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WO2018151243A1 - Agent protecteur de globules rouges - Google Patents

Agent protecteur de globules rouges Download PDF

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WO2018151243A1
WO2018151243A1 PCT/JP2018/005391 JP2018005391W WO2018151243A1 WO 2018151243 A1 WO2018151243 A1 WO 2018151243A1 JP 2018005391 W JP2018005391 W JP 2018005391W WO 2018151243 A1 WO2018151243 A1 WO 2018151243A1
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erythrocyte
hrg
solution
red blood
blood cell
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Japanese (ja)
Inventor
正洋 西堀
秀徳 和氣
輝 衷
秀治 森
阪口　政清
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Okayama University NUC
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Okayama University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/18—Erythrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics

Definitions

  • the present invention relates to a red blood cell protective agent. More specifically, the present invention relates to a erythrocyte protective agent containing a histidine-rich glycoprotein.
  • Blood products for transfusion include erythrocyte products, plasma products, platelet products, and whole blood products.
  • the red blood cell preparation is obtained by removing most of plasma, white blood cells and platelets from blood.
  • erythrocytes (Red ⁇ ⁇ blood ⁇ ⁇ cell: RBC) destroy the erythrocyte membrane, resulting in so-called hemolysis in which hemoglobin is released.
  • ACD solution containing acid-sodium citrate-dextrose and CPD solution containing sodium citrate-sodium phosphate-dextrose have been used for a long time. Has been used.
  • adenine has recently been added to the preservation solution.
  • the storage temperature is 2 to 3 ° C.
  • the effective period is 3 weeks after blood collection, and it is said that it can be extended to 6 weeks depending on the conditions.
  • metabolic waste ie, lactic acid and hydrogen ions
  • AS-1 Adsol (trade name)
  • AS-3 Nutricel (trade name)
  • AS-5 Optosol
  • Erythro-sol trade name
  • AS-2 AS-2 and AS-3
  • AS-5 saline
  • adenine adenine
  • AS-5 a small amount of citric acid and / or mannitol
  • a composition and method for the storage of red blood cells comprising adenine, dextrose, at least one non-metabolizable membrane protective sugar, and a pH buffer system (Patent Document 1).
  • Histidine-rich glycoprotein is a plasma protein having a molecular weight of about 80 kDa, identified in 1972 by Heimburger et al (1972).
  • HRG is known to be involved in the regulation of the coagulation / fibrinolysis system and the control of angiogenesis (Non-patent Document 1).
  • Patent Document 2 methods for inhibiting angiogenesis by administering HRG polypeptides, HRG polypeptides, antibodies and receptors that bind to HRG polypeptides, HRG-deficient plasma and polynucleotides, vectors and host cells encoding HRG polypeptides
  • Patent Document 3 a pharmaceutical composition and a product are disclosed.
  • Patent Document 4 a neutrophil-vascular endothelial cell adhesion inhibitor containing HRG as an active ingredient is also disclosed.
  • An object of the present invention is to provide an erythrocyte protective agent that can store an erythrocyte-containing solution, for example, an erythrocyte concentrate for a longer period of time and can be used more safely.
  • the inventors of the present application pay attention to the fact that phosphatidylserine (PS) is expressed on the erythrocyte membrane surface when erythrocytes are stored for a long period of time, and that vascular endothelial cells and collagen fibers of erythrocytes adhere from the expression of PS.
  • PS phosphatidylserine
  • this invention consists of the following. 1. A erythrocyte protective agent containing HRG as an active ingredient. 2. 2. The erythrocyte protective agent according to item 1 above, wherein the protection of erythrocytes suppresses the expression of phosphatidylserine on the erythrocyte membrane surface by HRG. 3. 2. The erythrocyte protective agent according to item 1 above, wherein the protection of erythrocytes reduces the calcium ion concentration in erythrocytes. 4). 2. The erythrocyte protective agent according to item 1 above, wherein the protection of erythrocytes suppresses the release of antioxidant enzymes in erythrocytes. 5).
  • 7. A method for stabilizing an erythrocyte-containing solution, which comprises adding the stabilizer according to item 5 or 6 to the erythrocyte-containing solution.
  • 8). A phosphatidylserine expression inhibitor on the surface of erythrocyte membrane, containing HRG as an active ingredient.
  • erythrocyte protective agent of the present invention erythrocyte deterioration due to erythrocyte storage can be suppressed.
  • Red blood cells can be protected more effectively by adding HRG as an active ingredient to existing blood storage solution, red blood cell storage solution, or blood development solution and red blood cell storage solution that will be developed in the future. .
