JP4763265B2 - Artificial oxygen carrier containing an anti-methanizing agent - Google Patents

Description

  The present invention relates to an anti-methanization agent containing L-tyrosine, and an artificial oxygen carrier containing the anti-methanization agent. Specifically, the present invention relates to an artificial oxygen carrier preparation that is suitable for long-term storage by preventing hemoglobin and the like encapsulated in a lipid vesicle having a bilayer structure from being oxidized and increasing the content of methemoglobin.

Current transfusion systems that inject allogeneic blood into veins have the following problems:
1) Possible infection (hepatitis, AIDS virus, etc.);
2) Red blood cell storage period is 3 weeks;
3) With the arrival of an aging society, the proportion of elderly patients among blood transfusion patients is increasing, while the total number of healthy blood donors continues to decline;
4) Risk of contamination during storage;
5) Not applicable to patients who refuse transfusions for religious reasons;
6) Inability to respond to emergency demand during a disaster;
7) A transfusion accident due to blood type incompatibility can occur.

  Therefore, there is a great demand for alternatives that can be applied at any time regardless of blood type, and as one of them, infusion preparations such as electrolyte infusion and colloid infusion have been widely used. However, since these infusion preparations do not have the most important function of blood, that is, the function of red blood cells that carry oxygen, the substances that substitute this oxygen carrying ability (oxygen infusion, artificial oxygen carrier) Development is an issue.

  Development of oxygen transfusions using hemoglobin that has the ability to dissociate oxygen (such as human hemoglobin, bovine hemoglobin, and genetically modified hemoglobin) is also underway. Intramolecular cross-linked hemoglobin, water-soluble polymer-bound hemoglobin, intermolecular cross-linked Clinical trials for polymerized hemoglobin are ongoing in Europe and the United States. As various side effects due to the non-cell type structure are pointed out in this clinical trial, the importance of the so-called cell type structure in which hemoglobin is encapsulated in the endoplasmic reticulum or capsule has become clear.

  It has been discovered that phospholipids, which are biological components, form lipid vesicles (liposomes), and Djordjevich and Miller (Fed. Proc. 36, 567, 1977) use phospholipid / cholesterol / fatty acid liposomes Since studying the endoplasmic reticulum, studies of hemoglobin endoplasmic reticulum have been developed in several groups, including our group.

  Hemoglobin endoplasmic reticulum can be used as it is without modifying hemoglobin; 2) viscosity, colloid osmotic pressure and oxygen affinity can be adjusted to arbitrary values; 3) retention time in blood is prolonged; 4) It has the advantage that various additives can be encapsulated in a high concentration in the endoplasmic reticulum aqueous phase. Of these, the advantage 4) is particularly important in the present invention. The present inventors have established an efficient method for preparing hemoglobin endoplasmic reticulum, and have obtained hemoglobin endoplasmic reticulum fluids whose physical properties are extremely close to blood, which is excellent in animal administration tests. Confirmed oxygen carrying capacity (Tsuchida ed. Blood Substitutes Present and Future Perspective, Elsevier, Amsterdam, 1998).

  Hemoglobin contains four hemes, which can bind oxygen reversibly when the heme iron is divalent iron (Fe (II)), but heme iron is oxidized trivalent iron (Fe (III) )) (This is called methation), methemoglobin (methemoglobin) cannot bind to oxygen. In addition, by the generation of superoxide radical anion accompanying the methanoylation of oxygen-bound hemoglobin (oxyhemoglobin), this acts as an oxidant and promotes the production of methemoglobin. There is a methemoglobin reduction system and a reactive oxygen scavenging system in erythrocytes, and a mechanism that does not increase the methemoglobin content works, but in the hemoglobin endoplasmic reticulum using purified hemoglobin, all these enzyme systems are removed during the purification process of hemoglobin Therefore, oxygenation of hemoglobin occurs during storage and after administration, resulting in a decrease in oxygen carrying capacity.

  As a method of suppressing this oxidation reaction, (i) a method of adding a reducing agent such as glutathione, homocysteine and / or ascorbic acid, and an enzyme which eliminates active oxygen such as catalase and / or superoxide dismutase (Sakai et al. , Bull. Chem. Soc. Jpn., 1994 (Non-Patent Document 1); Takeoka et al., Bioconjugate Chem., 8, 539-544, 1997 (Non-Patent Document 2)), (ii) Methylene blue as an electron mediator Is introduced into the endoplasmic reticulum membrane, and methemoglobin encapsulated in the endoplasmic reticulum is reduced by electron transfer from NADH added to the outer aqueous phase of the endoplasmic reticulum (Takeoka et al., Bull. Chem. Soc. Jpn., 70, 1171-1178, 1997 (non-patent document 3)), or (iii) a method for reducing methemoglobin by light irradiation in the near ultraviolet region (Sakai et al., Biochemistry, 39, 14595-14602, 2000 (non-patent) Document 4)) has been tried. In addition, as a method for stably storing hemoglobin endoplasmic reticulum for a long period of time (shelf storage), a method in which oxygen is completely removed and stored in a deoxy form has been attempted (Sakai et al., Bioconjugate Chem, 11, 425-). 432, 2000 (Non-Patent Document 5)).

