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WO2025074996A1 - Préparation pour prévenir une lésion d'ischémie-reperfusion lors d'interventions chirurgicales ou de transplantations - Google Patents

Préparation pour prévenir une lésion d'ischémie-reperfusion lors d'interventions chirurgicales ou de transplantations Download PDF

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WO2025074996A1
WO2025074996A1 PCT/JP2024/035097 JP2024035097W WO2025074996A1 WO 2025074996 A1 WO2025074996 A1 WO 2025074996A1 JP 2024035097 W JP2024035097 W JP 2024035097W WO 2025074996 A1 WO2025074996 A1 WO 2025074996A1
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organ
integer
active ingredient
formula
ischemia
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Japanese (ja)
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一広 ▲高▼橋
幸夫 長崎
竜也 小田
朋幸 杉
愛樹 丸島
奨 坂上
欽司 古屋
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University of Tsukuba NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • A61K31/787—Polymers containing nitrogen containing heterocyclic rings having nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to a preparation for blocking or ischemic blood flow to an organ, and further to suppressing organ damage caused 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 that contains a hydrophobic polymer segment having a cyclic nitroxyl radical in the side chain and a hydrophilic poly(ethylene glycol) (PEG) segment.
  • PEG poly(ethylene glycol)
  • Non-Patent Document 1 hepatoduodenal ligament blood flow interruption
  • This process of blood flow interruption and resumption causes severe damage to the organ as "ischemia-reperfusion injury," and hepatoduodenal ligament blood flow interruption for more than 15 minutes may cause irreversible damage to the liver, so blood flow interruption for more than 15 minutes is not currently performed. Furthermore, repeated blockage of blood flow, even for periods of less than 15 minutes, can cause irreversible damage to the liver and cause severe ischemia-reperfusion injury, leading to postoperative liver failure that can be fatal.
  • Non-Patent Document 2 also see Orci LA, et al. BJS 2013;100:600-609, hereafter sometimes referred to as Non-Patent Document 3.
  • these have not been shown to be effective, and no drugs have been used in clinical applications to date.
  • Non-Patent Document 4 ischemia-reperfusion injury in the field of organ transplantation cannot be solved by antioxidants.
  • the present inventors have developed self-assembled antioxidant nanoparticles or redox nanoparticles, in particular nanoparticles formed by self-assembly in an aqueous solution from amphiphilic block copolymers containing a hydrophobic polymer segment having a cyclic nitroxyl radical (particularly TEMPO (2,2,6,6-tetramethylpiperidinooxy radical)) in the side chain and a hydrophilic poly(ethylene glycol) (PEG) segment, which are known as preparations capable of scavenging free radicals generated by ischemia or 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).
  • a hydrophobic polymer segment having a cyclic nitroxyl radical particularly TEMPO (2,2,6,6-tetramethylpiperidinooxy radical)
  • PEG poly(ethylene glycol)
  • JP 2013-166711 A discusses 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.
  • N-TEMPO-RNP polymeric micellar nanoparticles
  • O-TEMPO-RNP polymeric micellar nanoparticles
  • the blood pressure fluctuations in test animals after administration of both RNPs are observed, and O-TEMPO-RNP, which is said not to cause a decrease in blood pressure, is used as the active ingredient.
  • O-TEMPO-RNP is intravenously administered to a beagle dog that has been placed in an ischemic state 45 minutes after the ischemia, reperfusion is performed 5 minutes after administration, and the heart is removed 4 hours later to measure the size of myocardial infarction. As a result, it is disclosed that the size of myocardial infarction is significantly reduced.
  • 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, as it does not exhibit a blood pressure lowering effect.
  • N-TEMPO-RNP can be used safely and can significantly and strongly suppress ischemia-reperfusion injury in the treated organ.
  • N-TEMPO-RNP accumulated in specific and significantly increased amounts in the target organ one hour after blood flow was resumed.
  • blood collection data collected six hours later confirmed that liver damage could be significantly and strongly suppressed compared to the control. No serious side effects such as kidney damage were observed when nanoparticles were administered preoperatively in this way.
