, and then the paraffin flakes are observed with an optical microscope (100 times) to obtain a hollow fiber. It can be determined by measuring the inner diameter of the membrane.

When the carrier is a non-woven fabric, the sample tube is filled with the carrier weighed in the range of 0.50 g to 0.55 g using “Accusorb 2100E” manufactured by Shimadzu Corporation, Japan or an equivalent specification machine. The surface area can be determined by performing degassing treatment with an accusorb body at a reduced pressure of 1 × 10 ⁻⁴ mmHg (at room temperature) for 20 hours, and then measuring at adsorption gas: nitrogen gas, adsorption temperature: liquid nitrogen temperature.

Even when the carrier is a particle, the carrier weighed in the range of 0.50 g to 0.55 g using “Accusorb 2100E” manufactured by Shimadzu Corporation, Japan, or an equivalent specification machine, as in the case of the nonwoven fabric. After filling the tube and degassing with accusorb body at a reduced pressure of 1 × 10 ⁻⁴ mmHg (at room temperature) for 20 hours, the surface area was measured by adsorption gas: nitrogen gas, adsorption temperature: liquid nitrogen temperature. Can be requested.

  Said base material is arrange | positioned in a blood extracorporeal circuit so that it may contact with the blood of treatment object. That is, said base material can also be called the base material for blood extracorporeal circuit arrangement. Examples of the extracorporeal blood circulation circuit include a dialysis device, a continuous slow blood filtration device, a plasma separation (exchange) device, a leukocyte removal device, a whole blood and plasma separation adsorption blood purification device, and a double filtration plasma exchange device.

(device)
The present invention also provides a device comprising a container having an inlet port through which blood flows in and an outlet port through which blood flows out, and the above-mentioned substrate filled in the container. FIG. 8 is a cross-sectional view illustrating one embodiment of the device. In FIG. 8, a device 100 is a module connected to an extracorporeal blood circulation circuit. A cap 150 having an inlet port 140 is screw-fitted into an opening at one end of the cylinder 110 via a packing 130 with a filter 120 on the inside, and a filter 120 ′ is stretched on the inside at the other end opening of the cylinder 110. A cap 170 having an outlet port 160 is screwed through 130 ′ to form a container, and the above-mentioned base material is filled and held in the gap between the filters 120 and 120 ′ to form a base layer 180 for suppressing organ inflammation. doing.

  The organ inflammation suppressing base material layer 180 may be filled with the above base material alone, or may be mixed or laminated with other base materials. As other base materials, for example, adsorbents of other malignant substances (antigens) such as DNA, activated carbon having a wide range of adsorption ability, and the like can be used. As a result, a wide range of clinical effects due to the synergistic effect of the base material can be expected. The volume of the organ inflammation suppressing base material layer 180 is suitably about 50 to 400 mL when used for blood purification using extracorporeal blood circulation.

  FIG. 9 is a cross-sectional view showing another embodiment of the device. In FIG. 9, a device 200 is a module connected to a blood extracorporeal circuit. The base material (hollow fiber) 220 is held by the potting agent 230 at both ends of the cylinder 210. Both end surfaces of the hollow fiber are open. A cap 250 having an inlet port 240 and a cap 250 ′ having an outlet port 260 are fitted into the cylinder 210 to fill and hold the substrate (hollow fiber) 220. Further, a dialysate inlet 270 and a dialysate outlet 280 are provided on the side surface of the cylinder 210.

  When used in a blood purification method by extracorporeal blood circulation, blood introduced from the body to be treated is introduced from the inlet port 240, passed through the inner space of the base material (hollow fiber) 220, through the outlet port 260, By returning to the body to be treated, a blood purification method by extracorporeal blood circulation can be performed. At that time, dialysis is performed during extracorporeal blood circulation by introducing the dialysate from the dialysate inlet 270, leading out the dialysate from the dialysate outlet 280, and circulating the dialysate in the device 200. Can do. Further, the dialysate may not be circulated, and the dialysate inlet 270 and the dialysate outlet 280 may be opened. In this case, blood is filtered. Further, by blocking the dialysate inlet 270 and the dialysate outlet 280 with a stopper or the like, it is possible to suppress blood filtration and perform a blood purification method by extracorporeal blood circulation.

