Preparation for preventing ischemia-reperfusion injury in surgical operations or transplant operations

Abstract

Disclosed is a preparation for effectively preventing ischemia-reperfusion injury in surgical operations or transplant operations. As an active ingredient in the preparation, a polymer micelle including a copolymer and having a nanosized average diameter as determined by dynamic light scattering (DLS) measurement in an aqueous solution is used, wherein the copolymer is characterized by being represented by formula (I) wherein, among main variable groups or moieties, L2 in L2-R1 represents -(CH2)a-NH-(CH2)a- or -(CH2)a-O-(CH2)a- wherein a's each independently represent an integer of 0 or 1 to 5, and R1 represents a cyclic nitroxide radical. In principle, the polymer micelle is parenterally administered to a patient or an organ donor prior to an organ exenteration in a surgery or transplant operation. In the case of a transplant operation, a removed organ is stored in a preservation solution containing the polymer micelle or is perfused with the preservation solution. Alternatively, in the case of a transplant operation, the polymer micelle is parenterally administered to a transplantation target patient. As a result, the ischemia-reperfusion-induced damage of an organ that is a target for the procedure of the operation is inhibited.

Description

Preparation for suppressing ischemia-reperfusion injury in surgery or transplantation

[0001] The present invention relates to a preparation for suppressing organ damage caused by blocking or ischemia of blood flow to an organ, and further by ischemia-reperfusion. More specifically, the present invention relates to a preparation that uses, as an active ingredient, a polymeric micelle formed from an amphiphilic block copolymer containing a hydrophobic polymer segment having a cyclic nitroxyl radical in its side chain and a hydrophilic poly(ethylene glycol) (PEG) segment.

[0002] <Techniques Related to Surgical Procedures, Mainly Liver Surgery> Hepatoduodenal ligament blood flow occlusion (Pringle method: see Mise Y, et al. Liver Cancer 2013;55-66 doi:10,1159 / 000346225, hereinafter sometimes referred to as Non-Patent Document 1) is a widely used method for reducing bleeding during liver resection. This technique involves temporarily occluding blood flow to the liver (approximately 15 minutes) while proceeding with liver parenchymal transection. Over the next five minutes, the transection is interrupted and blood flow to the liver is resumed, restoring oxygen and nutrients to the liver tissue. This blood flow occlusion-resumption process can cause significant organ damage known as "ischemia-reperfusion injury." Hepatoduodenal ligament blood flow occlusion for more than 15 minutes can potentially cause irreversible liver damage. Therefore, blood flow occlusion for longer than 15 minutes is not currently performed. Furthermore, repeated blockage of blood flow, even for less than 15 minutes, can cause irreversible damage to the liver and severe ischemia-reperfusion injury, leading to fatal postoperative liver failure.

[0003] Ischemia-reperfusion injury is caused by cell damage caused by so-called "bad" reactive oxygen species generated during ischemia. Previous reports have suggested that preoperative high-dose steroid administration or intraoperative prostaglandin E1 preparations can reduce liver damage (see Shigeru Maruhashi et al., J. Clinical Endocrinol. 1999;60:329-334, hereafter referred to as "Non-Patent Document 2"; see also Orci LA, et al., BJS 2013;100:600-609, hereafter referred to as "Non-Patent Document 3"). However, these methods have not proven effective, and no drugs have been clinically applied to date.

[0004] <Technologies for Improving the Success Rate of Organ Transplantation> In organ transplant surgery, donated organs (e.g., heart, lungs, kidneys, liver, pancreas, small intestine) are subjected to a period of ischemic stress before being implanted into the recipient's body and blood flow is restored. When blood flow resumes, tissue-damaging substances, primarily reactive oxygen species, are produced in large quantities by the cells that make up the transplanted organ, directly injuring the transplanted organ. Mitigating organ damage during the ischemic stress period and ischemia-reperfusion injury upon reperfusion of the organ after transplantation are major challenges affecting the success rate of transplants. Previous animal experiments have reported the effectiveness of antioxidants such as N-acetylcysteine ​​and edaravone to address these issues, but no clinical efficacy was observed (Orban JC, et al. Transplantation 2015;99:746-753, hereinafter sometimes referred to as Non-Patent Document 4). Therefore, it has been thought that ischemia-reperfusion injury in the field of organ transplantation cannot be resolved by antioxidants.

[0005] <Technology related to the elimination of reactive oxygen species> Recent research has revealed that reactive oxygen species cause tissue damage in various organs and are the cause of many diseases. In addition to "bad" reactive oxygen species that cause organ damage, there are also so-called "good" reactive oxygen species that are essential for redox reactions, including the mitochondrial electron transport chain in normal cells. In the past, there has been active development of antioxidants that suppress reactive oxygen species, but conventional antioxidants are also taken up by normal cells, disrupting the intracellular redox balance and suppressing the function of normal cells, resulting in severe side effects that have prevented them from being used in clinical applications. Under these circumstances, some of the present inventors have developed self-assembled antioxidant nanoparticles or redox nanoparticles, particularly nanoparticles formed into polymeric micelles through self-assembly in aqueous solution from amphiphilic block copolymers containing hydrophobic polymer segments having cyclic nitroxyl radicals (particularly TEMPO (2,2,6,6-tetramethylpiperidinooxy radical)) in their side chains and hydrophilic poly(ethylene glycol) (PEG) segments. These nanoparticles are known as preparations capable of scavenging free radicals generated by ischemia and inflammation (see WO 2009 / 133647 A, hereinafter sometimes referred to as Patent Document 1; see also WO 2016 / 052463 A, hereinafter sometimes referred to as Patent Document 2). Furthermore, Japanese Patent Laid-Open Publication No. 2013-166711 (JP 2013-166711 A) (hereinafter sometimes referred to as Patent Document 3) investigates the use of polymeric micellar nanoparticles, abbreviated as N-TEMPO-RNP and O-TEMPO-RNP, for the prevention or treatment of cardiac ischemia-reperfusion injury, as disclosed in Patent Document 1. In Patent Document 3, both RNPs were administered to test animals, and blood pressure changes in the animals were observed. O-TEMPO-RNP, which is not known to cause a decrease in blood pressure, was used as the active ingredient. More specifically, O-TEMPO-RNP was intravenously administered to beagle dogs induced into an ischemic state 45 minutes after the ischemia. Reperfusion was performed 5 minutes after administration, and the heart was removed 4 hours later, and the size of myocardial infarction was measured. As a result, it is disclosed that the size of myocardial infarction was significantly reduced.