  • N-TEMPO-RNP or O-TEMPO-RNP, which has a longer blood retention time than N-TEMPO-RNP, preoperatively. Therefore, the main themes of the present invention or disclosed in this specification include the following:
  • a formulation for suppressing ischemia-reperfusion injury in a treatment 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):
  • A represents unsubstituted or substituted C 1 -C 12 alkyl, the substituents when substituted being formyl or a group of formula R 1 R 2 CH-, where R 1 and R 2 independently represent C 1 -C 4 alkoxy or R 1 and R 2 together represent -OCH 2 CH 2 O-, -O(CH 2 ) 3 O- or -O(CH 2 ) 4 O-;
  • L 1 is a direct bond or or selected from -(CH 2 ) b S-, -CO ( CH 2 ) b S-, -(CH 2 ) b NH-, -(CH 2 )
  • Topic 2 The formulation of Aspect 1, wherein the organ comprises at least one selected from the group consisting of heart, lung, kidney, liver, pancreas, and small intestine.
  • the operation is a surgical operation comprising the steps of cutting off blood flow, followed by removal of a part or all of an organ, and then reperfusing the organ, and further comprising a step of parenterally administering the active ingredient to a patient or subject before cutting off blood flow, and wherein suppression of ischemia-reperfusion injury in the treated organ means suppression to a significantly higher degree than in a case not including a step of parenterally administering the active ingredient to a patient or subject.
  • the surgery is a transplant surgery, comprising a step of parenterally administering the active ingredient to an organ donor to be transplanted, and extracting the organ to be transplanted after spontaneous cardiac arrest in the donor, and then a step of preserving or perfusing the extracted organ with an organ preservation solution containing the active ingredient as a step simulated as reperfusion, 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.
  • the surgery is a transplant surgery, comprising a step of parenterally administering the active ingredient to a brain-dead organ donor to be transplanted, causing cardiac arrest, and then circulating an organ preservation solution containing the active ingredient throughout the donor's body, followed by a step simulated as reperfusion, in which the extracted organ is preserved or perfused with the organ preservation solution containing the active ingredient, and wherein suppression of ischemia-reperfusion injury in the treated organ means a significantly higher suppression than a step in which 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.
  • the surgery is a transplant surgery, which includes a step of parenterally administering the active ingredient to a transplant recipient patient, followed by implantation of the organ to be transplanted, and a step of blood reperfusion, and wherein suppression of ischemia-reperfusion injury in the treated organ means suppression to a significantly higher degree than in a case not including a step of parenterally administering the active ingredient to the transplant recipient;
  • 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):
  • A represents unsubstituted or substituted C 1 -C 12 alkyl, the substituents when substituted being formyl or a group of formula R 1 R 2 CH-, where R 1 and R 2 independently represent C 1 -C 4 alkoxy or R 1 and R 2 together represent -OCH 2 CH 2 O-, -O(CH 2 ) 3 O- or -O(CH 2 ) 4 O-;
  • L 1 is a direct bond or or selected from -(CH 2 ) b S-, -CO ( CH 2 ) b S-, -(CH 2 ) b NH-, -(CH 2 ) b CO-, -CO-
  • 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 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):
  • A represents unsubstituted or substituted C 1 -C 12 alkyl, the substituents when substituted being formyl or a group of formula R 1 R 2 CH-, where R 1 and R 2 independently represent C 1 -C 4 alkoxy or R 1 and R 2 together represent -OCH 2 CH 2 O-, -O(CH 2 ) 3 O- or -O(CH 2 ) 4 O-;
  • L 1 is a direct bond or or selected from -(CH 2 ) b S-, -CO ( CH 2 ) b S-, -(CH 2 )
  • the preparation is administered systemically to a patient or subject parenterally before a surgical or transplant operation, and the preparation can be selectively accumulated in the organ that has been the subject of ischemia, and it is possible to significantly suppress the organ damage caused by ischemia-reperfusion compared to a control. Therefore, for example, in a surgical operation involving liver resection, it is possible to reduce postoperative liver damage and the occurrence of liver failure. Furthermore, it is possible to extend the continuous blood flow occlusion time, and it is possible to shorten the total surgery time.
  • a preparation capable of suppressing ischemia-reperfusion injury during liver resection has not been developed in actual clinical practice, and if the safety of liver resection can be improved simply by administering it intravenously, it would be a major innovation in the history of liver surgery that has been running for more than 130 years.
  • the preparation may lead to improved graft function and survival due to its effect of suppressing ischemia-reperfusion injury.
  • a preparation capable of suppressing unavoidable ischemia-reperfusion injury has not been developed in the past. If it were possible to increase the success rate of transplant surgery simply by administering the drug 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 70-year-plus history of transplant surgery.
  • FIG. 1 is a schematic diagram showing the schedule of animal experiments in Test Example 1.