  When the base material is a hollow fiber, the blood passing speed in the blood purification method by extracorporeal blood circulation can be adjusted by changing the inner diameter of the hollow fiber. In this case, the larger the inner diameter, the higher the passing speed can be obtained. Moreover, when a base material is a particle | grain or a nonwoven fabric, a high passage speed can be obtained by enlarging a particle diameter and a fiber diameter, and making the filling degree of a base material low. As the blood passage speed is higher, a larger amount of body fluid treatment can be performed. The extracorporeal blood circulation may be performed continuously or intermittently according to clinical necessity or the like.

  The above device can be effectively used for suppressing or treating organ inflammation in diseases mediated by cytokines and chemokines. Specific diseases include sepsis, septic shock, toxic shock syndrome, ischemia reperfusion disorder, adult respiratory distress syndrome (ARDS), Paget's disease, osteoporosis, multiple myeloma, acute and chronic myelogenous leukemia, pancreas β-cell destruction, inflammatory bowel disease, psoriasis, Crohn's disease, ulcerative colitis, anaphylaxis, contact dermatitis, asthma, muscle degeneration, cachexia, Reiter syndrome, type I and type II diabetes, bone resorption, Examples include graft-versus-host reactions, atherosclerosis, brain trauma, multiple sclerosis, cerebral malaria, fever, and myalgia due to infection.

  The target organs that suppress inflammation by the blood purification method using blood external circulation using the above-mentioned base materials include lung, kidney, liver, large intestine, small intestine, duodenum, stomach, spleen, pancreas, brain, heart, blood vessels, etc. Can be mentioned. It is more preferable that the target organs are lungs, kidneys and livers because leukocytes are more likely to accumulate due to leukocyte migration by chemokines.

(Cytokine chemokine)
As described above, in the blood purification method based on the extracorporeal circulation of blood to be treated, the inflammation of the organ to be treated can be suppressed by bringing the base material into contact with the blood to be treated. Here, the GRO concentration in blood is 3500 [pg / mL] or higher, the concentration of MIP-1α is 5100 [pg / mL] or higher, the concentration of RANTES is 500 [pg / mL] or higher, or MIP If the concentration of −3α is 150 [pg / mL] or higher, the effect of suppressing organ inflammation is high. In particular, the concentration of GRO in blood is 3500 [pg / mL] or higher, or the concentration of MIP-1α Is more than 5100 [pg / mL], the effect of suppressing organ inflammation is higher.

  GRO is one of chemokines that are a group of cytokines. Chemokine is a basic protein that expresses its action through a G protein-coupled receptor, and is known to cause migration of leukocytes and the like and participate in the formation of inflammation. Chemokines are classified into CC chemokines, CXC chemokines, C chemokines and CX3C chemokines because of their structural differences. GRO is classified as a CXC chemokine. The concentration of GRO is the total concentration of the various GRO molecules. Rat GRO / KC, which is measured in Examples described later, is also a GRO molecule classified as a CXC chemokine, and the concentration of rat GRO is indicated by this concentration. There are three types of GRO molecules, GRO1, GRO2 and GRO3 in humans, and the total value of these is the concentration of GRO. Both human GRO and rat GRO can determine the inflammatory properties of organs, and the inflammatory level as a living body can be said to be almost the same. Therefore, the concentration of GRO to be treated can be determined at the same level.

  MIP-1α, RANTES, and MIP-3α are also chemokines that are a group of cytokines, and are classified as CC chemokines. Until now, about these chemokines, the expression state, function, etc. in SIRS were not fully known. The present inventors have found for the first time that GRO, MIP-1α, RANTES and MIP-3α are useful as markers for determining SIRS treatment timing, and that GRO and MIP-1α are particularly important. Similarly to GRO, both human MIP-1α and rat MIP-1α can determine the inflammatory properties of organs, and the level of inflammatory activity in the living body can be said to be almost the same. The concentration can be determined at the same level.

