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ARTIFICIAL RED BLOOD CELL HAVING ABILITY TO INHIBIT TRANSFORMATION OF HEMOGLOBIN INTO METHEMOGLOBIN meetings

Foreign code F170009262
File No. (S2016-0227-N0)
Posted date Oct 24, 2017
Country WIPO
International application number 2017JP008187
International publication number WO 2017150637
Date of international filing Mar 1, 2017
Date of international publication Sep 8, 2017
Priority data
  • P2016-040494 (Mar 2, 2016) JP
Title ARTIFICIAL RED BLOOD CELL HAVING ABILITY TO INHIBIT TRANSFORMATION OF HEMOGLOBIN INTO METHEMOGLOBIN meetings
Abstract The present invention inhibits, in an artificial red blood cell containing purified and enriched hemoglobin that substantially has no enzymatic activity to reduce methemoglobin, transformation of the hemoglobin into methemoglobin. Provided is an artificial red blood cell which comprises: an aqueous solution that contains NADH and/or NADPH and hemoglobin; and a capsule that includes the aqueous solution, wherein the aqueous solution and the capsule substantially have no enzymatic activity to reduce methemoglobin.
Outline of related art and contending technology BACKGROUND ART
Current in medicine, donation, blood transfusion system are essential in the art, are boasted a high safety. However, the possibility of infection by blood transfusion is not completely eliminated. In addition, also to threat of emerging infection is exposed. Different blood-blood medical or by administering to the patient are accident occurs. In addition, when an emergency need blood transfusions and blood type cross-matching is often difficult. After donation, concentrated red blood cell bag is, in a refrigerated Japan 3 weeks, 6 weeks and is less Europe, expired and must be discarded. The storage period is short, large-scale disaster or emergency situation such as when the demand for higher blood transfusion, blood transfusion cannot be sufficiently supplied to the can.
In order to overcome these problems, artificial blood has been formulation has been developed (non-patent document 1). Of the protein contained in the blood, most of the hemoglobin. - Hemoglobin oxygen dissociation reversibly binds the protein and, in other words the blood's primary function is oxygen delivery and, how the supply of oxygen is important to the maintenance of life is shown. Bind oxygen hemoglobin dissociation because it has been known for a long time, the artificial oxygen carrier (artificial red blood cells) have been developed as substances include, processed by a large number of hemoglobin. Is a so-called modified hemoglobin, hemoglobin sub-unit (i) to prevent dissociation of the hemoglobins and intra-molecular crosslinks, (ii) in order to increase the molecular weight of hemoglobin, such as glutaraldehyde or activation in a polymer obtained by intermolecular crosslinking of hemoglobin rafinozu, also, (iii) polyethylene glycol or dextran, albumin binding polymer is chemically bonded to the hemoglobin and the like. For a relatively simple method of manufacturing a modified hemoglobin is, in many cases progressed up to clinical trials, it was revealed that almost all toxic, interruption of the development. This is caused, hemoglobin vascular endothelial relaxing factor nitric oxide (NO) with a strong affinity, or reactive and, as a result of the deactivating NO cause a vasoconstriction, peripheral circulatory insufficiency cause, when active oxygen reacts with the byproducts resulting in direct contact with the vessel wall, or a muscle damage to the vessel wall, in addition, due to the small particle size easily leaks into the extravascular, and the like cause various side effects (non-patent document 2) as discussed above. That is, once the hemoglobin of red blood cells to be in the original red blood cells (hemolysis and) exits, is cytotoxic to is the same.
The inventors of the invention, having a format included in the liposome, the hemoglobin vesicle (Hb-V) has been developed. Is Hb-V, a high concentration hemoglobin (30-42 g/dL) high purity to the liposome containing a (non-patent document 3) artificial red blood cells. The components of the liposome, and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), and cholesterol, and 1,5-O-dihexadecyl-N-succinyl-L-glutamate (DHSG), 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-PEG5000 (DSPE-PEG5000) and the component 4. 10 g/dL Dispersion Hb-V and hemoglobin concentration, particle size can be controlled to 250-280 nm. Animal dose study by far high safety is confirmed, in addition, resuscitation from hemorrhagic shock, highly blood dilution, for administration to anemia, reduced infarct area cerebral infarction, oxygenation of the ischemic area, the compensation of the liquid in the extracorporeal circulation circuit, for organ perfusion liquid, as animal CO carrier effect is apparent from the experimental models.