  • FIG. 5 is a diagram showing the results of confirming the action of HRG or HSA added simultaneously on the erythrocyte membrane surface PS induced by Zn 2+ . It is a figure which shows the result observed that HRG suppresses PS expression more strongly compared with HSA.
  • FIG. 6 is a result of confirming the subsequent action of HRG or HSA on the erythrocyte membrane surface PS whose expression was induced in advance by Zn 2+ . It is a figure which shows the result observed that HRG suppresses PS expression more strongly compared with HSA. (Example 6) It is the result of confirming the ability to inhibit adhesion of unstimulated erythrocytes to collagen I by HRG.
  • FIG. 4 is a diagram showing the results of examining the adhesion ability to vascular endothelial cells by stimulating erythrocytes with Zn 2+ in advance and then adding HRG. The results show that the adhesion of erythrocytes to vascular endothelial cells was suppressed depending on the HRG concentration.
  • Example 8 It is a figure which shows the result of having confirmed the influence which it has on the erythrocyte membrane surface PS positive rate when HSA is added to the liquid for erythrocyte storage and it preserve
  • Example 9 It is a figure which shows the PS expression confirmation result of the CLP mouse
  • FIG. 10A shows the result that the amount of calcium in red blood cells increased in the group not added with HRG, but the free calcium concentration decreased in the group added with HRG.
  • FIG. 10B shows the inhibitory effect of HRG on the aggregation of red blood cells by Zn 2+ stimulation and the adhesion of the aggregated red blood cells to vascular endothelial cells.
  • Example 12 It is a figure which shows the result of having confirmed peroxiredoxin 2 (Prx2) release
  • FIG. 11A shows that Prx2 in the supernatant increases in a Zn 2+ concentration-dependent manner, and FIG.
  • FIG. 14A shows the Western blot of free hemoglobin and its quantification results
  • FIG. 14B shows the percentage of PS positive erythrocytes immediately after blood collection and after 4 hours of blood incubation
  • FIG. 14C shows the concentration of free calcium in erythrocytes after 4 hours of incubation
  • 14D shows the Zn content of the organ tissue.
  • the present invention relates to a erythrocyte protective agent, and more particularly to a erythrocyte protective agent containing histidine-rich glycoprotein (HRG).
  • HRG histidine-rich glycoprotein
  • the “red blood cell protective agent” is used to prevent red blood cells from being deteriorated in vitro, such as when red blood cells contained in blood or red blood cells separated from blood are suspended in a preservation solution or the like.
  • the erythrocyte protective agent of the present invention can contain a pharmacologically acceptable carrier.
  • the pharmaceutically acceptable carrier include excipients, disintegrants or disintegration aids, binders, lubricants, coating agents, dyes, diluents, bases, solubilizers or solubilizers, Examples include isotonic agents, pH adjusters, stabilizers, propellants, and pressure-sensitive adhesives.
  • the “blood storage solution” is a solution used for storing blood, for example, a storage solution used for blood products for blood transfusion, specifically, red blood cell preparations, plasma preparations, platelets. This refers to a preservative solution used for preparations and whole blood preparations.
  • the “blood storage solution” may be a storage solution known per se or any storage solution developed in the future.
  • an ACD solution or a CPD solution may be used.
  • it may be an erythrocyte storage additive solution (MAP solution) containing mannitol, adenine and phosphate, or a mixture of MAP solution and ACD solution or CPD solution.
  • MAP solution erythrocyte storage additive solution
  • the composition of the MAP solution include D-mannitol, adenine, crystalline sodium dihydrogen phosphate, sodium citrate, citric acid, butter sugar, sodium chloride and the like.
  • the “red blood cell storage solution” is a solution used for storing red blood cells, for example, a storage solution used for blood products for blood transfusion, specifically, storage used for red blood cell products and the like. Refers to working solution.
  • the “erythrocyte storage solution” may be a storage solution known per se or any storage solution developed in the future. Specifically, an ACD solution, a CPD solution, or a MAP solution may be used, or an ACD solution or a CPD solution may be mixed with a MAP solution.
  • the “erythrocyte-containing solution” is not particularly limited as long as it is a solution contained in a solution capable of storing red blood cells.
  • the blood storage solution described above contains red blood cells.