  When a mild to moderate inflammatory reaction is induced in vivo, the concentration of cytokines and chemokines in blood varies depending on the type of cytokines and chemokines, but generally, there are After exceeding a certain threshold value, it gradually increases with time, reaches a peak, then gradually decreases, and further decreases after a certain threshold value.

  In this case, the therapeutic effect is high when treated within a period (period) between the threshold value when the cytokine / chemokine concentration increases and the threshold value when it decreases. That is, if it is GRO, the threshold is 3500 [pg / mL] or more, and if it is MIP-1α, the threshold is 5100 [pg / mL] or more, which is uniquely effective for treatment. The period (period) is determined.

  On the other hand, when a strong inflammatory reaction occurs at a moderate or higher level, the cytokine and chemokines in the blood have different rising times depending on the types of the cytokines and chemokines, but the rising kinetics of the concentration is a certain threshold value. It will continue to rise over time until it reaches the death of the individual, or it will eventually lead to the death of the individual by continuing to remain at the peak level at a certain high concentration level. . Therefore, if inflammation occurs and the cytokines and chemokines in the blood rise and exceed a certain threshold, it is preferable to treat at the earliest possible stage, and furthermore, the cytokine and chemokines that rise above the threshold in the blood It is preferred to treat while the level is as low as possible.

  That is, in the case of GRO, the concentration range is preferably 3500 to 100,000 [pg / mL], and more preferably 3500 to 50000 [pg / mL]. In the case of MIP-1α, the concentration range is preferably in the range of 5100 to 100,000 [pg / mL], and more preferably in the range of 5100 to 50000 [pg / mL]. In the case of RANTES, the concentration range is preferably 500 to 50000 [pg / mL], more preferably 500 to 30000 [pg / mL]. In the case of MIP-3α, the concentration range is preferably 200 to 50000 [pg / mL], more preferably 200 to 30000 [pg / mL].

  Note that the series of blood chemokines described above is generally described in the examples described later in order to simultaneously measure enzyme immunoassay and various types of chemokines based on the principle of antigen-antibody reaction. And a method using multiplex bead technology represented by Bio-Plex (trademark) suspension array system (BioRad, USA).

  As one index for evaluating the inflammation control effect of the organ by the blood purification method (blood purification therapy), there is an evaluation of the inflammation state of the organ of the test animal. In particular, pulmonary tissue is rich in neutrophils even under normal conditions, and these neutrophils are activated by cytokines released by triggering biological invasion such as infection, trauma, and burns. It is said that the effect of the reaction is easily reflected. The lung tissue after the blood purification method is extracted, fixed in formalin, embedded in paraffin, sliced, and then stained with hematoxylin and eosin to evaluate the inflammatory state of the lung tissue.

  As an index for evaluating the inflammation suppression effect of the organ by the blood purification method, there is quantification of MDA (malondialdehyde) in the organ of the test animal. One factor that enhances the inflammatory response is active oxygen released from granulocytes by TNF-α, a cytokine released in the early stages of inflammation. This active oxygen activates Elastase, which is bound to α1-antitrypsin or α2-microglobulin and whose activity expression is suppressed. It is known that the activated elastase damages surrounding tissues and accelerates the inflammatory response. Since the measurement of the concentration of MDA in the organ is an index indicating the amount of active oxygen produced in the above process, it is an index indicating the degree of enhancement of the inflammatory reaction.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited at all by these Examples.

[Example 1]
Systemic inflammatory response syndrome model rats were prepared by administering lipopolysaccharide (LPS) 10 [mg / kg] to Wister male rats (body weight 250 to 300 g), which were used as test rats. After administration of LPS, rats were mixed with normal rats not administered LPS so that food and water could be consumed without restriction. Four types of chemokines (GRO / KC, MIP-1α, RANTES, MIP-3α) in blood collected from test rats (27 rats) and normal rats (3 mice) 6 hours after LPS administration were bio-plex system (Bio-Rad Bio-Plex Pro rat cytokine premix panel 23-Plex panel) was used. The results are shown in Table 1 and FIG. As a result, the blood levels of GRO / KC, MIP-1α, RANTES and MIP-3α in the test rats are higher than those in normal rats, in particular, GRO / KC and MIP-1α are extremely high, and GRO / KC and It has been clarified that MIP-1α is an effective marker for estimating the treatment timing of blood extracorporeal circulation. Further, from this result and the results of Examples 2 and 3 to be described later, when the substrate of the present invention is brought into contact with the blood to be treated in the blood purification method by extracorporeal blood circulation, the concentration of GRO in the blood to be treated Is 3500 [pg / mL] or higher or the concentration of MIP-1α is 5100 [pg / mL] or higher, it is shown that the effect of suppressing organ inflammation is high.

