WO2019070086A2 - Substitut sanguin transportant de l'oxygène obtenu à partir de sang de porc et son procédé de fabrication - Google Patents

Substitut sanguin transportant de l'oxygène obtenu à partir de sang de porc et son procédé de fabrication Download PDF

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Publication number
WO2019070086A2
WO2019070086A2 PCT/KP2018/000048 KP2018000048W WO2019070086A2 WO 2019070086 A2 WO2019070086 A2 WO 2019070086A2 KP 2018000048 W KP2018000048 W KP 2018000048W WO 2019070086 A2 WO2019070086 A2 WO 2019070086A2
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WO
WIPO (PCT)
Prior art keywords
haemoglobin
solution
blood
oxygen
polymer
Prior art date
Application number
PCT/KP2018/000048
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English (en)
Other versions
WO2019070086A3 (fr
Inventor
Won Nam Yun
Chol Hui HAN
Original Assignee
RIM, Chang Ho
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Publication date
Application filed by RIM, Chang Ho filed Critical RIM, Chang Ho
Publication of WO2019070086A2 publication Critical patent/WO2019070086A2/fr
Publication of WO2019070086A3 publication Critical patent/WO2019070086A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0026—Blood substitute; Oxygen transporting formulations; Plasma extender
    • 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
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/805—Haemoglobins; Myoglobins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to oxygen-canying blood substitute obtained from swine blood and the manufacturing method thereof!
  • Transfusion by whole blood is known to have various defects. As the number of donors rapidly decreases by increasing proportion of the old in population, the amount of whole blood required for transfusion is greatly lacked as compared with the demand, the transfusion by the whole blood causes side effects during the transfusion, and the attack rate of diseases by whole blood transfusion is high. Also, the shelf life of whole blood is short (42 days at 4oC) and it should not be shocked so as to avoid hemolysis in the course of its transfer.
  • Oxygen-carrying blood substitute which is recently developing has been known an ideal blood for transfusion which can overcome the defects in transfusion by whole blood.
  • the advantages of oxygen-carrying blood substitute obtained from bovine blood are that it does not require typing or screening blood before transfusion due to the absence of blood group* there is no danger of generating disease by transfusion, it is convenient to store and transport and it has rich source.
  • many researches for manufacturing oxygen-carrying blood substitute from bovine blood and using only the blood substitute or the same together with the whole blood in hospital stage as well as pre-hospital stage are being progressed.
  • HBOC haemoglobin-based oxygen carriers
  • the present invention provides oxygen-carrying blood substitute obtained from swine blood and the manufacturing method thereof.
  • oxygen-carrying blood substitute obtained, from swine blood is that it has oxygen-carrying ability and supplies oxygen of 1.5 times as compared with the whole blood, thereby being very effective in treatment of shock by bleeding, bleeding diseases and hypoxia.
  • Oxygen-carrying blood substitute according to the present invention is obtained from swine blood instead of whole blood, its cost is low and there is no limit of source of blood.
  • the present invention provides oxygen-carrying blood substitute obtained from swine blood.
  • the present invention also provides a method for manufacturing oxygen-canying blood substitute from swine blood.
  • the said method comprises a process for removing plasma protein from swine blood, a process for separating haemoglobin, a process for purifying haemoglobin with strong acid cation exchange resin, a process for polymerizing haemoglobin with glutaraldehyde, a process for ultrafiltering haemoglobin polymer, a process for inactivating virus in haemoglobin polymer and packaging process.
  • the present invention provides oxygen-carrying blood substitute obtained from swine blood.
  • composition of oxygen-carrying blood substitute obtained from swine blood according to the present invention is as follows.
  • the half -life of the said oxygen-carrying blood substitute is IS ⁇ 20 hours and it can be stored at less than 15oC for at least a year.
  • the present invention provides a method for manufacturing oxygen-carrying blood substitute from swine blood.
  • the said method comprises a process for removing plasma protein from swine blood, a process for separating haemoglobin, a process for purifying haemoglobin with strong acid cation exchange resin, a process for polymerizing haemoglobin with glutaraldehyde, a process for uhrafiltering haemoglobin polymer, a process for inactivating virus in haemoglobin polymer and packaging process.
  • the blood serum is removed by centrifugal separating swine blood by freezing centrifuge at 2500 ⁇ 3500rpm and at 4 - 10°C for 10 - 20 minutes. Then concentrated red blood cell is diluted, with cleaning solution (0.85 ⁇ 1% saline solution) to be 1: 3 - 5v/v in volume ratio and the diluted solution is centrifugal separated at 2500 ⁇ 3500rpm and at 4 - 10°C for 10 ⁇ 20 minutes to remove plasma protein. The said process is repeated 7 - 10 times. The content of plasma protein in the obtained red blood cell is less than 1.5ng/mL.
  • Sterile distilled water is added to the concentrated red blood cell so that the volume ratio of concentrated red blood cell and distilled water is 1: 2 - 5v/v, maintaining the solution at 4 - 10°C for 1 -2 hours to haemolyze and centrifugal separating the solution at 8000 ⁇ 12000 ⁇ and at 4 ⁇ 10°C for 10 ⁇ 20 minutes to remove membrane matrix, thereby separating haemoglobin.