  • An example of an erythrocyte-containing solution is a erythrocyte concentrate which is an erythrocyte preparation.
  • the erythrocyte concentrate is a dark red solution containing a small amount of CPD solution mixed with about 46 mL of MAP solution in a red blood cell layer from which most of leukocytes and plasma have been removed from 200 mL of human blood mixed with 28 mL of CPD solution. It is. In the examples described later, it may be referred to as an erythrocyte solution sample.
  • the “stabilizer for erythrocyte-containing solution” refers to a erythrocyte-containing solution such as a erythrocyte concentrate, which is used for preventing and stabilizing red blood cells.
  • the stabilizer for erythrocyte-containing solution of the present invention can be prepared by adding HRG to a erythrocyte storage solution such as MAP solution.
  • a solution containing HRG of 10 to 100 ⁇ g / mL, preferably 50 to 100 ⁇ g / mL, more preferably 100 ⁇ g / mL to the erythrocyte storage solution is suitable.
  • the red blood cell storage solution that can be used as the stabilizer for the red blood cell-containing solution of the present invention is not limited to the MAP solution.
  • the erythrocyte-containing solution stabilizer of the present invention may contain a pharmacologically acceptable carrier.
  • the pharmaceutically acceptable carrier include excipients, disintegrants or disintegration aids, binders, lubricants, coating agents, dyes, diluents, bases, solubilizers or solubilizers, Examples include isotonic agents, pH adjusters, stabilizers, propellants, and pressure-sensitive adhesives.
  • “HRG” as an active ingredient such as a erythrocyte protective agent and a erythrocyte-containing solution stabilizer of the present invention is a method of isolation and purification from biological components, a method of preparation using genetic recombination technology, or a method of synthesis. can do. For example, it can be purified and / or isolated from biological components such as blood such as plasma and serum, spinal fluid and lymph. Suitable biological components are blood components such as plasma and serum.
  • a method for isolation / purification from a biological component a method known per se or any method developed in the future can be applied. For example, it can be prepared by passing plasma through an affinity column prepared using Ni-NTA (nickel-nitrilotriacetic acid) agarose resin.
  • a full-length cDNA encoding HRG or a cDNA encoding a portion having HRG activity can be cloned into an expression vector and prepared.
  • it may be a protein biosynthesized from all or part of the nucleotide specified by GenBank Accession No. NM000412.
  • a full-length cDNA encoding the amino acid sequence of mature HRG (SEQ ID NO: 1) or a cDNA encoding a portion thereof can be cloned into an expression vector and prepared.
  • the HRG as the active ingredient of the present invention may be the entire HRG protein, or a partial protein or peptide having HRG activity. Furthermore, it may contain a sugar chain or a sugar chain may not be added.
  • ⁇ Mature HRG is composed of four main regions: cystatin-like regions 1, 2, His / Pro region, and C-terminal region after being cleaved from the signal peptide by proteolytic enzymes.
  • the His / Pro region is very rich in proline and histidine residues, including, for example, about 12 tandem repeats in which the pentapeptide GHHPH (SEQ ID NO: 2) is conserved in the human form.
  • it is specified by the amino acid sequence shown at positions 330 to 389 of the amino acid sequence specified by SEQ ID NO: 1.
  • HRG used in the erythrocyte protective agent of the present invention has an action of suppressing the expression of phosphatidylserine (PS) on the surface of the erythrocyte membrane.
  • PS on the surface of the erythrocyte membrane is stimulated by erythrocytes stored for a long period of time or under energy depletion conditions, or by Prostaglandin E 2 (PGE 2 ), Platelet activating factor (PAF), retinoic acid or certain drugs. It is said that it will appear when It has also been suggested that vascular endothelial cells are damaged by the interaction between erythrocytes expressing PS and vascular endothelial cells.
  • the present invention extends to a PS expression inhibitor on the erythrocyte membrane surface containing HRG as an active ingredient, and also to a method for protecting red blood cells, which comprises adding HRG to a red blood cell-containing solution. Furthermore, the present invention extends to a method for suppressing PS expression on the surface of an erythrocyte membrane, which comprises adding HRG to an erythrocyte-containing solution.
  • the HRG used for the erythrocyte protective agent of the present invention further has an action of lowering the intracellular calcium concentration and suppressing the release of important enzymes in the cell.