  • 1 is a graph showing the results of biochemical evaluation part 1 in Test Example 1.
  • 1 is a graph showing the results of biochemical evaluation part 2 in Test Example 1.
  • 1 is a black-and-white photograph, instead of a microscope image, of liver tissue in histological evaluation part 1 in Test Example 1, taken after hematoxyline-eosin staining.
  • 1 is a black-and-white photograph, instead of a micrograph, taken after TUNEL staining of liver tissue cells in histological evaluation part 2 in Test Example 1.
  • 1 is a graph showing the results of histological evaluation part 2 in Test Example 1.
  • FIG. 1 is a schematic diagram showing the schedule of animal experiments in Test Example 2.
  • FIG. 1 is a photograph in place of a fluorescent microscope image of liver tissue taken after treatment in Test Example 2.
  • FIG. 1 is a schematic diagram showing the schedule of animal experiments in Test Example 3.
  • 1 is a graph showing the measurement results of biological indicators when refrigerated storage was performed in biological evaluation part 1 of Test Example 3.
  • 1 is a graph showing the measurement results of biological indicators when thermal preservation was performed in biological evaluation part 1 of Test Example 3.
  • 1 is a graph showing the measurement results of biological indicators when refrigerated storage was performed in biological evaluation part 2 of Test Example 3.
  • 13 is a graph showing the measurement results of biological indicators when thermal preservation was performed in biological evaluation part 2 of Test Example 3.
  • 13 is a graph showing the measurement results of biological indicators when refrigerated storage was performed in biological evaluation part 3 of Test Example 3.
  • FIG. 13 is a graph showing the measurement results of biological indicators when thermal preservation was performed in biological evaluation part 3 of Test Example 3.
  • 1 is a black-and-white photograph, instead of a microscope image, of liver tissue sampled during cold storage in the histological evaluation of Test Example 3, taken after staining with Hematoxyline-Eosin.
  • 1 is a black-and-white photograph, instead of a microscope image, of liver tissue sampled during thermal preservation in the histological evaluation of Test Example 3, taken after staining with Hematoxyline-Eosin.
  • FIG. 1 is a schematic diagram showing the schedule of animal experiments in Test Example 4.
  • 1 is a graph showing the measurement results of biological indicators up to the time when heart rate resumed after heart transplantation in physiological evaluation part 1 of Test Example 4.
  • 1 is a graph showing the measurement results of biological indicators of cardiac output strength in physiological evaluation part 1 of Test Example 4.
  • 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 standard values in healthy individuals, or the histological evaluation of the organ declines. However, in a broader sense, it is not limited to these, and refers to some kind of damage to the organ that is the subject of surgery or to the surrounding organs, organs, or tissues caused by the surgery.
  • a "patient or subject” is a person from whom at least a part of an organ is to be removed, extracted, or transplanted; in the case of transplant surgery, this may be either the organ donor or the transplant recipient.
  • 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 is no limitation on the form or type of means employed.
  • transplant surgery such cutting off of blood flow is performed in organ donor patients before the transplant target organ is extracted or harvested, and in transplant recipients before the organ corresponding to the transplanted organ is harvested.
  • transplant recipients 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 a state of occlusion is also included in the above-mentioned cutting off of blood flow.
  • the patient Before liver transection begins, the patient is intravenously administered a fixed dose of polymeric micelles containing a copolymer represented by formula (I) and having a nano-sized average diameter in an aqueous solution as measured by dynamic light scattering (DLS), or polymeric micelles having a nano-sized average diameter formed from a copolymer represented by formula (I) (these polymeric micelles are sometimes abbreviated as "nanoparticles”), or a formulation containing nanoparticles as an active ingredient.
  • the patient is allowed to wait several minutes, for example about 3 minutes, until the nanoparticles are distributed throughout the body, and then the hepatoduodenal ligament blood flow is blocked to begin liver transection.
  • Organ removal from donors in a cardiac arrest state (heart, lungs, liver, kidneys, pancreas, small intestine)
  • 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.
  • a fixed dose e.g. 90 mg/kg
  • 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, and the antioxidant nanoparticle formulation is filled into the graft.
  • organ preservation solution e.g., 1.0 mg/mL
  • 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.
  • a fixed dose e.g. 90 mg/kg
  • 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.
  • organ preservation solution e.g., 1.0 mg/mL
  • Each transplant organ is removed and manually perfused with the organ preservation solution containing the self-assembling antioxidant nanoparticle formulation to fill the graft with the antioxidant nanoparticle formulation.