[0006] The preparations used or considered for use in the aforementioned surgical procedures, primarily liver surgery, and procedures for improving the success rate of organ transplants to suppress ischemia-reperfusion injury have not necessarily demonstrated satisfactory effects. Therefore, if the use of N-TEMPO-RNP or O-TEMPO-RNP in surgical procedures or organ transplants could potentially suppress ischemia-reperfusion injury simply and effectively, it would be worth investigating their use.

[0007] For example, Patent Document 3 examines the possibility of using a preparation containing N-TEMPO-RNP and O-TEMPO-RNP as a pharmaceutical preparation for preventing or treating cardiac ischemia-reperfusion injury, and confirms the effectiveness of only the latter, O-TEMPO-RNP, from the viewpoint that it does not exhibit a blood pressure lowering effect.

[0008] However, in this study, we demonstrated that N-TEMPO-RNP can be safely administered to animals in an animal model mimicking ischemia-reperfusion during surgical or transplant surgery, and that it significantly and strongly suppresses ischemia-reperfusion injury in the target organ. Specifically, when N-TEMPO-RNP was administered to test animals prior to ischemia-reperfusion injury, specific and significantly increased accumulation of N-TEMPO-RNP in the target organ was confirmed one hour after resumption of blood flow. Furthermore, blood sampling data collected six hours later confirmed that liver damage was significantly and strongly suppressed compared to controls. No serious side effects, such as kidney damage, were observed with this preoperative administration of nanoparticles. Based on the results of these animal experiments, it is believed that preoperative administration of both N-TEMPO-RNP and O-TEMPO-RNP, which has a longer blood retention time than N-TEMPO-RNP, may be able to suppress ischemia-reperfusion injury in human patients undergoing liver surgery or organ transplantation. Therefore, the main subject matter of the present invention and disclosed herein can be as follows:

[0009] [Subject 1] A preparation for suppressing ischemia-reperfusion injury in a target organ during surgery or transplantation, comprising a copolymer represented by the following formula (I) and containing, as an active ingredient, polymeric micelles having a nano-sized average diameter in an aqueous solution as measured by dynamic light scattering (DLS): Formula (I): where A is an unsubstituted or substituted C-C 12 The substituents, when substituted, are formyl groups or groups of the formula R １ R ２ represents a CH- group, where R １ and R ２ are independently C1-C4 alkoxy or R １ and R ２ together represent —OCH2CH2O—, —O(CH2)3O— or —O(CH2)4O—, L1 is a direct bond or or -(CH ２ ) ｂ S-, -CO(CH ２ ) ｂ S-, -(CH ２ ) ｂ NH-, -(CH ２ ) ｂ selected from CO—, —CO—, —OCOO—, and —CONH—, where each b is independently an integer of 1 to 5 (when these groups represent different structures depending on the direction of the bond, they are incorporated into formula (I) in the direction described herein); L ２ -R １ Is, L ２ Ga-(CH ２ ) ａ -NH-(CH ２ ) ａ - or - (CH ２ ) ａ -O-(CH ２ ) ａ -, where each a is independently 0 or an integer from 1 to 5; R １ But, the formula (wherein R' is methyl), and R ２ is chloro, bromo or hydroxyl, ２ -R１ and R ２ The repeating units in the polymer backbone having L ２ -R １ The unit p having the formula: is an integer ranging from 2 to 100, and R ２ The unit q having the formula (I) is absent (zero) or is an integer ranging from 1 to 20, provided that the total number of these units is n; Z is H, SH, or S(C=S)-Ph, where Ph represents phenyl optionally substituted with one or two methyl or methoxy groups; m represents an integer from 2 to 10,000; and n represents an integer from 3 to 100.

[0010] [Subject 2] The formulation according to Subject 1, wherein the organ includes at least one selected from the heart, lung, kidney, liver, pancreas, and small intestine.

[0011] [Subject 3] The formulation of Subject 1, wherein the organ is the liver.

[0012] [Subject 4] A preparation for suppressing ischemia-reperfusion injury in a treated organ during the surgical or transplant operation described in Subject 1 or 2, the preparation comprising a constituent feature selected from the following (i) to (iv): (i) the operation is a surgical operation that includes the steps of partially or completely resecting an organ after blocking blood flow, followed by blood reperfusion, and includes a step of parenterally administering the active ingredient to a patient or subject before blocking blood flow, and suppressing ischemia-reperfusion injury in the treated organ means suppressing ischemia-reperfusion injury to a significantly greater extent than in a case that does not include the step of parenterally administering the active ingredient to a patient or subject. (ii) The surgery is a transplant surgery, comprising a step of parenterally administering the active ingredient to a donor of an organ to be transplanted, and extracting the organ to be transplanted after spontaneous cardiac arrest in the donor, followed by a step of preserving or perfusing the extracted organ with an organ preservation solution containing the active ingredient as a simulated reperfusion step, and the suppression of ischemia-reperfusion injury in the treated organ means a significantly higher suppression than a step of not administering the active ingredient to the organ donor and preserving the extracted organ in an organ preservation solution not containing the active ingredient. (iii) The surgery is a transplant surgery, comprising a step of parenterally administering the active ingredient to a brain-dead organ donor to induce cardiac arrest, followed by circulating an organ preservation solution containing the active ingredient throughout the donor's body, and then, as a step simulated as reperfusion, preserving or perfusing the extracted organ with an organ preservation solution containing the active ingredient, wherein the suppression of ischemia-reperfusion injury in the treated organ means a significantly greater suppression than in a case where the active ingredient is not administered to the organ donor and the extracted organ is preserved or perfused with an organ preservation solution not containing the active ingredient. (iv) The surgery is a transplant surgery, comprising a step of parenterally administering the active ingredient to a patient to be transplanted, followed by implanting the organ to be transplanted, and a blood reperfusion step, wherein the suppression of ischemia-reperfusion injury in the treated organ means a significantly greater suppression than in a case where the active ingredient is not administered to the transplant recipient.