  However, the conventional reduction method or preservation method of the oxidized hemoglobin vesicle described above leaves room for improvement in the following points.

  First, when blood is used as a raw material, virus inactivation is required in the hemoglobin purification step, and thus hemoglobin is desired to be heated at 60 ° C. for 10 hours or longer as with the albumin preparation. At that time, the methemoglobin reductase system present in the erythrocytes is also denatured and inactivated. If mild purification by hypotonic hemolysis is performed to utilize the activity of these enzyme systems, the oxidation rate of the resulting hemoglobin endoplasmic reticulum can be suppressed, but it is difficult to achieve virus inactivation. It is. Moreover, since an enzyme system is chemically unstable, there exists a possibility that activity may fall during the preservation | save for a long period of time.

  As described above, by incorporating a reducing agent such as glutathione or homocysteine in the hemoglobin endoplasmic reticulum, the formed methemoglobin can be reduced, and the oxidation reaction can be relatively suppressed. However, even if methemoglobin does not exist, the reducing agent is oxidized and gradually deactivated by reacting with oxygen in the air (auto-oxidation). Furthermore, the superoxide radical anion or hydrogen peroxide generated by this reaction On the other hand, the methanoylation of hemoglobin is promoted by reactive oxygen species such as.

In addition, when hemoglobin is stored for a long period of time, by completely removing oxygen, methemoglobin formation of hemoglobin endoplasmic reticulum can be suppressed only in a sealed state. However, in the case of actually using hemoglobin vesicles as oxygen carriers, naturally it is used in the state of oxyhemoglobin in the presence of oxygen, so this method is a means to solve the methemocytosis of hemoglobin vesicles. Must not. For example, when hemoglobin endoplasmic reticulum is used as a perfusate for transplanted organs or when it is used as an extracorporeal circulating fluid, it is exposed to the atmosphere for a certain period of time.
Sakai et al., Bull. Chem. Soc. Jpn., 1994 Takeoka et al., Bioconjugate Chem., 8, 539-544, 1997 Takeoka et al., Bull. Chem. Soc. Jpn., 70, 1171-1178, 1997 Sakai et al., Biochemistry, 39, 14595-14602, 2000 Sakai et al., Bioconjugate Chem, 11, 425-432, 2000

  Therefore, there is a demand for a dispersion system that can suppress the methemoglobin formation rate of hemoglobin vesicles in the presence of oxygen, and unlike the reducing agent, the additive is present stably without reacting with oxygen.

  The inventors have conducted systematic research on artificial oxygen carriers for many years and have made efforts to develop a method for suppressing the methemoglobin formation rate of hemoglobin endoplasmic reticulum. As a result, the inventors have arrived at the present invention that can solve the above problems. Is.

That is, the present invention is as follows.
(1) (1) An anti-methanization agent containing tyrosine. An example of tyrosine is L-tyrosine.
The concentration of L-tyrosine is 0.01 mM to 20 mM, preferably 1 mM to 20 mM, and more preferably 8 mM to 20 mM.
(2) An artificial oxygen carrier comprising a lipid vesicle encapsulating the methation inhibitor and heme protein according to (1) above.
(3) A method for producing an artificial oxygen carrier comprising encapsulating the anti-methetization agent and heme protein according to (1) above in a lipid vesicle.
(4) A method for preventing methemocytosis of a heme protein, comprising encapsulating the methetogenesis inhibitor and the heme protein according to (1) above in a lipid endoplasmic reticulum.
(5) A method for preserving an artificial oxygen carrier comprising encapsulating the anti-methetization agent and heme protein according to (1) above in a lipid vesicle.
(6) In the methods described in (3) to (5) above, examples of the hemoprotein include hemoglobin. In the method of the present invention, enzymes (for example, catalase, methemoglobin, etc.) can be further encapsulated in the lipid vesicles.