  • simple cooling preservation or machine perfusion preservation is performed with the preservation solution containing the antioxidant nanoparticle formulation.
  • the nanoparticle formulation (e.g., 90 mg/kg) is administered intravenously or intraportally to the recipient patient, and the patient is allowed to wait for several minutes, e.g., about 3 minutes, until the nanoparticles are distributed throughout the body. Then, each graft is implanted and blood is reperfused.
  • the nanoparticles include or consist 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-size is in the order of nanometers, and may be in the range of 5 nm to 500 nm, 10 nm to 300 nm, 10 nm to 100 nm, or 10 nm to 60 nm, but is not limited thereto.
  • L 2 is -(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 nano-size refers to the average diameter when the nanoparticles are measured by dynamic light scattering (DLS) in an aqueous solution.
  • 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.
  • C 1 -C 12 alkyl includes straight chain, branched or cyclic alkyl having 1 to 12 carbon atoms, typically including, but 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, etc.
  • the nanoparticles thus formed collapse upon being protonated in a low pH environment, and therefore typically accumulate specifically in the liver where blood flow has been blocked, which is in a low pH environment, and in some cases, in the surrounding organs or organs, or in transplanted organs, and collapse in situ to expose cyclic nitroxide radicals that exert a redox effect, which are understood to be effective in removing the "bad" active oxygen involved in the injury caused by ischemia and the subsequent reperfusion of blood flow.
  • nanoparticles equivalent to O-TEMPO-RNP in which the -NH- portion is -O-, have a half-life several times longer than N-TEMPO-RNP, and can remain or remain in the blood circulation or storage solution for a longer period of time, according to pharmacokinetic studies. Therefore, it is presumed that either N-TEMPO-RNP or O-TEMPO-RNP nanoparticles will act effectively depending on the type or circumstances of the surgery or transplant operation.
  • the nanoparticles formed as described above can be dissolved or uniformly dispersed in an aqueous medium (an aqueous solution that may contain physiological salt or a pH adjuster, if necessary), and can be reconstituted with water, so that they can be provided as a preparation in various forms. Therefore, for example, they can be made into parenteral preparations in various forms, including parenteral preparations (including intravenous injections, subcutaneous administration agents, etc.). Such parenteral preparations can contain a typical pharma- ceutically acceptable medium, such as water, glycol, sucrose, lactose, etc., as long as it does not adversely affect the stability of the nanoparticles in the aqueous medium.
  • 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, but the dose can be determined by a specialist based on data obtained through small-scale clinical trials, etc.
  • 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.
  • the nanoparticles may be effective over a wide range of dosages, so that, for example, in the treatment of an adult, the dosage may be about 50 to about 1000 mg per kg of body weight or about 100 to about 500 mg per kg of body weight.
  • the nanoparticles used in the following examples are copolymers produced by the method described in Patent Document 2, and are formed, for example, as follows from a block copolymer (PEG-b-PMNT) represented by formula (I), in which A is methyl, 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.
  • PEG-b-PMNT block copolymer represented by formula (I), in which A is methyl, 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
  • PEG-b-PMNT was dissolved in dimethylformamide (DMF) at a concentration of 150 mg/mL, 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 collected after 24 hours. PBS with a 10-fold concentration was added to the solution in the dialysis membrane so that the final concentration was 1x PBS concentration.
  • the particle size and particle size distribution were measured using dynamic light scattering (DLS), and the particle size (Z-Ave) was 26.5 nm, the polydispersity index (PDI) was 0.12, and the particle size distribution was uniform.
  • DFS dynamic light scattering
  • Biochemical evaluation part 1 As a biochemical evaluation in Test Example 1, the 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, which is the case where the preparation was not used.
  • 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. As can be seen from Figure 3, in the cases where the preparation of the present invention was used, a significant decrease in TBRS, an oxidative stress marker, was observed compared to the control where no preparation was used.
  • Histological evaluation 1 was performed by staining the resected liver tissue 6 and 24 hours after the operation in Test Example 1 with hematoxylin-eosin. Black and white photographs taken after staining, instead of microscopic images, are shown in FIG. 4. The original microscopic photographs in this organ transplant are color images. In order to obtain clearer information, we are prepared to submit the color images if necessary.
  • FIG. 4 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. In addition, in the control group, extensive necrosis was observed after 24 hours of ischemia, but the area of necrosis was small in the RNP-administered group.