[0013] [Subject 5] A polymeric micelle for suppressing ischemia-reperfusion injury in a treated organ during surgery or transplantation, comprising a copolymer represented by the following formula (I), and having a nano-sized average diameter in an aqueous solution as measured by dynamic light scattering (DLS): Formula (I): where A is an unsubstituted or substituted C-C 12 The substituents, when substituted, are formyl groups or groups of the formula R １ R ２ represents a CH- group, where R １ and R ２ are independently C1-C4 alkoxy or R １ and R ２ together represent —OCH2CH2O—, —O(CH2)3O— or —O(CH2)4O—, L1 is a direct bond or or -(CH ２ ) ｂ S-, -CO(CH ２ ) ｂ S-, -(CH ２ ) ｂ NH-, -(CH ２ ) ｂ selected from CO—, —CO—, —OCOO—, and —CONH—, where each b is independently an integer of 1 to 5 (when these groups represent different structures depending on the direction of the bond, they are incorporated into formula (I) in the direction described herein); L ２ -R １ Is, L ２ Ga-(CH ２ ) ａ -NH-(CH ２ ) ａ - or - (CH ２ ) ａ -O-(CH ２ ) ａ -, where each a is independently 0 or an integer from 1 to 5; R １ But, the formula (wherein R' is methyl), and R ２ is chloro, bromo or hydroxyl, ２ -R １and R ２ The repeating units in the polymer backbone having L ２ -R １ The unit p having the formula: is an integer ranging from 2 to 100, and R ２ The unit q having the formula (I) is absent (zero) or is an integer ranging from 1 to 20, provided that the total number of these units is n; Z is H, SH, or S(C=S)-Ph, where Ph represents phenyl optionally substituted with one or two methyl or methoxy groups; m represents an integer from 2 to 10,000; and n represents an integer from 3 to 100.

[0014] [Subject 6] A method for suppressing ischemia-reperfusion injury in a treated organ during surgery or transplantation, the method comprising the step of administering to a patient or subject in need thereof an effective amount of polymeric micelles containing a copolymer represented by the following formula (I), which has a nano-sized average diameter in an aqueous solution as measured by dynamic light scattering (DLS): Formula (I): where A is an unsubstituted or substituted C-C 12 The substituents, when substituted, are formyl groups or groups of the formula R １ R ２ represents a CH- group, where R １ and R ２ are independently C1-C4 alkoxy or R １ and R ２ together represent —OCH2CH2O—, —O(CH2)3O— or —O(CH2)4O—, L1 is a direct bond or or -(CH ２ ) ｂ S-, -CO(CH ２ ) ｂ S-, -(CH ２ ) ｂ NH-, -(CH ２ ) ｂ selected from CO—, —CO—, —OCOO—, and —CONH—, where each b is independently an integer of 1 to 5 (when these groups represent different structures depending on the direction of the bond, they are incorporated into formula (I) in the direction described herein); L ２ -R１ Is, L ２ Ga-(CH ２ ) ａ -NH-(CH ２ ) ａ - or - (CH ２ ) ａ -O-(CH ２ ) ａ -, where each a is independently 0 or an integer from 1 to 5; R １ But, the formula (wherein R' is methyl), and R ２ is chloro, bromo or hydroxyl, ２ -R １ and R ２ The repeating units in the polymer backbone having L ２ -R １ The unit p having the formula: is an integer ranging from 2 to 100, and R ２ The unit q having the formula (I) is absent (zero) or is an integer ranging from 1 to 20, provided that the total number of these units is n; Z is H, SH, or S(C=S)-Ph, where Ph represents phenyl optionally substituted with 1 or 2 methyl or methoxy groups; m represents an integer ranging from 2 to 10,000; and n represents an integer ranging from 3 to 100.

[0015] According to the present invention, by systemically parenterally administering the formulation to a patient or subject prior to surgery or transplantation, the formulation can be selectively accumulated in the organ affected by ischemia, potentially significantly suppressing ischemia-reperfusion-induced organ damage compared to controls. Therefore, for example, in surgical procedures involving liver resection, it may be possible to alleviate postoperative liver damage and reduce the incidence of liver failure. Furthermore, it may be possible to extend the duration of continuous blood flow occlusion, potentially shortening the total surgical time. No formulation capable of suppressing ischemia-reperfusion injury during liver resection has been developed in clinical practice to date. For example, if the safety of liver resection could be improved simply by intravenous administration, it would be a significant innovation in the more than 130-year history of liver surgery. Furthermore, in the field of organ transplantation, the formulation's inhibitory effect on ischemia-reperfusion injury may lead to improved graft function and survival. No formulation capable of suppressing unavoidable ischemia-reperfusion injury has been developed in the field of organ transplantation to date. If it were possible to increase the success rate of transplant surgery simply by administering it intravenously to organ donors and transplant recipients, or by adding it to the preservation solution after submission, it would be a major innovation that stands out in the more than 70-year history of transplant surgery.