  • An important enzyme in the cell is an antioxidant enzyme, specifically peroxidase, and more specifically peroxiredoxin (Prx). Prx is an important antioxidant enzyme in maintaining the physiological function of cells because it has a strong antioxidant action and exists in large amounts in cells.
  • the present invention also extends to a method for stabilizing erythrocytes, which comprises adding the “erythrocyte protective agent” or “erythrocyte-containing solution stabilizer” of the present invention to an erythrocyte-containing solution. That is, the present invention extends to a stabilization method using a erythrocyte protective agent containing HRG as an active ingredient or a erythrocyte-containing solution stabilizer containing HRG as an active ingredient.
  • a stabilization method using a erythrocyte protective agent containing HRG as an active ingredient or a erythrocyte-containing solution stabilizer containing HRG as an active ingredient.
  • PS is a phospholipid component, and is usually held on the inner leaf (cytoplasmic side) of the cell membrane by an enzyme called flippase.
  • flippase an enzyme that causes apoptosis.
  • PS becomes exposed on the cell surface.
  • erythrocytes when nuclei are separated from reticulocytes, the nuclei are said to expose PS on the cell surface.
  • PS is a lipid expressed on the surface of apoptotic cells and is known to be exposed on the cell membrane only in dead cells.
  • PS is thought to be exposed on the cell surface during the process of DNA degradation during apoptosis and nucleation of erythrocytes, so it was considered that PS is also exposed on the erythrocyte surface during the process of erythrocyte decay.
  • the solution for storing red blood cells containing HRG is the red blood cell contained in the red blood cell-containing solution. It can be used as a protective agent.
  • Example 1 Purification of human plasma-derived histidine-rich glycoprotein (HRG)
  • HRG human plasma-derived histidine-rich glycoprotein
  • human plasma-derived HRG was purified. Using human plasma (240 mL) as a starting material, HRG protein was purified using Ni-NTA affinity chromatography and high performance liquid chromatography (anion exchange column (monodisperse hydrophilic polymer beads: Mono Q)). A purified HRG sample was obtained with a molecular weight of about 80 kDa.
  • Example 2 Production of recombinant human HRG The recombinant human HRG was cultured in 10% FCS-containing GIBCO (R) Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12). Cells (Chinese Hamster Ovary cells) were co-transfected with an HRG expression vector, a transposase expression vector, and a drug resistance gene expression vector at a DNA amount of 5: 4: 1 using FuGENE (trademark) -HD (gene introduction reagent). After the gene was introduced and cultured for 48 hours, 10 ⁇ g / mL Puromycin (antibiotic) was added, and drug selective culture was performed for 3 weeks while changing the medium once every 3 days.
  • the culture supernatant containing recombinant human HRG was collected.
  • QIAGEN (R) Ni-NTA agarose gel gel with Ni-NTA bound to Sepharose CL-6B support ) pre-washed with 30 mL of PBS (-) was added to the culture supernatant and rotated at 4 ° C for 2 hours. and it was bound recombinant human HRG in QIAGEN (R) Ni-NTA agarose gel.
  • Example 3 Protective agent for erythrocyte concentrate per 50 mL of D-mannitol (728.5 mg), adenine (7.0 mg), sodium dihydrogen phosphate (47.0 mg), sodium citrate hydrate (75.0 mg), HRG and human serum albumin (HSA) prepared from human plasma in a commercially available erythrocyte preservation solution MAP solution (MAP solution bag) containing acid hydrate (10.0 mg), glucose (360.5 mg) and sodium chloride (248.5 mg) Were added so that the final concentration would be 100 ⁇ g / mL, respectively, and used as a protective agent for erythrocyte-containing solutions.
  • MAP solution bag containing acid hydrate (10.0 mg), glucose (360.5 mg) and sodium chloride (248.5 mg)
  • the expression rate of PS on the erythrocyte surface when Zn 2+ was added to the prepared erythrocyte solution sample was measured. After adding Zn 2+ to 0, 5, 10, 15 and 20 ⁇ M and incubating at 37 ° C. for 1 hour, the PS expression level on the surface of erythrocytes was measured.
  • the expression level of PS was determined by adding 4 ⁇ L of FITC-labeled annexin V reaction solution (manufactured by Funakoshi) to 400 ⁇ L of erythrocyte solution sample, allowing to stand at room temperature for 15 minutes, adding 400 ⁇ L of 4% paraformaldehyde (PFA), fixing, and performing FACS analysis. These operations were performed according to the instruction manual of the FITC-labeled annexin V assay kit (Funakoshi).