  • Histological evaluation part 2 The second histological evaluation was performed by TUNEL staining to evaluate apoptosis of the resected hepatocytes 6 and 24 hours after the operation in Test Example 1.
  • the black and white photographs taken after TUNEL staining are shown in FIG. 5, and the percentage (%) of the TUNEL positive area is shown in FIG. 6.
  • the original microscopic photographs of the organ transplant are color images. If necessary, we are prepared to submit the color images to obtain clearer information. In the cases where this preparation was used (RNP group), the TUNEL staining positive area was reduced 6 and 24 hours after the operation compared to the cases where this preparation was not used (control group). These results show that cell death was suppressed by administration of RNP.
  • Test Example 2 According to the schematic diagram of the schedule of the animal experiment shown in FIG. 7, experimental animals were administered RNP (90 mg/kg) and divided into an IR- group in which the hepatoduodenal ligament blood flow was not blocked, and an IR+ group in which the hepatoduodenal ligament blood flow was blocked. Fluorescent microscopic images of liver tissue were taken one hour after the end of the blockage to evaluate the accumulation of RNP in the liver. A black and white photograph instead of the fluorescent photograph is shown in FIG. 8. The original of the microscope image is a color image. If necessary, we are prepared to submit the color image to obtain clearer information.
  • organ preservation solution Wisconsin solution, hereafter abbreviated as UW solution
  • the liver was immediately removed, and the former was simply cooled and preserved at 4°C in UW solution without RNP added, and the latter was simply heated and preserved at 37°C in UW solution (1.0 mg/ml) with RNP added. After preservation, biochemical and histological evaluations were performed on the preservation solution and the preserved liver.
  • Biochemical evaluation part 2 In the second biochemical evaluation, the concentration of glutathione (hereinafter abbreviated as GSH) contained in liver tissue collected 6, 12, and 24 hours after the start of storage in the cold storage of Test Example 3 was measured. The results are shown in FIG. 12. Since GSH exhibits antioxidant activity by reducing reactive oxygen species, a low GSH level can be interpreted as a strong oxidative stress. From FIG. 12, the control group to which the preparation was not added showed a decrease in GSH, an antioxidant, compared to the case to which the preparation of the present invention was added. Next, the concentration of GSH contained in liver tissue collected 6 and 12 hours after the start of storage in the warm storage of Test Example 3 was measured. The results are shown in FIG. 13. From FIG.
  • the control to which the preparation was not added showed a tendency for GSH, an antioxidant, to decrease after 6 hours of storage, compared to the case to which the preparation 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 oxidative stress of preserved organs was reduced by adding RNP to the preservation solution during cold or warm preservation.
  • dROM Diacron-Reactive Oxygen Metabolites, a derivative (R-OOH) of
  • the histological evaluation was carried out by staining the liver tissue collected 6 and 24 hours after the start of cold storage in Test Example 3 with Hematoxyline-Eosin. Black-and-white photographs in place of the microscope images taken after staining are shown in FIG. 16. The original microscope photographs in the organ transplantation are color images. In order to obtain clearer information, the color images are available if necessary. From FIG. 16, the morphology of the liver tissue was maintained in both the control group and the RNP-added group 6 hours after storage, but after 24 hours, the liver cells in the control group were destroyed and the sinusoids were enlarged, whereas in the RNP group, the destruction of the liver cells was small and the sinusoids were hardly enlarged.
  • the histological evaluation in the warm storage was carried out by staining the liver tissue collected 3 and 12 hours after the start of storage with Hematoxyline-Eosin. Black-and-white photographs in place of the microscope images taken after staining are shown in FIG. 17.
  • the original microscope photographs in the organ transplantation are color images. In order to obtain clearer information, we are prepared to submit color images if necessary.
  • hepatocytes began to decompose and the sinusoids expanded from 3 hours after storage, whereas in the RNP group, the morphology of liver tissue was maintained.
  • Fig. 18 shows a schematic diagram of the schedule of the animal experiment.
  • Donor experimental animals C57BL/6 mice
  • the control group was perfused with 4 ml of organ preservation solution (UW solution)
  • the heart graft was removed, and the excised heart graft was washed with 4 ml of UW solution.
  • the RNP group was perfused with 4 ml of RNP-added UW solution (1 mg/ml), the heart graft was removed, and the excised heart graft was washed with 4 ml of RNP-added UW solution. Subsequently, the heart grafts in the control group were cold-preserved in UW solution at 4°C for 24 hours, while the RNP group was cold-preserved in UW solution containing RNP (1 mg/dl) for 24 hours at 4°C.