  • the said separated haemoglobin solution is filtered through 100kDa of ultrafilter.
  • Strong acid cation exchange resin Dowex 50Wx8 (H+type) is added to the filtered solution to pH of 5.5 ⁇ 6.0, centrifugal separating it at 8000 ⁇ 12000rpm and at 4 ⁇ 10°C for 10 ⁇ 20 minutes and removing sediment.
  • the concent of phospholipid in the purified haemoglobin solution is less than 0.02ng/mL.
  • 1 ⁇ 2M of carbonate buffer is added to the said purified haemoglobin solution to pH of 8.0 ⁇ 9.5.
  • the said haemoglobin solution is deoxygenated by forming 200 ⁇ 400pa of sound pressure for 20 ⁇ 40 minutes.
  • 5 ⁇ 10% of glutaraldehyde solution is added to the deoxygenated haemoglobin solution in the amount of 25 ⁇ 50mL per lOOg of haemoglobin and reacting at 4 ⁇ 10°C for 8 ⁇ 10 hours.
  • the solution is filtered through lOkDa of ultrafilter to dialyze the remaining glutaraldehyde, and adding NaBRt in the amount of 400 ⁇ 600mg per lOOg of haemoglobin to reduce and remove the remaining glutaraldehyde.
  • the solution is centrifugal separated at 8000 ⁇ 12000rpm and at the 4 ⁇ 10°C for 30 ⁇ 40 minutes, removing the sediment and catching the supernatant (haemoglobin polymer solution) in the sterilized container.
  • the said haemoglobin polymer solution is filtered through 100kDa of ultrafilter to remove haemoglobin which is not polymerized, and filitering the said haemoglobin polymer solution through 300kDa of ultrafilter to remove high polymer, thereby obtaining the solution of haemoglobin polymer with molecular weight of 100 ⁇ 300kDa.
  • the haemoglobin polymer solution is deoxygenated by forming 100 ⁇ 200pa of sound pressure for 40 ⁇ 60 minutes, maintaining the deoxygenated haemoglobin polymer solution at 60 ⁇ 65°C for 8 ⁇ 10 hours to inactivate virus.
  • the inactivated haemoglobin polymer solution is diluted so that the concentration of haemoglobin polymer is 3% and the concentration of glucose is 5%. Then, pH of the obtained solution is regulated as 7.4 ⁇ 7.8 by adding 0.1M of phosphate buffer. The obtained solution is filtered through 0.2um of filter. The filtered solution is deoxygenated by forming 100 ⁇ 200pa of sound pressure for 40 ⁇ 60 minutes, dividing the solution by 250mL and packaging it.
  • the content of phospholipid in oxygen-carrying blood substitute obtained from the said method is as follows.
  • the yield of haemoglobin in each process for manufacturing oxygen-carrying blood substitute is as follows.
  • Embodiment 1 The result , of inactivating virus in haemoglobin polymer was evaluated.
  • control group 0.1 mL of undiluted solution of influenza virus was added to haemoglobin polymer and inoculated into cell strain MDCK(1.0*10 5 /rnL) at 37°C for 1 hour.
  • the table below shows stability of oxygen-carrying blood substitute according to shelflife.
  • Embodiment 2 The test on acute toxicity of oxygen-carrying blood substitute was carried out.
  • Mouses of 18g ⁇ 22g were divided into 6 groups by 5 mouses, in each group, different amounts of haemoglobin polymer was injected into the adborninal cavity in order to test acute toxicity by Berens-kerver's test.
  • Median lethal dose of oxygen-carrying blood substitute is 13,8g/kg in the amount of haemoglobin.
  • Embodiment 3 The test of nontoxicity on oxygen-canying blood substitute was carried out
  • Embodiment 4 Anaphylactic test on oxygen-canying blood substitute was carried out
  • bovine serum albumin was used.
  • Embodiment 5 After injection of oxygen-carrying blood substitute, pathologicoanatomic test was carried out.
  • White rat was injected with oxygen-rarrying blood substitute in the amount of 1mL/100g via tail's vein weekly and repeated the said injection 10 times. As the result of pathologicoanatomic test, abnormal finding was not observed.
  • Embodiment 6 When exchange transfusing 50% of dog blood, change in tissue oxygen pressure was measured.
  • tissue oxygen pressure in the experiment group is significantly higher compared with the control group.
  • Embodiment 7 When exchange transfusing more than 80% of white rat blood with oxygen-carrying blood substitute, influence on survival rate of white rat was observed.
  • Embodiment 8 The half life of oxygen-carrying blood substitute was measured.
  • the half life is calculated according to the following formula.
  • K ⁇ lnC 1 -lnC 2 y(t 2- t 1 )
  • Embodiment 9 Clinical efficacy of oxygen-carrying blood substitute was tested. In various hemorrhagic shocks and chronic anemic diseases patients, clinical efficacy of oxygen-carrying blood substitute was tested. Table 11. After transfusion of oxygen-carrying blood substitute, tissue oxygen pressure and oxygen saturation
  • tissue oxygen pressure and oxygen saturation were remarkdely increased compared with before transfusion.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Developmental Biology & Embryology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Dermatology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un substitut sanguin transportant de l'oxygène obtenu à partir de sang de porc. La présente invention concerne un procédé de fabrication d'un substitut sanguin transportant de l'oxygène à partir de sang de porc. Ledit procédé comprend un processus d'élimination de la protéine plasmatique du sang de porc, un processus de séparation de l'hémoglobine, un processus de purification de l'hémoglobine avec une résine d'échange de cation fortement acide , un processus de polymérisation d'hémoglobine avec du glutaraldéhyde, un processus d'ultrafiltration de polymère d'hémoglobine, un processus d'inactivation de virus dans un polymère d'hémoglobine et un processus d'emballage.
PCT/KP2018/000048 2017-09-20 2018-11-20 Substitut sanguin transportant de l'oxygène obtenu à partir de sang de porc et son procédé de fabrication WO2019070086A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KP201720003509 2017-09-20
KP201720003509 2017-09-20