    In the present invention, examples of the hemoprotein include hemoglobin capable of reversibly binding oxygen. The lipid endoplasmic reticulum can further contain enzymes (for example, catalase). Furthermore, methemoglobin also shows peroxidase activity using tyrosine as a substrate and can be included in this. Furthermore, the lipid vesicles are composed of a single layer or a multilayer film, and the membranes of these lipid vesicles may be modified with polyethylene glycol or the like. Furthermore, in the artificial oxygen carrier or method of the present invention, when the lipid vesicle encapsulating the methation inhibitor and heme protein described in (1) above is left at 37 ° C. and an oxygen partial pressure of 5 to 300 Torr for 60 hours. The metation rate is preferably 50% or less. Moreover, it is preferable that the metation rate after adding 60 minutes after adding hydrogen peroxide to the lipid vesicle encapsulating the methetization inhibitor and heme protein described in (1) is 20% or less.

  INDUSTRIAL APPLICABILITY According to the present invention, there are provided an anti-methanization agent containing L-tyrosine, and an artificial oxygen carrier comprising an endoplasmic reticulum encapsulating the anti-methetization agent and heme protein. The artificial oxygen carrier of the present invention can prevent oxyhemoglobin encapsulated in lipid vesicles having a membrane structure from being oxidized and increase the methemoglobin content, so that the artificial oxygen carrier having a long effective period of use is used. Useful as a body.

Hereinafter, the method of the present invention will be described in detail. The present invention has been completed by paying attention to the point that tyrosine (particularly L-tyrosine) has the property of preventing methation, and the tyrosine is intended to be applied to an artificial oxygen carrier or an agent for preventing methemolysis in blood. Is. Here, methation means that the central iron of protoheme, which is a prosthetic group of hemoglobin, is oxidized from divalent (Fe ²⁺ ) to trivalent (Fe ³⁺ ).

  The purpose of use of tyrosine to prevent methemoglobin in red blood cells (including prevention of increased methemolysis) is not particularly limited. For example, preoperative blood dilution, extracorporeal circulation, organ preservation, fluid ventilation In addition, sickle cell anemia, stroke, carbon monoxide poisoning, cancer treatment, poisoning due to accidental ingestion, and other clinical medicine can be used. However, it is not limited to these purposes.

  In order to apply tyrosine for the above purpose, tyrosine can be encapsulated in a lipid vesicle such as a liposome.

  In the present invention, “lipid endoplasmic reticulum” means interaction between molecules (hydrophobic interaction, electrostatic interaction, hydrogen bonding, etc.) in an aqueous medium without a covalent bond by lipid and / or lipoprotein. ) Is a molecular assembly having an endoplasmic reticulum structure, and the film forms a single layer or multiple layers (for example, two layers).

  The lipid vesicles in the present invention can be used as phospholipids alone or as a mixed lipid of phospholipids and cholesterol or fatty acids. The endoplasmic reticulum can be prepared by a method already published by the present inventors (Sakai et al., Biotechnol. Progress, 12, 119-125, 1996; Bioconjugate Chem., 8, 23-30, 1997). Specifically, first, an appropriate amount of pyridoxal 5 ′ phosphate and L-tyrosine as allosteric factors is added to the purified hemoglobin solution, and the mixed lipid powder is mixed and hydrated. This hemoglobin lipid mixed solution is stepped through a filter with a pore size of 3 μm to 0.22 μm using a high-pressure extrusion device to control the particle size, and unencapsulated hemoglobin is removed by centrifugation to prepare hemoglobin vesicles. .

 In the present invention, in addition to the above methods, general methods for producing endoplasmic reticulum, for example, ultrasonic irradiation method, forced stirring (homogenizer) method, vortex method, freeze-thaw method, organic solvent injection method, surfactant removal Method, reverse phase evaporation method, microfluidizer method and the like can be employed. For example, in the freeze-thaw method, pyridoxal 5 'phosphate and L-tyrosine are added to the purified hemoglobin solution, mixed with the mixed lipid powder, hydrated, and then frozen (-197 ° C) and thawed (40 ° C) Repeat three times to prepare hemoglobin vesicles. In addition, in the organic solvent injection method, the mixed lipid is dissolved in chloroform or diethyl ether / methanol mixed solvent, injected into a purified hemoglobin solution to which pyridoxal 5 'phosphate and L-tyrosine are added, and the solvent is removed under reduced pressure. 3. Prepare hemoglobin vesicles. In the ultrasonic irradiation method, pyridoxal 5 'phosphate and L-tyrosine are added to the purified hemoglobin solution, the mixed lipid powder is mixed and hydrated, and then irradiated using a probe type ultrasonic irradiation device. Prepare vesicles.