[0016] 1 is a conceptual diagram of the schedule for the animal experiment in Test Example 1. FIG. 2 is a graphical representation of the results of biochemical evaluation part 1 in Test Example 1. FIG. 3 is a graphical representation of the results of biochemical evaluation part 2 in Test Example 1. FIG. 4 is a black-and-white photograph in place of a micrograph taken after hematoxyline-eosin staining of liver tissue in histological evaluation part 1 in Test Example 1. FIG. 5 is a black-and-white photograph in place of a micrograph taken after TUNEL staining of liver tissue cells in histological evaluation part 2 in Test Example 1. FIG. 6 is a conceptual diagram of the schedule for the animal experiment in Test Example 2. FIG. 7 is a photograph in place of a fluorescent micrograph of liver tissue taken after treatment in Test Example 2. FIG. 8 is a conceptual diagram of the schedule for the animal experiment in Test Example 3. FIG. 9 is a graphical representation of the results of measurement of biological indicators when cold preservation was performed in biological evaluation part 1 in Test Example 3. FIG. 10 is a graphical representation of the results of measurement of biological indicators when warm preservation was performed in biological evaluation part 1 in Test Example 3. 1 is a graph showing the measurement results of biological indices when cold preservation was performed in biological evaluation part 2 of Test Example 3. FIG. 2 is a graph showing the measurement results of biological indices when warm preservation was performed in biological evaluation part 2 of Test Example 3. FIG. 3 is a graph showing the measurement results of biological indices when cold preservation was performed in biological evaluation part 3 of Test Example 3. FIG. 4 is a graph showing the measurement results of biological indices when warm preservation was performed in biological evaluation part 3 of Test Example 3. FIG. 5 is a black-and-white photograph in place of a microscope image of liver tissue collected during cold preservation in the histological evaluation of Test Example 3, taken after hematoxyline-eosin staining. FIG. 6 is a black-and-white photograph in place of a microscope image of liver tissue collected during warm preservation in the histological evaluation of Test Example 3, taken after hematoxyline-eosin staining. FIG. 7 is a conceptual diagram of the schedule of the animal experiment in Test Example 4. FIG. 8 is a graph showing the measurement results of biological indices up to the time when heart rate resumed after heart transplantation in physiological evaluation part 1 of Test Example 4. 10 is a graph showing the measurement results of biological indicators of cardiac output strength in physiological evaluation part 1 of Test Example 4. Detailed Description of the Invention

[0017] Terms used in this specification are to be construed as having the meanings commonly used in the art unless otherwise specified.

[0018] "Ischemia-reperfusion injury in surgery or organ transplantation" refers primarily to a phenomenon in which biochemical parameters that indicate the function of an organ that has been resected, extracted, or transplanted deteriorate compared to preoperative values ​​or compared to reference values ​​in healthy individuals, or the histological evaluation of the organ deteriorates. However, in a broader sense, it is not limited to these, and refers to any damage to the organ that is the target of surgery or to the surrounding organs, organs, or tissues that is caused by the surgery.

[0019] A "patient or subject" is a person from whom at least a part of an organ is to be removed, extracted, or transplanted, and in the case of transplant surgery, may be either an organ donor or a transplant recipient.

[0020] "Cutting off blood flow to an organ" is primarily a means of reducing bleeding during the resection, transection, or extraction of an organ, and there are no limitations on the form or type of means employed. In transplant surgery, such cutoff of blood flow is performed in organ donor patients before the transplant target organ is extracted or harvested, and in transplant recipients before the corresponding organ is harvested. Furthermore, before the harvested organ is implanted in the patient, the patient's blood vessels and the blood vessels of the transplanted organ are anastomosed, and blood flow is stopped until blood flow is resumed. Such an ischemic state is also included in the above-mentioned cutoff of blood flow.

[0021] Surgery, mainly liver surgery and transplant surgery, is carried out as outlined below in <Techniques related to surgery, mainly liver surgery> and <Techniques related to improving the success rate of organ transplants>, but is not limited to these, and briefly and specifically, may be carried out as follows: <Techniques related to surgery, mainly liver surgery>

[0022] Before liver transection begins, a patient is intravenously administered a fixed dose of polymeric micelles containing a copolymer of formula (I) and having a nanosized average diameter in aqueous solution as determined by dynamic light scattering (DLS) measurement, or polymeric micelles formed from a copolymer of formula (I) having a nanosized average diameter (these polymeric micelles are sometimes abbreviated as "nanoparticles"), or a formulation containing nanoparticles as an active ingredient. After waiting several minutes, e.g., about 3 minutes, until the nanoparticles are distributed throughout the body, hepatoduodenal ligament blood flow is blocked, and liver transection is initiated.

[0023] <Technologies for improving the success rate of organ transplants> 1. Removal of organs (heart, lungs, liver, kidneys, pancreas, small intestine) from organ donors under cardiac arrest. The nanoparticle formulation is intravenously administered to the organ donor at a fixed dose (e.g., 90 mg / kg) timed to coincide with heparin administration. After spontaneous cardiac arrest occurs, each organ is removed, and the graft is manually perfused with an organ preservation solution (e.g., 1.0 mg / mL) containing the nanoparticle formulation, filling the graft with the antioxidant nanoparticle formulation. The graft is then subjected to simple cooling or machine perfusion preservation in a preservation solution containing the antioxidant nanoparticle formulation.

[0024] 2. Organ removal (heart, lungs, liver, kidneys, pancreas, small intestine) from brain-dead organ donors: The nanoparticle formulation is intravenously administered to the organ donor at a fixed dose (e.g., 90 mg / kg) in accordance with the timing of heparin administration. After cardiac arrest is induced in the organ donor by open heart surgery, the self-assembling antioxidant nanoparticle formulation is perfused throughout the donor's body with an organ preservation solution (e.g., 1.0 mg / mL) containing the self-assembling antioxidant nanoparticle formulation. Each transplant organ is removed and manually perfused with the organ preservation solution containing the self-assembling antioxidant nanoparticle formulation, filling the graft with the antioxidant nanoparticle formulation. Subsequently, the organ is preserved by simple cooling or machine perfusion with the preservation solution containing the antioxidant nanoparticle formulation.

[0025] 3. Implantation of the excised organ: The nanoparticle formulation (e.g., 90 mg / kg) is administered intravenously or intraportally to the recipient, and the patient is allowed to wait several minutes, e.g., about 3 minutes, until the nanoparticles are distributed throughout the body. Thereafter, the graft is implanted and blood is reperfused.

[0026] <Self-assembled antioxidant nanoparticles> The nanoparticles contain or are composed of a copolymer represented by formula (I), and their preparation methods are described in Patent Document 1 or Patent Document 2. The average diameter of the nano-sized nanoparticles is in the order of nanometers, and may be within the range of, but is not limited to, 5 nm to 500 nm, 10 nm to 300 nm, 10 nm to 100 nm, or 10 nm to 60 nm. In the present invention, among the polymer micelles described in Patent Document 1 or Patent Document 2, in the copolymer represented by the above formula (I), L ２ Ga-(CH ２ ) ａ -NH-(CH ２ ) ａ - or - (CH ２ ) ａ -O-(CH ２ ) ａ -, where each a is independently 0 or an integer of 1 to 5 or 1 to 3, and preferably each a is the integer 0. The nanosize refers to the average diameter of the nanoparticles when measured by dynamic light scattering (DLS) in an aqueous solution.