  • Example 4 Action of HRG on PS on erythrocytes induced to induce expression by metal ions Only HBSS (Hank's Balanced Salt Solutions) or ZnCl 2 (20 ⁇ M) in HBSS was prepared for the erythrocyte solution sample prepared in the same manner as in Reference Example 1. , MgCl 2 (20 ⁇ M), CaCl 2 (20 ⁇ M), ZnCl 2 (20 ⁇ M) + HRG (100 ⁇ g / mL), ZnCl 2 (20 ⁇ M) + HSA (100 ⁇ g / mL), HRG alone (100 ⁇ g / mL) or HSA alone (100 ⁇ g / mL) was incubated at 37 ° C. for 1 hour, and then the PS expression level on the erythrocyte surface was measured. The PS expression level was measured by the same method as in Reference Example 1.
  • PS was expressed on the erythrocyte surface by the addition of Zn 2+ , but suppression of PS expression was observed by the addition of HRG or a system containing HSA (FIG. 2).
  • Example 6 Action on PS on erythrocytes pre-induced in expression by Zn 2+
  • ZnCl 2 (20 ⁇ M) was added to the erythrocyte solution sample prepared in the same manner as in Reference Example 1 and incubated at 37 ° C. for 1 hour. Red blood cells treated with ZnCl 2 (20 ⁇ M) were designated as RBC (Z).
  • RBC Red blood cells treated with ZnCl 2 (20 ⁇ M) were designated as RBC (Z).
  • PBS was added instead of ZnCl 2 (20 ⁇ M) and incubated at 37 ° C. for 1 hour (RBC (s)).
  • each concentration of HRG or HSA was added and incubated at 37 ° C. for 15 minutes.
  • the PS expression level on the erythrocyte surface was measured by the same method as in Reference Example 1.
  • Example 7 Inhibition of adhesion of erythrocytes to collagen I by HRG
  • HRG Inhibition of adhesion of erythrocytes to collagen I was confirmed.
  • Red blood cell solution sample stored in PBS containing the above HRG (1, 10, 100 ⁇ g / mL) on a commercially available cell culture plate coated with collagen I (Corning Biocoat Collagen I Cellware) (Red blood cell count: 4 ⁇ 10 6 / mL) ) And seeded at 37 ° C. for 30 minutes.
  • a control PBS was seeded with erythrocytes stored only in PBS without HRG. As a result, it was observed that red blood cells were inhibited from adhering to collagen I depending on the HRG concentration (FIG. 5).
  • Example 8 Inhibition of adhesion of erythrocytes to collagen I by HRG
  • a commercial cell culture plate coated with collagen I (Corning Biocoat Collagen I Cellware) was used in the same manner as in Example 6 or 7.
  • a erythrocyte solution sample (erythrocyte count: 4 ⁇ 10 6 / mL) stimulated with zinc in advance was seeded under the dependence of HRG (1, 10, 100 ⁇ g / mL), and cultured at 37 ° C. for 30 minutes. Thereafter, the number of red blood cells adhered to the plate was measured by the same method as in Example 7.
  • erythrocytes treated with PBS by the same method as in Example 6 were designated as RBC (s), and erythrocytes treated with PBS containing various concentrations of ZnCl 2 were designated as RBC (Z).
  • RBC erythrocytes treated with PBS containing various concentrations of ZnCl 2
  • erythrocytes treated with PBS containing various concentrations of ZnCl 2 were designated as RBC (Z).
  • Example 9 Confirmation of erythrocyte protecting action of erythrocyte storage solution
  • the erythrocyte protecting action of HRG against an erythrocyte solution sample was confirmed.
  • the red blood cell solution sample red blood cell count: 8 ⁇ 10 9 / mL
  • the proportion of PS-positive erythrocytes was calculated by FACS.
  • the proportion of PS-positive erythrocytes gradually increased according to the number of storage days, but it was confirmed that the PS-positive rate was suppressed in the group added with HRG (FIG. 7).
  • the erythrocyte solution sample used in this example was prepared by the following method.
  • a blood collection bag, a bag containing a MAP liquid, and a red blood cell concentrated liquid storage bag (Terumo Corporation) including a tube formed by connecting them were used.
  • a red blood cell concentrated liquid storage bag (Terumo Corporation) including a tube formed by connecting them were used.
  • 500 ⁇ L of HRG after filter sterilization was added to the erythrocyte concentrated solution storage bag and mixed so that the final concentrations of HRG and HSA were 100 ⁇ / mL, respectively, to prepare a erythrocyte storage solution.
  • Example 10 Confirmation of PS expression on erythrocyte surface of sepsis model mouse
  • erythrocytes collected from a cecal ligation and puncture (CLP) sepsis model mouse (hereinafter also referred to as "CLP mouse") were used. The effect of HRG on it was confirmed.
  • CLP mouse cecal ligation and puncture
  • HRG 10 mg / kg
  • HSA 10 mg / kg
  • PBS 100 ⁇ L
  • Mice were anesthetized 24 hours after the operation, and 200 ⁇ L of blood was collected from the heart using 10% ACD solution as an anticoagulant.
  • the collected blood was centrifuged at 400 g, and the plasma and buffy coat were removed by suction.
  • the obtained erythrocyte solution was washed with HBSS 400 g for 5 minutes three times. 1 ⁇ L of washed erythrocytes was added to 1 ⁇ mL of HBSS.
  • Example 11 Zn 2+ -induced phosphatidylserine (PS) expression and inhibition by HRG
  • PS phosphatidylserine
  • the PS expression cell rate on the erythrocyte surface was 40% in the group not added with HRG, whereas the PS expression cell rate was suppressed to 10% or less in the group added with HRG (1 ⁇ g / mL). (FIG. 9).