However, the two subunits of the hemoglobin 1 molecule is 4 (α2 β2) composed, each sub-unit 1 to the heme oxygen binding site exists on one. The center of the heme iron is divalent (ferrous, Fe2+) of reversibly binds oxygen when. In the divalent state iron center in a bonded state oxygen oxyhemoglobin (HbO2) 、is not bound to the oxygen state referred to as deoxyhemoglobin (deoxyHb). Bound oxygen HbO2 is, gradually auto-oxidation by oxygen does not bind trivalent iron (ferric, Fe3+) of methemoglobin (metHb) changes. At this time of the following chemical reaction expressed by the formula super-oxide anions (O2-,) release. HbO2 →metHb + O2-, O2-, is, disproportionation reaction and by hydrogen peroxide, HbO2 and promote the oxidation of deoxyHb. In the red blood cells, in order to suppress these reactions, a mechanism for reducing metHb, included in a mechanism of removing active oxygen. A system for reducing metHb is known to include the following. (i) A reducing agent such as ascorbic acid or glutathione directly reacts with the reduced metHb, oxidized glutathione oxidized reduced by dehydroascorbic acid is returned to the body. (ii) The base is nicotinamide adenine dinucleotide (NADH) as NADH methemoglobin reductase system, is has been reported NADH-methemoglobin reductase, NADH-cytochrome b this5 reductaseto the action of the cytochrome b5 metHb is an electronic medium and mechanism for the reduction of the revealed. Oxidation type NAD+ NADH Embden Myerhof pathway is restored. NADH cytochrome b5 reductaseis, and red blood cells in the film, the film is dissolved in the red blood cells. (Iii) the base is nicotinamide adenine dinucleotide phosphate (NADPH) is reduced by the action of the NADPH methemoglobin reductase metHb, oxidation type NADP+ NADPH is restored to the pentose phosphate pathway. (iv) The red blood cells, O2-, superoxide dismutase (SOD) is converted into hydrogen peroxide, hydrogen peroxide is erased containing catalase (CAT).
And feedstock for the production of artificial oxygen carrier is hemoglobin, human red blood cells, isolated purified from livestock. A general purification method includes, first, anti-coagulant blood containing red blood cells are centrifuged to settle. The supernatant plasma layer and platelets, buffy coat (white blood cells) was removed, recovering the precipitated red blood cells. The addition of saline to this purge red blood cells are dispersed, is subjected to centrifugal separation, collecting the red blood cells and the supernatant was removed. This operation was 2, washed with 3 times of repetition of red blood cells can be obtained. Distilled water is gradually added to red blood cells hemolysis, hemoglobin is liberated. The solution was referred to as haemolyzed blood (hemolysate). Stroma (erythrocyte membrane) in order to remove the component, a molecular weight of about 1000kDa (i) ultrafiltration membrane in ultrafiltration processing, and transmitting only the water-soluble substance, stromal component is removed, or, by ultracentrifugation (ii) stromal component removed as precipitate. The hemoglobin solution may be obtained, stroma-free hemoglobin (stroma free hemoglobin, SFHb) is referred to as a, the erythrocytes and the hemoglobin is the main component but also the water-soluble co-enzyme systems. Then, the molecular weight fraction is dialyzed with an ultrafiltration membrane in 8-10kDa, concentrated. Enzyme system containing stroma-free hemoglobin is, if an adding a substrate for the enzyme, can be restored since metHb-reduction system, the metmyoglobin formation described above can be suppressed. In addition, a solution of crude hemoglobin (SFHb) substrate was added and the enzyme system, which is included in a capsule, in the case where the artificial red blood cells, by rotating metHb reductase system of metmyoglobin formation of the artificial red blood cells can be delayed.
The use of enzymes containing the 'disadvantage' stroma-free hemoglobin, virus inactivation, removing step becomes an incomplete state. If the hemoglobin derived from human red blood cells, artificial oxygen carrier made therefrom corresponding to a particular bio-based formulation. Derived from a single animal red blood cells, corresponding to the bio-based formulation. In any case, in the step of purified, inactivated virus, can be introduced into the removing step, the rate in its inactivation, removal rate compared to a predetermined value (Log Reduction Value) be sufficient obtained. Virus inactivation steps may include, a liquid heating processing (60°C, time 10) or S/D (organic solvent/surfactant treatment) processing is. These are, by modifying deactivating the virus. In addition, processing and removing step nanofiltration virus, (for example 15nm) than the pore diameters of the filters is removed because it can be a major virus can be transmitted. Wherein the globular protein and hemoglobin is structurally stable, enzymes may be unstable. Normal hemoglobin heme oxygen are bonded together, be converted to carbon monoxide (HbCO) it, or, the oxygen is removed from the charged deoxyHb is converted to heat resistance, a liquid heating processing becomes possible and the, to be made to the inactivation of the virus, almost all of the enzymes may be modified during heating phenomenon (non-patent document 4, 5). In addition, the case of using the nanofiltration is, larger than the hole diameter and nanofiltration enzymes are eliminated.