[0027] In addition, in formula (I), m represents an integer of 2 to 10,000, but may represent an integer of 5 to 1,000 or 10 to 500, while n represents an integer of 3 to 100, but may represent an integer of 5 to 100 or 5 to 60.

[0028] In formula (I), C is defined for any variable group or moiety. １ -C １２ Alkyl, C １ -C ４ Regarding the alkyl moiety in alkoxy etc., for example, C １ -C １２ Alkyl includes straight-chain, branched, or cyclic alkyl having 1 to 12 carbon atoms, and typical examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, heptyl, octyl, isooctyl, 2-ethylhexyl, nonyl, isononyl, decyl, isodecyl, undecyl, dodecyl, and the like.

[0029] The copolymer represented by formula (I), which is contained in or constitutes the nanoparticles corresponding to N-TEMPO-RNP described above, accumulates and associates as a plurality of molecules in an aqueous medium at a pH higher than neutral, where the -NH- moiety does not undergo protonation, to form self-assembled antioxidant nanoparticles. While the scope of the present invention is not limited by theory, the nanoparticles thus formed disintegrate upon protonation in a low pH environment. Therefore, they typically accumulate specifically in the liver, which is subject to blood flow interruption and, in some cases, in surrounding organs or organs, or in transplanted organs, which are subject to a low pH environment. The redox-active cyclic nitroxide radicals exposed by the in situ disintegration are understood to be effective in removing "bad" reactive oxygen species involved in injury caused by ischemia and subsequent reperfusion of blood flow. On the other hand, nanoparticles corresponding to O-TEMPO-RNP, in which the -NH- moiety is -O-, have been shown in vivo to have a half-life several times longer than that of N-TEMPO-RNP, and can remain or persist longer in the blood circulation or in a preservation solution. Therefore, it is presumed that either N-TEMPO-RNP or O-TEMPO-RNP nanoparticles will be effective depending on the type and circumstances of the surgical or transplant operation.

[0030] <Formulations> The nanoparticles formed as described above can be dissolved or uniformly dispersed in an aqueous medium (an aqueous solution that may optionally contain physiological salt or a pH adjuster) and can be reconstituted with water, allowing them to be provided as formulations in various forms. Therefore, they can be made into various forms of parenteral formulations, including parenteral formulations (including intravenous infusions, subcutaneous administration agents, etc.). Such parenteral formulations can contain common pharmaceutically acceptable vehicles, such as water, glycol, sucrose, lactose, etc., as long as they do not adversely affect the stability of the nanoparticles in aqueous media.

[0031] The optimal dose of such preparations varies depending on the type of surgery and the condition of the patient or subject undergoing surgery, so a uniform dose cannot be specified. However, the dose can be determined by a specialist based on data obtained through small-scale clinical trials, etc. While not limited to this, the nanoparticles may be contained as an active ingredient in an amount of at least 5% by weight. The proportion of the active ingredient in these preparations or compositions may vary and may conveniently be between about 30% and about 90% by weight based on the total weight of the preparation or composition. Since the nanoparticles may be effective over a wide range of dosages, for example, the dose for treating an adult may be about 50 to about 1,000 mg or about 100 to about 500 mg per kg of body weight.

[0032] The present invention will be explained in more detail below using specific examples, but the present invention is not limited to these examples.

[0033] The nanoparticles used in the following examples are copolymers prepared by the method described in Patent Document 2, and are represented by formula (I), in which A is methyl and L １ is paraxylylene, m is about 40, p is 15, q is 4, a is 0, Z is S(C=S)-Ph, Ph is unsubstituted phenyl, and R １ is the residue shown at the left end of the three types of cyclic nitroxy radicals listed above: 2,2,6,6-tetramethylpiperidine-1-oxyl, and is formed, for example, as follows from a block copolymer (PEG-b-PMNT):

[0034] PEG-b-PMNT was dissolved in dimethylformamide (DMF) at a concentration of 150 mg / mL, and the solution was placed in a dialysis membrane, sealed, and dialyzed against distilled water to produce nanoparticles. The distilled water was replaced every 2, 4, 8, and 20 hours after the start of dialysis, and the solution in the dialysis membrane was recovered after 24 hours. PBS with a 10x concentration was added to the solution in the dialysis membrane to achieve a final concentration of 1x PBS. Dynamic light scattering (DLS) measurements of particle size and particle size distribution revealed a particle size (Z-Ave) of 26.5 nm and a polydispersity index (PDI) of 0.12, indicating a uniform particle size distribution.

[0035] Test Example 1: According to the conceptual diagram of the animal experiment schedule shown in Figure 1, experimental animals (SD rats, 10-12 weeks old) were divided into a control group (n = 5 or 6) administered with PBS and an RNP group (n = 5 or 6) administered with the nanoparticles (hereinafter referred to as RNP, 90 mg / kg), and 10 minutes after administration, the hepatoduodenal ligament was subjected to simultaneous occlusion of blood flow to induce hepatic ischemia-reperfusion for 20 minutes. Liver damage was evaluated biochemically and histologically 6 and 24 hours after surgery.

[0036] (1) Biochemical Evaluation Part 1: As a biochemical evaluation in Test Example 1, changes in GPT, GOT, and LDH, which are indicators of liver function, were measured. The results are shown in Figure 2. As can be seen from Figure 2, in the case where the preparation of the present invention was used, a significant decrease was observed in all of the indicators related to the degree of liver inflammation compared to the control case where the preparation was not used.

[0037] (2) Biochemical Evaluation Part 2: The results of measuring the serum concentration of TBRS (2-thiobarbituric acid reactive substances), an oxidative stress marker, 24 hours after surgery in Test Example 1 are shown in Figure 3. Figure 3 shows that in cases where the preparation of the present invention was used, there was a significant decrease in TBRS, an oxidative stress marker, compared to the control, which did not use the preparation.