Publications (2)

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WO2019070086A2 true WO2019070086A2 (fr) 2019-04-11
WO2019070086A3 WO2019070086A3 (fr) 2019-06-13

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113509488A (zh) * 2021-06-29 2021-10-19 西安交通大学 一种携氧仿生纳米解毒剂及其制备方法和应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6023084B2 (ja) * 1979-07-11 1985-06-05 味の素株式会社 代用血液
EP2026824A2 (fr) * 2006-05-09 2009-02-25 The United States Of America As Represented By the Sectretary of the Navy Substitut sanguin plurifonctionnel
RU2361608C1 (ru) * 2008-03-18 2009-07-20 Общество С Ограниченной Ответственностью "Научно-Производственная Компания "Медбиофарм" Кровезаменитель с функцией переноса кислорода, фармацевтическая композиция (варианты)
CN107137699B (zh) * 2017-06-15 2021-02-09 中国医学科学院输血研究所 一种天然血红蛋白类血液代用品的脱氧方法和制备工艺

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113509488A (zh) * 2021-06-29 2021-10-19 西安交通大学 一种携氧仿生纳米解毒剂及其制备方法和应用
CN113509488B (zh) * 2021-06-29 2022-05-20 西安交通大学 一种携氧仿生纳米解毒剂及其制备方法和应用

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WO2019070086A3 (fr) 2019-06-13

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