  • Example 12 Increase in intracellular calcium by Zn 2+ and inhibition by HRG
  • the amount of calcium in red blood cells when HRG was added to a Zn 2+ stimulated erythrocyte solution sample was confirmed.
  • the sample of erythrocyte solution prepared in Reference Example 1 with Zn 2+ added to a concentration of 20 ⁇ M was added to an HRG concentration of 1 ⁇ g / mL, incubated at 37 ° C. for 1 hour, and then the amount of calcium in the erythrocytes. was measured.
  • red blood cells As a result, it was confirmed that the amount of calcium in red blood cells increased in the group not added with HRG, but was suppressed in the group added with HRG (1 ⁇ g / mL) (FIG. 10A). In the group without HRG, red blood cells aggregate and red blood cells adhere to vascular endothelial cells, whereas in the group to which HRG was added, red blood cells did not aggregate and adhesion to vascular endothelial cells was not observed (Fig. 10B).
  • Prx2 peroxiredoxin 2 release amount was confirmed for erythrocyte solution samples stimulated with Zn 2+ or calcium ionophore.
  • Prx is a kind of peroxidase having a strong antioxidant action, and is present in a large amount in the cell. Therefore, Prx is an important antioxidant enzyme for maintaining the physiological function of the cell. By measuring the amount of Prx2 released from erythrocytes, it becomes an index for measuring the cytotoxicity of erythrocytes.
  • Prx2 release by Zn 2+ and inhibition by HRG In this example, the characteristics of Prx2 release from erythrocytes when HRG was added to a Zn 2+ stimulated erythrocyte solution sample were confirmed. Add Zn 2+ to the erythrocyte solution sample to 50 ⁇ M, and further incubate Prx2 in the erythrocyte solution sample supernatant after incubation for 1 hour at 37 ° C in a system to which 100 ⁇ g / mL HSA or HRG has been added. And confirmed by electrophoresis. As a result, a decrease in Prx2 was observed in the group added with HRG, and the effect was more remarkable than in the group added with HSA (FIG. 12).
  • mice were anesthetized 24 hours after CLP operation, and 200 ⁇ L of blood was collected from the heart using 10% ACD solution as an anticoagulant. The collected blood was centrifuged at 400 g to obtain plasma, and Prx2 contained in the plasma was confirmed by electrophoresis under non-reducing conditions. As a result, Prx2 was observed in the plasma of CLP mice, but was hardly observed in SHAM (FIG. 13).
  • Example 14 Confirmation in CLP mouse
  • free hemoglobin in blood collected from the CLP mouse prepared by the method shown in Example 10 PS on the surface of erythrocytes, free calcium concentration in erythrocytes, contained in various organs The zinc content was confirmed.
  • HLP (20 mg / kg, iv), HSA (20 mg / kg, iv), PBS (200 ⁇ l, iv) were administered to CLP mice 10 minutes after CLP induction, and the mice were anesthetized 24 hours after surgery, and 10% 200 ⁇ L of ACD solution was collected from the heart as an anticoagulant. After plasma separation, plasma free hemoglobin levels in each group were detected by Western blot. Western blot results were quantified by densitometry to produce the bottom bar graph.
  • HLP (20 mg / kg, i.v.), HSA (20 mg / kg), and PBSPBS (200 ⁇ l, i.v.) as a control were administered to CLP mice 10 minutes after CLP induction. Mice were anesthetized 24 hours after the operation, and 200 ⁇ L of blood was collected from the heart using 10% ACD solution as an anticoagulant. The collected blood was centrifuged at 400 g, and the plasma and buffy coat were removed by suction. The obtained erythrocyte solution was washed with HBSS 400 g for 5 minutes three times. 1 ⁇ L of washed erythrocytes was added to 1 ⁇ mL of HBSS.
  • FITC-conjugated AnnexinTCV 4 ⁇ L was added to 400 ⁇ L of the suspension and incubated at room temperature for 15 minutes. Thereafter, 400 ⁇ L of 4% PFA resin solution was added and fixed for 15 minutes. After fixation, the number of PS positive cells was measured by FACS. 1 ⁇ L of separately washed red blood cells added to 1 ⁇ mL of HBSS was incubated at 37 ° C. for 4 hours, followed by FITC staining in the same manner, and the number of PS positive cells was measured.
  • CLP mice had increased Zn 2+ content in kidneys and lungs compared to intact mice, but there was no difference in spleen.
  • erythrocyte protective agent of the present invention erythrocyte deterioration due to storage can be suppressed.
  • Red blood cells can be protected more effectively by adding HRG as an active ingredient to existing red blood cell storage solution, blood storage solution, or later developed red blood cell storage solution or blood storage solution. . Thereby, it is possible to provide a higher quality preparation for the red blood cell preparation and the whole blood preparation in the blood preparation for transfusion.