The inventors have found that, to eliminate all infectious agents can be artificial oxygen carrier (artificial red blood cells) be understood that conditions, purifying hemoglobin from red blood cells to incorporating a heating treatment and nanofiltration. Thus, meet the criteria of the virus is Log Reduction Value, degree of safety of the formulation can be significantly improved. However, the enzymatic system by heat treatment and nanofiltration for disappearing, as a result HbO2 metHb is trivalent iron metmyoglobin formation and, iron divalent state cannot be reduced, the oxygen-carrying function gradually decreases. Therefore, virus inactivation, through the removal step, substantially does not contain the enzyme system in the case of using concentrated purified hemoglobin, this formulation are enclosed in the development of artificial red blood cells, a method of inhibiting metmyoglobin formation described above is required.
Various non-enzymatic metHb far reduction system has been studied. (i) Thiols such as cysteine and glutathione is, metHb react directly with the reduction of the (non-patent document 6). The method, the deoxygenized state when stored for a long time, the remaining oxygen are completely erased, completely metHb is suitable for reducing (patent document 1, non-patent document 7). However, a high concentration of oxygen is present in a state of not metHb and thiols react directly with the dissolved oxygen and the active oxygen species is generated, on the contrary is the case of promoting metmyoglobin formation. (ii) And an electron transfer agent such as flavin, glucose as the electron donor added to the concentrated hemoglobin solution purified by visible light irradiation is devised a method of reducing metHb, and light irradiation method is limited, which is not a practical method (patent document 2). (Iii) the amino acid tyrosine methemoglobin coexist, pseudo-catalase activity hydrogen peroxide is erased, the rate of metmyoglobin to some extent is observed suppression, which is not a practical method (patent document 3). (iv) With respect to the artificial red blood cells (hemoglobin vesicle), was added from the outside and methylene blue NADH, reduced glutathione metHb in artificial red blood cells (non-patent document 8). This is, is methylene blue Leukomethylene blue and reacts with the reduced form NADH, diffuses into this artificial red blood cells is believed to reduce the metHb. Further, artificial red blood cells (hemoglobin vesicle) (v) after intravasal administration of at the time when the increase in the content of metHb, very little administration of methylene blue, methylene blue and red blood cells in the blood stream to react with the reduced form NADH and NADPH Leukomethylene blue, artificial red blood cells diffuses into this reducing metHb, clear effect, was found to be practical (non-patent document 9). However, Leukomethylene blue reacts with oxygen to generate active oxygen in a case where, the color tone of the skin becomes a problem that the blue. Therefore, substantially free of enzymatic systems and artificial red blood cells containing hemoglobin preparations, metmyoglobin formation with another means to retard or suppress the development of a technique is demanded.
In addition, from the vessel wall within the vessel is always as vascular endothelial relaxing factor is nitric oxide (NO) is being released, this very high reactivity of hemoglobin, to facilitate the metmyoglobin formation. Further, ischemia reperfusion injury in the inflammatory response, by induction of iNOS in the blood vessel or when increased production of NO, activation of the neutrophils or macrophages, the production of superoxide by the action of the oxidase NADPH - enhance, and disproportionation of hydrogen peroxide in this, these are also reacts with hemoglobin to promote metmyoglobin formation in question. Therefore, with respect to the oxidative stress in the body, artificial red blood cells as a function of the formulation, excess endogenous active oxygen, for a new NO held defense system has been demanded. This need is a conventional hemoglobin or encapsulates in the development of artificial red blood cells, has not been recognized.