[0038] (3) Histological Evaluation 1: Histological evaluation 1 was performed by hematoxylin-eosin staining of liver tissues resected 6 and 24 hours after surgery in Test Example 1. Figure 4 shows black-and-white photographs, instead of microscopic images, taken after staining. The original microscopic photographs for this organ transplant are color images. We are prepared to provide these color images, if necessary, to obtain clearer information. Figure 4 shows that in the control group, hepatocytes around the hepatic vein began to become hollow after 6 hours of ischemia, whereas hollowing was mild in the RNP-administered group. Furthermore, while extensive necrosis was observed in the control group after 24 hours of ischemia, the extent of necrosis was small in the RNP-administered group.

[0039] (4) Histological Evaluation 2: Histological evaluation 2 was performed by TUNEL staining to evaluate apoptosis of hepatocytes resected in Test Example 1 at 6 and 24 hours postoperatively. Figure 5 shows black-and-white photographs taken after TUNEL staining, replacing the micrographs, and Figure 6 shows a graphical representation of the percentage of TUNEL-positive areas. The original micrographs of this organ transplant were color images. We are prepared to provide these color images, if necessary, to obtain clearer information. In cases where this formulation was used (RNP group), the TUNEL-stained positive areas were reduced at 6 and 24 hours postoperatively compared to cases where this formulation was not used (control group). These results demonstrate that cell death was suppressed by RNP administration.

[0040] Test Example 2: In accordance with the conceptual diagram of the animal experiment schedule shown in Figure 7, experimental animals were administered RNP (90 mg / kg) and divided into an IR- group (without hepatoduodenal ligament blood flow occlusion) and an IR+ group (with hepatoduodenal ligament blood flow occlusion). Fluorescence microscopic images of liver tissue were taken 1 hour after the end of occlusion to evaluate RNP hepatic accumulation. Figure 8 shows black-and-white photographs in place of the fluorescent images. The original microscopic images are color images. If necessary, we are prepared to provide color images to obtain clearer information. Comparison of the photographs between the two groups revealed increased RNP accumulation in the hepatic sinusoids in the IR+ group (with hepatoduodenal ligament blood flow occlusion). This phenomenon is thought to result in RNP specifically accumulating in the injured liver, thereby effectively reducing liver damage.

[0041] Test Example 3: To verify the effectiveness of organ preservation in organ transplantation, experimental animals (SD rats) were divided into a control group (n = 3 to 6) administered saline and an RNP group (n = 3 to 6) administered RNP (90 mg / kg) according to the schematic diagram of the animal experiment schedule shown in Figure 9. Ten minutes after administration, each group underwent abdominal surgery. The former group underwent systemic perfusion via the aorta with 40 ml of organ preservation solution (Wisconsin solution, hereafter referred to as UW solution) without RNP, while the latter group underwent systemic perfusion via the aorta with UW solution (1.0 mg / ml) with RNP. After this, blood was drained via incision in the inferior vena cava, and the entire organ was filled with UW solution. The livers were then immediately removed and simply preserved at 4°C or 37°C in UW solution (1.0 mg / ml) without RNP. After preservation, biochemical and histological evaluations were performed on the preservation solution and the preserved liver.

[0042] (1) Biochemical Evaluation Part 1: During cold storage, changes in LDH and GPT were measured as biochemical indicators of liver damage. The results are shown in Figure 10. Figure 10 shows that LDH and GPT, which are associated with liver damage, were reduced in the cases where the formulation of the present invention was added, compared to the control, which did not use the formulation. Furthermore, the concentration of the stress marker TBARS in the preservation solution was also reduced in the cases where the formulation of the present invention was used, compared to the control, which did not use the formulation. Meanwhile, changes in LDH and GPT, which are biochemical indicators of liver damage, were also measured during warm storage. The results are shown in Figure 11. Figure 11 shows that LDH and GPT, which are associated with liver damage, were reduced in the cases where the formulation of the present invention was used, compared to the control, which did not use the formulation. Furthermore, the concentration of the stress marker TBARS in the preservation solution was significantly reduced in the cases where the formulation of the present invention was used, compared to the control, which did not use the formulation. These findings demonstrate that the addition of RNP to the preservation solution reduces organ damage in preserved organs during cold or warm storage.

[0043] (2) Biochemical Evaluation Part 2: In Biochemical Evaluation Part 2, glutathione (GSH) concentrations were measured in liver tissue samples collected 6, 12, and 24 hours after the start of cold storage in Test Example 3. The results are shown in Figure 12. GSH exhibits antioxidant properties by reducing reactive oxygen species, etc., so low GSH levels can be interpreted as indicating severe oxidative stress. Figure 12 shows that the control group, to which the formulation of the present invention was not added, showed a decrease in GSH, an antioxidant, compared to the group to which the formulation of the present invention was added. Next, GSH concentrations were measured in liver tissue samples collected 6 and 12 hours after the start of warm storage in Test Example 3. The results are shown in Figure 13. Figure 13 shows that the control group to which the formulation of the present invention was not added showed a tendency for GSH, an antioxidant, to decrease after 6 hours of storage, compared to the group to which the formulation of the present invention was added, and a significant decrease in GSH was observed after 12 hours. From the above, it can be seen that the addition of RNP to the preservation solution during cold or warm preservation reduced oxidative stress in the preserved organs.