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Abstract

L'invention concerne un agent protecteur de globules rouges qui permet un stockage à plus long terme et une utilisation plus sûre d'une solution contenant des globules rouges (par exemple, un concentrat de globules rouges). Lorsque des globules rouges sont stockés pendant une longue période de temps, de la phosphatidylsérine (PS) est exprimée sur la surface des membranes de globules rouges. Cet agent protecteur de globules rouges contient une glycoprotéine riche en histidine (HRG) en tant que principe actif. La HRG supprime l'expression de la PS sur les surfaces des globules rouges et permet un stockage à plus long terme et une utilisation plus sûre d'une solution contenant des globules rouges.
PCT/JP2018/005391 2017-02-20 2018-02-16 Agent protecteur de globules rouges Ceased WO2018151243A1 (fr)

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JP2018568621A JP6983420B2 (ja) 2017-02-20 2018-02-16 赤血球保護剤

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JP2017028738 2017-02-20
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WO2018151243A1 true WO2018151243A1 (fr) 2018-08-23

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CN113980894A (zh) * 2021-12-27 2022-01-28 深圳博雅感知医疗科技有限公司 制备骨髓浓缩细胞的方法及其用于治疗卵巢早衰的用途
CN117426372A (zh) * 2023-12-20 2024-01-23 中国人民解放军军事科学院军事医学研究院 一种保护红细胞的组合物、制备方法及应用

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WO2013183494A1 (fr) * 2012-06-06 2013-12-12 国立大学法人 岡山大学 Agent thérapeutique, méthode de traitement et procédé d'essai pour des maladies associées à l'activation de granulocytes neutrophiles

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WO2013183494A1 (fr) * 2012-06-06 2013-12-12 国立大学法人 岡山大学 Agent thérapeutique, méthode de traitement et procédé d'essai pour des maladies associées à l'activation de granulocytes neutrophiles

Non-Patent Citations (2)

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Title
WAKE, H ET AL.: "Histidine-Rich Glycoprotein Prevents Septic Lethality through Regulation of Immunothrombosis and Inflammation", EBIOMEDICINE, vol. 9, 2016, pages 180 - 194, XP055535875, ISSN: 2352-3964 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113980894A (zh) * 2021-12-27 2022-01-28 深圳博雅感知医疗科技有限公司 制备骨髓浓缩细胞的方法及其用于治疗卵巢早衰的用途
CN117426372A (zh) * 2023-12-20 2024-01-23 中国人民解放军军事科学院军事医学研究院 一种保护红细胞的组合物、制备方法及应用

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JPWO2018151243A1 (ja) 2019-12-12

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