  The phospholipid that is a constituent of the endoplasmic reticulum may be either a saturated phospholipid or an unsaturated phospholipid (Japanese Patent No. 2936109). For example, egg yolk lecithin, hydrogenated lecithin, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dilinoleoylphosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and the like can be used. . These phospholipids are selected from polymerizable phospholipids having a polymerizable group such as ene (double bond), in (triple bond), diene, diyne, and styrene. Examples of such polymerizable phospholipids include 1,2-di (octadeca-trans-2, trans-4-dienoyl) phosphatidylcholine, 1,2-di (octadeca-2,4-dienoyl) phosphatidic acid, 1 , 2-biseleostearoylphosphatidylcholine and the like. As the fatty acid, a saturated or unsaturated fatty acid having 12 to 20 carbon atoms is used. For example, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, octadeca-2,4-dienoic acid and the like.

  In the present invention, an appropriate additive can be added to the membrane of the lipid molecule assembly to modify the membrane. Examples of the additive for modifying the membrane include sialic acid, sugar-bonded fatty acid, polyoxyethylene-bonded phospholipid, polyoxyethylene-bonded fatty acid, and the like, and preferably modified with polyoxyethylene (polyethylene glycol). . The molecular weight of polyethylene glycol is about 400 Da to 12000 Da, preferably 1000 Da to 5000 Da.

  Examples of the hemoprotein encapsulated in the lipid endoplasmic reticulum include hemoglobin, myoglobin, and albumin-heme. Purified hemoglobin can be obtained by methods known in the art (edited by the Biochemical Society of Japan, Second Biochemistry Experiments, Volume 8 “Blood”, Tokyo Kagaku Dojin, 1987; Methods in Enzymology, Volume 76, 1981, Academic Press, New York; The Chromatograph of Hemoglobin, 1983, Dekker, New York, etc.). For example, when purifying hemoglobin by the hemolysis method, a hypotonic solution is added to the washed erythrocytes to cause hemolysis by osmotic pressure difference, and erythrocyte membrane components are removed by centrifugation. Then, high-purity hemoglobin is obtained using an ultrafiltration method, a crystallization method, an HPLC method, or the like.

  Further, by binding carbon monoxide to hemoglobin (HbCO), methotization can be suppressed and high-temperature stability can be improved. In this case, it is effective to completely remove the residual solvent when purified by solvent treatment (for example, carbon tetrachloride, toluene, chloroform, diethyl ether, etc.). Furthermore, proteins present accompanying hemoglobin can be removed by heating. Since HbCO is stable to heating, it can inactivate contaminating proteins and coexisting viruses.

  In the present invention, an endoplasmic reticulum encapsulating hemoglobin as a water-soluble substance is referred to as a hemoglobin endoplasmic reticulum. Hereinafter, the hemoglobin vesicle encapsulating L-tyrosine will be described as an example, but the present invention is not limited thereto.

  In the present invention, when L-tyrosine is applied, it is preferable that L-tyrosine is encapsulated in the hemoglobin endoplasmic reticulum. Therefore, the L-tyrosine encapsulated in the hemoglobin vesicle of the present invention can be mixed with a water-soluble substance after preparing the hemoglobin vesicle, but when preparing the hemoglobin vesicle, the water-soluble substance (dispersion) It is preferable that L-tyrosine is contained in the liquid) in advance because the rate of inhibition of endoplasmic reticulum metogenesis is high. In the present invention, it is preferable to use L-tyrosine as a monomer.

  In addition, the effect of hemoglobin endoplasmic reticulum on methetogenesis increases with increasing L-tyrosine concentration. Therefore, in the present invention, the addition concentration when L-tyrosine is used as an anti-methanization agent is preferably as high as possible. In the present invention, the addition concentration of L-tyrosine is at least 0.01 mM, preferably 1.0 mM or more. Furthermore, addition of 8.0 mM or more is more preferable, and for example, L-tyrosine can be dissolved up to about 20 mM.

  In using the anti-methetization agent of the present invention, a dispersion of hemoglobin vesicles is diluted with a physiological salt solution to a predetermined component concentration (for example, a hemoglobin concentration of 5 g / dL). At this time, even if the hemoglobin endoplasmic reticulum dispersion is diluted, the component concentration of the aqueous phase of the endoplasmic reticulum is maintained without being diluted. This is extremely advantageous in applying the method of the present invention. Assuming that it is used as an extracorporeal circulation fluid or tissue culture fluid, the rate of methotrelation of hemoglobin endoplasmic reticulum encapsulating L-tyrosine can be determined by shaking at 37 ° C in the atmosphere or under low oxygen partial pressure. It is suppressed as compared with unencapsulated hemoglobin endoplasmic reticulum. For example, the metation rate when left for 60 hours under low oxygen partial pressure conditions is 50% or less. Here, the low oxygen partial pressure condition (low oxygen partial pressure condition) is preferably 40 Torr in the case of 5 to 300 Torr at 37 ° C.