[0044] (3) Biochemical evaluation 3: Biochemical evaluation 3 evaluated the oxidative stress level (dROM: Diacron-Reactive Oxygen Metabolites: derivatives of hydrogen peroxide (H₂O₂) (R-OOH), which measures peroxidized lipids such as lipid peroxidation (L-OOH), proteins, amino acids, nucleic acids, etc.; hereinafter referred to as dROM) contained in liver tissue collected 6, 12, and 24 hours after the start of cold storage in Test Example 3; antioxidant potential (BAP: Biological Antioxidant Potential: the reduction power of ferric iron to ferrous iron by substances with reducing power in blood and tissues (glutathione, thiols, ascorbic acid, bilirubin, uric acid, etc.), i.e., antioxidant potential; hereinafter referred to as BAP); and the relative oxidative stress level (Oxidative stress index: dROM / BAP*100; hereinafter referred to as OSI) calculated from dROM and BAP (n=3 per group). In this experiment, the supernatant obtained by centrifuging liver tissue homogenate was used to measure dROM and BAP. The results are shown in Figure 14. Figure 14 confirms that the cases to which the formulation of the present invention was added (RNP group) tended to have lower oxidative stress levels (dROM) at each time point, higher antioxidant power (BAP), and lower relative oxidative stress levels (OSI) compared to the control group without the formulation. Next, the levels of dROM, BAP, and OSI in liver tissue samples collected 6 and 12 hours after the start of thermal preservation in Test Example 3 were measured (n = 3 per group). The results are shown in Figure 15. Figure 15 also confirms that, similar to cold ischemia, the cases to which the formulation of the present invention was added (RNP group) tended to have lower oxidative stress levels (dROM) at each time point, higher antioxidant power (BAP), and lower relative oxidative stress levels (OSI) compared to the control group without the formulation. BAP at 12 hours after warm ischemia was significantly higher in the cases to which the formulation of the present invention was added (RNP group). From the above, it can be seen that adding RNP to the preservation solution during cold or warm preservation suppresses peroxidation of the tissues of preserved organs, maintains the antioxidant power of the tissues, and reduces the oxidative stress that occurs in the tissues during organ preservation.

[0045] (4) Histological Evaluation: Histological evaluation was performed by hematoxyline-eosin staining of liver tissue collected 6 and 24 hours after the start of cold storage in Test Example 3. Figure 16 shows black-and-white photographs, instead of microscopic images, taken after staining. The original microscopic photographs for this organ transplant were color images. We are prepared to provide these color images, if necessary, to obtain clearer information. As shown in Figure 16, the morphology of liver tissue was maintained in both the control and RNP-added groups after 6 hours of storage. However, after 24 hours, hepatocytes in the control group had collapsed and sinusoidal expansion was observed, whereas in the RNP group, hepatocyte collapse was minimal and sinusoidal expansion was barely observed. Next, histological evaluation of warm storage was performed by hematoxyline-eosin staining of liver tissue collected 3 and 12 hours after the start of storage. Figure 17 shows black-and-white photographs, instead of microscopic images, taken after staining. The original microscopic photographs of this organ transplant are color images. We are prepared to submit color images if necessary to obtain clearer information. In the control group, hepatocytes began to decay and sinusoids expanded three hours after storage, whereas the morphology of liver tissue was maintained in the RNP group. After 12 hours, most hepatocytes in the control group had collapsed and the sinusoids had expanded widely, whereas in the RNP-treated group, hepatocytes had begun to decay but the sinusoids were only slightly expanded. These findings demonstrate that adding RNP to the preservation solution during cold or warm storage reduces organ damage in preserved organs.

[0046] Test Example 4: To verify the inhibitory effect of ischemia during organ preservation and subsequent ischemia-reperfusion injury following organ transplantation, a schematic diagram of the animal experiment schedule is shown in Figure 18. Donor experimental animals (C57BL / 6 mice) were divided into a control group (n = 2) administered intravenously with saline and an RNP group (n = 2) administered with RNP (90 mg / kg). Five minutes after saline or RNP administration, the control group underwent systemic perfusion with 4 ml of organ preservation solution (UW solution), followed by removal of the heart graft and subsequent irrigation with 4 ml of UW solution. In contrast, the RNP group underwent systemic perfusion with 4 ml of RNP-supplemented UW solution (1 mg / ml), followed by removal of the heart graft and subsequent irrigation with 4 ml of RNP-supplemented UW solution. Subsequently, the heart grafts in the control group were cold-preserved in UW solution at 4°C for 24 hours, while those in the RNP group were cold-preserved in UW solution (1 mg / dL) containing RNP for 24 hours. Five minutes before the start of transplantation, recipients in the control group received intravenous saline, while recipients in the RNP group received intravenous RNP (90 mg / kg) before intraperitoneal transplantation of the heart graft. Physiological evaluations of the recipients and heart grafts in each group were performed.

[0047] (1) Physiological Evaluation Part 1: Figure 19 shows the time from resumption of blood flow to resumption of cardiac beat in each group. In the cases where the formulation of the present invention was used (RNP group), the time until resumption of cardiac beat was shortened compared to the control group, which did not use the formulation. Next, Figure 20 shows the cardiac beat intensity at the time of resumption of cardiac beat after blood perfusion and 6 hours after surgery in each group, based on the Stanford Cardiac Surgery Laboratory Graft Scoring System (maximum score of 4). In the cases where the formulation of the present invention was used, an increase in cardiac beat intensity was observed at each time point compared to the control group, which did not use the formulation. From the above, it can be seen that the cardiac beat of the cardiac graft after transplantation in the cases where the formulation of the present invention was used was more advantageous in terms of both duration and intensity than in the cases where the formulation of the present invention was not used.

[0048] According to the present invention, a preparation for suppressing ischemia-reperfusion injury of a target organ in a surgical operation or transplant operation is provided, and therefore the present invention is applicable at least to industries that manufacture or sell such preparations.

Claims

1. A preparation for suppressing ischemia-reperfusion injury in a target organ during surgery or transplantation, comprising a copolymer represented by the following formula (I) and a polymeric micelle having a nano-sized average diameter in an aqueous solution as determined by dynamic light scattering (DLS) measurement as an active ingredient. Formula (I): In the above formula, A is an unsubstituted or substituted C-C 12 The substituent, when substituted, is a formyl group or a group of the formula R １ R ２ represents a CH- group, where R １ and R ２ are independently C1-C4 alkoxy or R １ and R ２ together represent -OCH2CH2O-, -O(CH2)3O- or -O(CH2)4O-; L1 is a direct bond or or -(CH ２ ) ｂ S-, -CO(CH ２ ) ｂ S-, -(CH ２ ) ｂ NH-, -(CH ２ ) ｂ selected from CO-, -CO-, -OCOO-, -CONH-, where each b is independently an integer from 1 to 5; ２ -R １ L ２ - (CH ２ ) ａ -NH-(CH ２ ) ａ - or - (CH ２ ) ａ -O-(CH ２ ) ａ -, where each a is independently 0 or an integer from 1 to 5; R １ But, the formula (wherein R' is methyl), ２ is chloro, bromo or hydroxyl, ２ -R １ and R ２ The repeat units in the polymer backbone having L ２ -R １ p is an integer ranging from 2 to 100, R ２ The unit q having the formula: is absent (zero) or is in the range of an integer from 1 to 20, provided that the total number of these units is n; Z is H, SH, or S(C=S)-Ph, where Ph represents phenyl optionally substituted with 1 or 2 methyl or methoxy; m represents an integer from 2 to 10,000; and n represents an integer from 3 to 100.