[Example 2]
(Production of base materials and devices)
A hollow fiber membrane made of an ethylene-vinyl alcohol copolymer (EVAL) (hollow fiber inner diameter 175 μm, film thickness 25 μm, water ultrafiltration performance (UFR) 12 [mL / hr / 0.13 kPa / m ² ]) 50 cm ² Was encapsulated in a polycarbonate resin housing having a dialysate inlet and outlet, and both ends were fixed to the housing with urethane resin. After the urethane resin was cured, the device was obtained by cutting at the end face of the housing to open the hollow fiber, and then adhering lids having an inlet port and an outlet port for blood to both end faces of the housing.

(Implementation of blood purification by extracorporeal circulation)
Fifteen test rats were prepared in the same manner as in Example 1. A catheter was inserted into the external jugular vein and femoral artery of the test rat to secure the blood inlet / outlet of blood extracorporeal circulation and blood of the rat was collected. GRO / KC, MIP-1α Was measured. The results are shown in Table 2. A blood purification method by extracorporeal blood circulation was performed for 30 minutes at a blood flow rate of 1 [mL / min] using the above device. At this time, blood filtration replenisher BSG (Fuso Pharmaceutical Co., Ltd.) was supplied to the dialysate inlet at a flow rate of 8 [mL / hour]. The amount of water filtered from the blood was calculated from the amount of fluid replenished for 30 minutes. After extracorporeal circulation, the same amount of physiological saline was replenished in the test rat. After the extracorporeal circulation, the test rats were placed in an environment where they could eat and drink without limitation, and the survival test was performed 3 hours after the extracorporeal circulation (9 hours after LPS administration) and 18 hours (24 hours after LPS administration). . As a result of calculating the survival rate of 24 hours after LPS administration, it was 80% (the number of surviving rats was 12 / the number of test rats was 15). Test rats that survived 18 hours after blood extracorporeal circulation (24 hours after LPS administration) were killed, and lung tissue samples were collected. The collected lung tissue specimens were fixed in formalin, embedded in paraffin, and sectioned with a microtome. The prepared sections were stained with hematoxylin and eosin and observed. FIG. 2 is a photograph showing the results of lung tissue observation. There was no congestion, edema or bleeding in the lung tissue, and inflammation was very mild.

Example 3
(Evaluation of inflammatory condition after implementation of blood purification method)
Rats that survived 24 hours after execution of the blood purification method of Example 2 were sacrificed, and evaluation of a sample (HE-stained sample) in which an organ was stained with hematoxylin and eosin, and evaluation of the concentration of MDA (Malonaldehyde) in the organ The inflammatory status of the organs was evaluated.

(Evaluation of HE-stained sample)
After the rats that survived 24 hours after the blood purification method of Example 2 were sacrificed, the lungs were removed, fixed in formalin, embedded in paraffin, sliced into about 6 μm using a microtome, and thinned. Cut samples were stained with hematoxylin and eosin. Subsequently, the degree of inflammation of the lung tissue was evaluated together with the samples of Comparative Examples 2 and 4 described later as follows.

  In Comparative Example 2 described later, a blood purification method was performed on rats using a device comprising a base material that is a polysulfone hollow fiber membrane made of polysulfone and polyvinylpyrrolidone, and a HE-stained sample of lung tissue was prepared in the same manner as described above. did. In Comparative Example 4 to be described later, extracorporeal blood circulation was performed on rats using only a circuit, and a HE-stained sample of lung tissue was prepared in the same manner as described above.