  • the recipients in the control group were intravenously administered saline, and the recipients in the RNP group were intravenously administered RNP (90 mg/kg), and then the heart grafts were intraperitoneally transplanted.
  • Physiological evaluations were performed on the recipients and heart grafts in each group.
  • Physiological evaluation part 1 The time from resumption of blood flow to resumption of heartbeat in each group of grafted heart pieces is shown in Figure 19.
  • the preparation of the present invention was used (RNP group)
  • the time until resumption of heartbeat was shortened compared to the control group not using the preparation.
  • the heartbeat intensity at the time of resumption of heartbeat after blood perfusion and 6 hours after surgery in each group is shown in Figure 20 according to the Stanford Cardiac Surgery Laboratory Graft Scoring System (maximum 4 points).
  • an increase in heartbeat intensity was observed at each time compared to the control not using the preparation. From the above, it can be seen that the heartbeat of the heart graft after transplantation is more advantageous in terms of time and intensity in the case where the preparation of the present invention is used than in the case where the preparation of the present invention is not used.

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  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Urology & Nephrology (AREA)
  • Vascular Medicine (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Epidemiology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

Est divulguée une préparation pour prévenir efficacement une lésion d'ischémie-reperfusion lors d'interventions chirurgicales ou de transplantations. Une micelle polymère comprenant un copolymère et présentant un diamètre moyen nanométrique tel que déterminé par une mesure de diffusion dynamique de lumière (DLS) dans une solution aqueuse est utilisée en tant que principe actif dans la préparation, le copolymère étant caractérisé en ce qu'il est représenté par la formule (I) dans laquelle, parmi des principaux groupes ou fractions variables, L2 dans L2-R1 représente -(CH2)a-NH-(CH2)a- ou -(CH2)a-O-(CH2)a-, les a représentant chacun indépendamment un nombre entier de 0 ou de 1 à 5, et R1 représente un radical nitroxyde cyclique. En principe, la micelle polymère est administrée par voie parentérale à un patient ou à un donneur d'organe avant une exentération d'organe lors d'une intervention chirurgicale ou d'une transplantation. Dans le cas d'une transplantation, un organe retiré est stocké dans une solution de conservation contenant la micelle polymère ou est perfusé avec la solution de conservation. En variante, dans le cas d'une transplantation, la micelle polymère est administrée par voie parentérale à un patient cible de transplantation. Par conséquent, les lésions induites par ischémie-reperfusion d'un organe qui est une cible pour la procédure de l'intervention sont inhibées.
PCT/JP2024/035097 2023-10-02 2024-10-01 Préparation pour prévenir une lésion d'ischémie-reperfusion lors d'interventions chirurgicales ou de transplantations Pending WO2025074996A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013166711A (ja) * 2012-02-14 2013-08-29 Univ Of Tsukuba 高分子化ニトロキシドラジカル化合物を含有する虚血再還流障害の処置剤
WO2023068362A1 (fr) * 2021-10-22 2023-04-27 ＣｒｅｓｔｅｃＢｉｏ株式会社 Utilisation de nanoparticules redox pour le traitement de cellules

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013166711A (ja) * 2012-02-14 2013-08-29 Univ Of Tsukuba 高分子化ニトロキシドラジカル化合物を含有する虚血再還流障害の処置剤
WO2023068362A1 (fr) * 2021-10-22 2023-04-27 ＣｒｅｓｔｅｃＢｉｏ株式会社 Utilisation de nanoparticules redox pour le traitement de cellules

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FUKAI HARA, HAGA SANAE, OZAKI MICHITAKA: "Signal transduction mechanisms of ischemia-reperfusion injury in liver transplantation", JAPANESE JOURNAL OF CLINICAL CHEMISTRY, vol. 37, no. 2, 30 April 2008 (2008-04-30), JP , pages 131 - 140, XP093299877, ISSN: 0370-5633, DOI: 10.14921/jscc1971b.37.2_131 *
NAGASAKI YUKIO: "Designing nanomedicine to combat ischemia-reperfusion injury", DRUG DELIVERY SYSTEM, vol. 30, no. 4, 25 September 2015 (2015-09-25), JP, pages 327 - 335, XP093299873, ISSN: 0913-5006, DOI: 10.2745/dds.30.327 *

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