Scope of claims (In Japanese)[請求項1]
NADH及び/又はNADPHとヘモグロビンとを含む水溶液と、該水溶液を内包するカプセルとからなり、前記水溶液及びカプセルはメトヘモグロビンを還元する酵素活性が実質的にない、人工赤血球。
[請求項2]
ヘモグロビンの50%メト化時間が72時間以上である、請求項1に記載の人工赤血球。
[請求項3]
前記カプセルに内包された水溶液中のヘモグロビン濃度は10g/dL~45g/dL(1.6mM~7.0mM)であり、該水溶液中のNADH及び/又はNADPHのモル濃度はヘモグロビンのモル濃度の0.5~10倍である、請求項1又は2に記載の人工赤血球。
[請求項4]
第2の水溶液中のヘモグロビンのモル濃度の0.5~3倍のモル濃度のpyridoxal 5'-phosphateを第2の水溶液中に含む、請求項1ないし3のいずれか1項に記載の人工赤血球。
[請求項5]
前記カプセルは、リポソーム、ポリマーソーム及び高分子薄膜からなる群から選択される少なくとも1つである、請求項1ないし4のいずれか1項に記載の人工赤血球。
[請求項6]
前記リポソームは、1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)、
cholesterol、
1,5-O-dihexadecyl-N-succinyl-L-glutamate (DHSG)及び1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-PEG50000 (DSPE-PEG5000)
からなる、請求項5に記載の人工赤血球。
[請求項7]
請求項1ないし6のいずれか1項に記載の人工赤血球を含む輸血代替用製剤。
[請求項8]
請求項1ないし6のいずれか1項に記載の人工赤血球を水溶液中に分散された状態で含み、さらに、該水溶液中に、電解質、糖質、アミノ酸、膠質、NADH及びNADPHからなる群から選択される少なくとも1種類の化合物を生理的に許容される濃度で含む、請求項7に記載の輸血代替用製剤。
[請求項9]
NADH及び/又はNADPHの水溶液を内包するカプセルを含む、NO、H2O2及びO2-・からなるグループから選択される少なくとも1つの物質の消去剤。
[請求項10]
人工赤血球の製造方法であって、
(a)ヘモグロビンを含む第1の水溶液からメトヘモグロビン還元する酵素活性を実質的に除去するステップと、
(b)第1の水溶液にNADH及び/又はNADPHを溶解して、実質的にメトヘモグロビンを還元する酵素活性がないヘモグロビンと、NADH及び/又はNADPHとを含む第2の水溶液を用意するステップと、
(c)第2の水溶液をカプセルに内包して、第2の水溶液とカプセルとからなる人工赤血球を得るステップとを含む、人工赤血球の製造方法。
[請求項11]
前記ステップ(a)は、第1の水溶液を60~65℃にて1~12時間加熱することを含む、請求項10に記載の人工赤血球の製造方法。
[請求項12]
第2の水溶液中のヘモグロビン濃度は10g/dL~45g/dL(1.6mM~7.0mM)であり、第2の水溶液中のNADH及び/又はNADPHのモル濃度はヘモグロビンのモル濃度の0.5~10倍である、請求項10又は11に記載の人工赤血球の製造方法。
[請求項13]
前記ステップ(b)において、第2の水溶液中のヘモグロビンのモル濃度の0.5~3倍のモル濃度のpyridoxal 5'-phosphateを第2の水溶液中に含む、請求項10ないし12のいずれか1項に記載の人工赤血球の製造方法。
[請求項14]
前記ステップ(b)は、pyridoxal 5'-phosphateを第1の水溶液又は第2の水溶液に溶解することを含む、請求項13に記載の人工赤血球の製造方法。
[請求項15]
前記カプセルは、リポソーム、ポリマーソーム及び高分子薄膜からなる群から選択される少なくとも1つである、請求項10ないし14のいずれか1項に記載の人工赤血球の製造方法。
[請求項16]
前記リポソームは、1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)、
cholesterol、
1,5-O-dihexadecyl-N-succinyl-L-glutamate (DHSG)及び1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-PEG5000(DSPE-PEG5000)
からなる、請求項15に記載の人工赤血球の製造方法。
[請求項17]
請求項1ないし6のいずれか1項に記載の人工赤血球を含む、敗血症の治療、亜硝酸塩の大量摂取の治療及びNO吸入療法におけるメトヘモグロビン血症予防のための医薬組成物。
[請求項18]
請求項7又は8に記載の輸血代替用製剤を含む、請求項17に記載の医薬組成物。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • NARA MEDICAL UNIVERSITY
  • Inventor
  • SAKAI Hiromi
  • YAMADA Magohei
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG

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