  With respect to the HE-stained samples of the lung tissues of Example 3, Comparative Example 2, and Comparative Example 4, organ observation was performed for each of 8 visual fields at 100 to 400 times using an optical microscope. Then, each of the four items of congestion, edema, inflammatory cells, and major bleeding was ranked from a visual field with a low degree of inflammation, and the sum of the ranks of each item was used as the histology score of each HE-stained sample.

  The results are shown in FIG. A rat lung tissue (indicated as “EVAL” in Example 3 and FIG. 3) in which blood purification was performed with a device provided with a base material that is a hollow fiber membrane made of an ethylene vinyl alcohol copolymer (EVAL). Lung tissue of a rat subjected to blood purification using a device comprising a substrate, which is a polysulfone hollow fiber membrane composed of polysulfone and polyvinylpyrrolidone (Comparative Example 2, indicated as “PS” in FIG. 3), extracorporeal blood circulation The histology scores were improved for all the evaluation items as compared with the lung tissue of rats that were subjected to blood purification using only the circuit (Comparative Example 4, shown as “circuit only” in FIG. 3).

(Measurement of MDA concentration in organs)
The measurement of the concentration of MDA (Malonaldehyde) in lung tissue was performed by the following method. After the rats that survived 24 hours after the blood purification method of Example 2 were sacrificed, the lungs were removed, frozen in liquid nitrogen, pulverized and dissolved in a potassium chloride solution. Using this dissolved solution as a sample, the concentration of MDA was measured with an MDA measurement kit (TBARS method) (manufactured by Japan Aging Control Laboratory). At the same time, the total amount of protein in the sample was quantified using a BCA protein assay kit (Thermo), and the concentration of MDA in lung tissue was normalized by the value per unit protein amount.

  The results are shown in FIG. Rat lung tissue (shown as “EVAL” in FIG. 4) subjected to blood purification using a device comprising a base material that is a hollow fiber membrane made of ethylene vinyl alcohol copolymer (EVAL) is polysulfone described later. Compared with rat lung tissue (indicated as “PS” in FIG. 4) that was subjected to blood purification with a device comprising a base material that is a polysulfone hollow fiber membrane made of polypyrrole and polyvinylpyrrolidone, the inflammatory state was improved. It was.

[Comparative Example 1]
(Production of base materials and devices)
Polysulfone hollow fiber membrane made of polysulfone and polyvinylpyrrolidone (hollow fiber inner diameter 185 μm, film thickness 45 μm, water ultrafiltration performance (UFR) 250 [mL / hour / 0.13 kPa / m ² ]) as a substrate A device was fabricated in the same manner as in Example 2 except for the above.

(Implementation of blood purification by extracorporeal circulation)
12 test rats were prepared in the same manner as in Example 1. A catheter was inserted into the external jugular vein and femoral artery of the test rat to secure the blood inlet / outlet of the blood extracorporeal circulation and blood of the rat was collected, and GRO / KC, MIP− in blood was collected in the same manner as in Example 1. 1α was measured. The results are shown in Table 3. A blood purification method by extracorporeal circulation was carried out for 30 minutes at a blood flow rate of 1 [mL / min] using a device in which the substrate was not coated with a vitamin E derivative. At this time, blood filtration replenisher BSG (Fuso Pharmaceutical Co., Ltd.) was supplied to the dialysate inlet at a flow rate of 8 [mL / hour]. The amount of water filtered from the blood was calculated from the amount of fluid replenished for 30 minutes. After extracorporeal circulation, the same amount of physiological saline was replenished in the test rat. After the extracorporeal circulation, the test rats were placed in an environment where they could eat and drink without limitation, and the survival test was performed 3 hours after the extracorporeal circulation (9 hours after LPS administration) and 18 hours (24 hours after LPS administration). . The survival rate at 24 hours after LPS administration was calculated to be 50% (6 surviving rats / 12 test rats). Test rats that survived 18 hours after blood extracorporeal circulation (24 hours after LPS administration) were killed, and lung tissue samples were collected. The collected lung tissue specimens were fixed in formalin, embedded in paraffin, and sectioned with a microtome. The prepared sections were stained with hematoxylin and eosin and observed. FIG. 5 is a photograph showing the results of lung tissue observation. There was no congestion or edema in the lung tissue, but the lung tissue was enlarged and a mildly inflamed area was observed.