2. The preparation according to claim 1, wherein the organ comprises at least one organ selected from the group consisting of heart, lung, kidney, liver, pancreas, and small intestine.

3. The formulation of claim 1, wherein the organ is the liver.

4. A preparation for suppressing ischemia-reperfusion injury in a treated organ during a surgical procedure or transplant procedure as described in claim 1 or 2, comprising a constituent feature selected from the following (i) to (iv): (i) the procedure is a surgical procedure that includes the steps of partially or completely resecting an organ after blocking the blood flow, followed by blood reperfusion, and includes a step of parenterally administering the active ingredient to a patient or subject before blocking the blood flow, and suppressing ischemia-reperfusion injury in the treated organ means suppressing ischemia-reperfusion injury to a significantly higher extent than a procedure that does not include a step of parenterally administering the active ingredient to a patient or subject. (ii) The surgery is a transplant surgery, comprising a step of parenterally administering the active ingredient to a donor of an organ to be transplanted, and extracting the organ to be transplanted after spontaneous cardiac arrest in the donor, and then, as a step simulated as reperfusion, a step of preserving or perfusing the extracted organ with an organ preservation solution containing the active ingredient, and the suppression of ischemia-reperfusion injury in the treated organ means a significantly higher suppression than a step including a step of not administering the active ingredient to the organ donor and preserving the extracted organ in an organ preservation solution not containing the active ingredient. (iii) The surgery is a transplantation surgery, comprising a step of parenterally administering the active ingredient to a brain-dead organ donor to cause cardiac arrest, circulating an organ preservation solution containing the active ingredient throughout the donor's body, and then, as a step simulated as reperfusion, preserving or perfusing the extracted organ with an organ preservation solution containing the active ingredient, and the suppression of ischemia-reperfusion injury in the treated organ means a significantly higher suppression than a step not including administration of the active ingredient to the organ donor and preserving or perfusing the extracted organ with an organ preservation solution not including the active ingredient. (iv) The surgery is a transplantation surgery, comprising a step of parenterally administering the active ingredient to a patient to be transplanted, implanting the organ to be transplanted, and a blood reperfusion step, and the suppression of ischemia-reperfusion injury in the treated organ means a significantly higher suppression than a step not including administration of the active ingredient to the transplant recipient.

5. A polymeric micelle for suppressing ischemia-reperfusion injury in a treated organ during surgery or transplantation, comprising a copolymer represented by the following formula (I), and having a nano-sized average diameter in an aqueous solution as measured by dynamic light scattering (DLS). Formula (I): In the above formula, A is an unsubstituted or substituted C-C 12 The substituent, when substituted, is a formyl group or a group of the formula R １ R ２ represents a CH- group, where R １ and R ２ are independently C1-C4 alkoxy or R １ and R ２ together represent -OCH2CH2O-, -O(CH2)3O- or -O(CH2)4O-; L1 is a direct bond or or -(CH ２ ) ｂ S-, -CO(CH ２ ) ｂ S-, -(CH ２ ) ｂ NH-, -(CH ２ ) ｂ selected from CO-, -CO-, -OCOO-, -CONH-, where each b is independently an integer from 1 to 5; ２ -R １ L ２ - (CH ２ ) ａ -NH-(CH ２ ) ａ - or - (CH ２ ) ａ -O-(CH ２ ) ａ -, where each a is independently 0 or an integer from 1 to 5; R １ But, the formula (wherein R' is methyl), ２ is chloro, bromo or hydroxyl, ２ -R １ and R ２ The repeat units in the polymer backbone having L ２ -R １ p is an integer ranging from 2 to 100, R ２ The unit q having the formula: is absent (zero) or is in the range of an integer from 1 to 20, provided that the total number of these units is n; Z is H, SH, or S(C=S)-Ph, where Ph represents phenyl optionally substituted with 1 or 2 methyl or methoxy; m represents an integer from 2 to 10,000; and n represents an integer from 3 to 100.

6. A method for suppressing ischemia-reperfusion injury in a treated organ during surgery or transplantation, comprising the step of administering to a patient or subject in need thereof an effective amount of polymeric micelles containing a copolymer represented by the following formula (I) and having a nano-sized average diameter in an aqueous solution as measured by dynamic light scattering (DLS). Formula (I): In the above formula, A is an unsubstituted or substituted C-C 12 The substituent, when substituted, is a formyl group or a group of the formula R １ R ２ represents a CH- group, where R １ and R ２ are independently C1-C4 alkoxy or R １ and R ２ together represent -OCH2CH2O-, -O(CH2)3O- or -O(CH2)4O-; L1 is a direct bond or or -(CH ２ ) ｂ S-, -CO(CH ２ ) ｂ S-, -(CH ２ ) ｂ NH-, -(CH ２ ) ｂ selected from CO-, -CO-, -OCOO-, -CONH-, where each b is independently an integer from 1 to 5; ２ -R １ L ２ - (CH ２ ) ａ -NH-(CH ２ ) ａ - or - (CH ２ ) ａ -O-(CH ２ ) ａ -, where each a is independently 0 or an integer from 1 to 5; R １ But, the formula (wherein R' is methyl), ２ is chloro, bromo or hydroxyl, ２ -R １ and R ２ The repeat units in the polymer backbone having L ２ -R １ p is an integer ranging from 2 to 100, R ２ The unit q having the formula: is absent (zero) or is within the range of an integer from 1 to 20, provided that the total number of these units is n; Z is H, SH, or S(C=S)-Ph, where Ph represents phenyl optionally substituted with 1 or 2 methyl or methoxy groups; m represents an integer from 2 to 10,000; and n represents an integer from 3 to 100.

Images