[Comparative Example 2]
(Evaluation of inflammatory state of organs after blood purification)
Rats that survived 24 hours after the blood purification method of Comparative Example 1 were sacrificed, and the inflammation state of the organ was evaluated by evaluating the HE staining sample of the organ and evaluating the concentration of MDA in the organ.

(Evaluation of HE-stained sample)
In the same manner as in Example 3, eight fields of lung tissue stained with hematoxylin and eosin were observed, and a hollow fiber membrane composed of an ethylene vinyl alcohol copolymer (EVAL) for four items of congestion, edema, inflammatory cells, and major bleeding. A device comprising a base material that is a hollow fiber membrane made of EVAL, after ranking from a visual field with a low degree of inflammation, in combination with a lung tissue observation result with a base material and a lung tissue observation result with a circuit alone The total of the respective ranks of only the device and the circuit provided with the base material which is a polysulfone hollow fiber membrane composed of polysulfone and polyvinylpyrrolidone was calculated. The results are shown in FIG. The lung tissue of a rat (indicated as “PS” in FIG. 3) subjected to blood purification using a device having a base material, which is a polysulfone hollow fiber membrane composed of polysulfone and polyvinylpyrrolidone, is only a blood extracorporeal circuit to be described later. The histology score was improved in comparison with the lung tissue of the rat subjected to the blood purification method in FIG. 3 (indicated as “circuit only” in FIG. 3). However, all the evaluation items were compared with the lung tissue of the rat (shown as “EVAL” in FIG. 3) in which the blood purification method was performed with the above-described device provided with the base material that is a hollow fiber membrane made of EVAL. About histology score was getting worse.

(Measurement of MDA concentration in organs)
In the same manner as in Example 3, a lung tissue lysate was prepared, and the concentration of MDA was measured. The results are shown in FIG. The lung tissue (shown as “PS” in FIG. 4) of a rat subjected to blood purification using a device comprising a base material, which is a polysulfone hollow fiber membrane composed of polysulfone and polyvinylpyrrolidone, is the above-described ethylene vinyl alcohol co-polymer. Compared with rat lung tissue (indicated as “EVAL” in FIG. 4) that was subjected to blood purification with a device comprising a base material that is a hollow fiber membrane made of a polymer (EVAL), the concentration of MDA was higher. It was.

[Comparative Example 3]
(Execution of extracorporeal blood circulation with circuit only)
12 test rats were prepared in the same manner as in Example 1. A catheter was inserted into the external jugular vein and femoral artery of the test rat to secure the blood inlet / outlet of the blood extracorporeal circulation and blood of the rat was collected, and GRO / KC, MIP− in blood was collected in the same manner as in Example 1. 1α was measured. The results are shown in Table 4. Only a blood extracorporeal circuit was connected without connecting a device, and blood extracorporeal circulation was performed for 30 minutes at a blood flow rate of 1 [mL / min]. After the extracorporeal circulation, the test rats were placed in an environment where they could eat and drink without limitation, and the survival test was performed 3 hours after the extracorporeal circulation (9 hours after LPS administration) and 18 hours (24 hours after LPS administration). . The survival rate of 24 hours after LPS administration was calculated to be 33% (3 surviving rats / 12 test rats). Test rats that survived 18 hours after blood extracorporeal circulation (24 hours after LPS administration) were killed, and lung tissue samples were collected. The collected lung tissue specimens were fixed in formalin, embedded in paraffin, and sectioned with a microtome. The prepared sections were stained with hematoxylin and eosin and observed. FIG. 6 is a photograph showing the results of lung tissue observation. There was congestion in the lung tissue, severe tissue enlargement, and severe inflammation.

[Comparative Example 4]
(Evaluation of inflammatory condition after implementation of blood purification method)
Rats that survived 24 hours after the blood purification method of Comparative Example 3 were sacrificed, and the inflammation state of the organ was evaluated by evaluating the HE staining sample of the organ.

(Evaluation of HE-stained sample)
In the same manner as in Example 3, eight fields of lung tissue stained with hematoxylin and eosin were observed, and a hollow fiber membrane composed of an ethylene vinyl alcohol copolymer (EVAL) for four items of congestion, edema, inflammatory cells, and major bleeding. In addition to the lung tissue observation result on the base material, and the lung tissue observation result on the base material which is a polysulfone hollow fiber membrane made of polysulfone and polyvinylpyrrolidone, ranking was performed from a visual field with a low degree of inflammation, and then from EVAL The total of the respective ranks of the device including the substrate that is the hollow fiber membrane, the device including the substrate that is the polysulfone hollow fiber membrane composed of polysulfone and polyvinylpyrrolidone, and the circuit was calculated. The results are shown in FIG. The lung tissue (shown as “circuit only” in FIG. 3) of the rat subjected to the blood purification method using only the extracorporeal blood circuit is a hollow fiber membrane made of the above-described ethylene vinyl alcohol copolymer (EVAL). Blood purification using a device comprising a substrate, which is a polysulfone hollow fiber membrane composed of polysulfone and polyvinylpyrrolidone. Compared with the lung tissue of the rat subjected to the method (indicated as “PS” in FIG. 3), the histology scores for all the evaluation items were deteriorated.

[Comparative Example 5]
(Execution of extracorporeal blood circulation with circuit only)
Catheters were inserted into the external jugular vein and femoral artery of normal rats not administered LPS.
Only a blood extracorporeal circuit was connected without connecting a device, and blood extracorporeal circulation was performed for 30 minutes at a blood flow rate of 1 [mL / min]. After extracorporeal circulation, rats were placed in an environment where they could eat and drink without restriction, and were sacrificed 18 hours after extracorporeal circulation, and lung tissue samples were collected. The collected lung tissue specimens were fixed in formalin, embedded in paraffin, and sectioned with a microtome. The prepared sections were stained with hematoxylin and eosin and observed. FIG. 7 is a photograph showing the results of lung tissue observation. There was no congestion, edema, or bleeding in the lung tissue, and there was no inflammation such as enlargement of the lung tissue.

  The organ inflammation-suppressing substrate and device of the present invention are diseases that develop or worsen mediated by inflammatory cytokines / chemokines, particularly septic shock, acute organ damage, organ failure, and organ inflammation caused by ischemia reperfusion. Useful for treatment.

  100, 200 ... device, 110, 210 ... cylinder, 120, 120 '... filter, 130, 130' ... packing, 140, 240 ... inlet port, 150, 170, 250, 250 '... cap, 160, 260 ... outlet port , 180 ... base layer for organ inflammation suppression, 220 ... base material (hollow fiber), 230 ... potting agent, 270 ... dialysate inlet, 280 ... dialysate outlet.

Claims (9)

  An organ inflammation inhibitor comprising a water-insoluble carrier and an ethylene vinyl alcohol copolymer coated on at least a part of the surface of the water-insoluble carrier, and disposed in a blood extracorporeal circuit so as to come into contact with blood to be treated. Base material.   A base material for organ inflammation suppression, which is made of an ethylene vinyl alcohol copolymer and is disposed in a blood extracorporeal circuit so as to come into contact with blood to be treated.   The base material according to claim 1, wherein the water-insoluble carrier is a hollow fiber, a particle, or a nonwoven fabric.   The base material according to claim 3, wherein the water-insoluble carrier is a hollow fiber.   The base material according to claim 2, wherein the ethylene vinyl alcohol copolymer is in the form of a hollow fiber, a particle or a nonwoven fabric.   The base material according to claim 5, wherein the ethylene vinyl alcohol copolymer is in the form of a hollow fiber.   The group according to any one of claims 1 to 6, wherein the GRO concentration in the blood to be treated is 3500 [pg / mL] or more, or the MIP-1α concentration is 5100 [pg / mL] or more. Wood.   The concentration of GRO in the blood to be treated is 3500 to 100,000 [pg / mL] or the concentration of MIP-1α is 5100 to 100,000 [pg / mL]. Base material.   A device comprising a container having an inlet port through which blood flows in and an outlet port through which blood flows out, and a substrate according to any one of claims 1 to 8 filled in the container.