  As described above, L-tyrosine used in the present invention can suppress the methation rate of hemoglobin endoplasmic reticulum, so that the time for functioning as an oxygen carrier can be extended over a long period of time. For example, in various applications such as blood dilution fluid, extracorporeal circulation fluid, tissue culture fluid, etc., when the hemoglobin vesicle is used, the function time as an oxygen carrier is greatly extended because the rate of its methation is suppressed. Can be used for a long time. In addition, as a method for preserving hemoglobin vesicles in an oxy state, by using the hemoglobin vesicles, an increase in methemoglobin concentration can be suppressed over a long period of time.

  As described above, according to the present invention, the hemoglobin endoplasmic reticulum encapsulating L-tyrosine is suppressed in its methation rate.

  It is known that when hemoglobin in the oxy state is oxidized to methemoglobin, hydrogen peroxide is generated, which promotes the methemoglobin formation. Recent studies by the inventors have revealed that methemoglobin has an enzyme activity that consumes hydrogen peroxide when specifically oxidizing L-tyrosine to dityrosine, so-called peroxidase activity. For this reason, since the hydrogen peroxide density | concentration in a system falls, the mechanism in which the meth- odation of hemoglobin by hydrogen peroxide is considered at present.

  Therefore, when an appropriate amount of methemoglobin is encapsulated in an endoplasmic reticulum encapsulating L-tyrosine and oxy-state hemoglobin in advance, the meth- odation of oxy-state hemoglobin can be significantly suppressed.

  Moreover, it is considered that L-tyrosine and hemoglobin do not interact directly. Actually, even when the binding heat generated by the interaction (binding) between L-tyrosine and hemoglobin was measured by using an isothermal titration type microcalorimetry, it was hardly observed. From the oxygen dissociation curve of hemoglobin mixed with L-tyrosine, no particular influence on the allosteric effect was observed, and no change in oxygen affinity was observed.

  Furthermore, in the present invention, various enzymes can be encapsulated in the hemoglobin endoplasmic reticulum. Examples of the enzymes include catalase and superoxide dismutase. The amount added is 10,000 units / mL to 50,000 units / mL for catalase and 1000 units / mL to 10000 units / mL for superoxide dismutase, which effectively acts to suppress methation.

A comparison of the methemolysis rate of a hemoglobin solution mixed with L-tyrosine and catalase and a hemoglobin solution mixed with catalase showed that the former showed a further suppression of methetolation rate. By hydrogen scavenging ability. For example, after 60 minutes have passed since hydrogen peroxide was added to a lipid vesicle encapsulating a methetogenesis inhibitor and a heme protein, the metation rate was 20% or less (see Examples), and after 420 minutes had passed. Even in this case, the metation rate is 40% or less.

  Further, after shaking the hemoglobin solution mixed with L-tyrosine at 37 ° C. and analyzing by UV-vis spectrum measurement, fluorescence measurement, and HPLC, a slight amount of dityrosine was confirmed. When this experiment was performed by adding hydrogen peroxide to a methemoglobin solution mixed with L-tyrosine, a large amount of dityrosine was confirmed. This is due to the peroxidase activity of methemoglobin. Furthermore, we observed changes in methemoglobin concentration during refrigerated storage (4 ° C). In the hemoglobin endoplasmic reticulum that contained L-tyrosine ([L-tyrosine] = 1 mM) (inclusive system), In addition, the metation rate at the time of preparation was 3.0%), 4.4 and 9.3% at 1 month, 10.2 and 24.3% at 3 months, respectively, and the methanation was greatly suppressed in the inclusion system. This indicates that the hemoglobin endoplasmic reticulum can be stably stored for a long period of time.

  As described above, according to the present invention, in the hemoglobin endoplasmic reticulum encapsulating L-tyrosine, the methation rate is suppressed and the oxygen transport time can be extended.

  In the present invention, tyrosine may be added to an appropriate buffer solution to prepare an injection preparation (a liquid preparation for intravenous, arterial or subcutaneous injection or a liquid preparation for extracorporeal treatment). Various additives can be added to the preparation. Examples of the additive include a preservative, a buffer, a solvent, and the like.

  In the present invention, when tyrosine is administered to a patient for the purpose of clinical medicine, the dose as an active ingredient is 100 μg / kg to 1000 mg / kg, preferably 500 μg / kg to 10 mg / kg per day. .

  Next, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Preparation of L-tyrosine-encapsulated hemoglobin endoplasmic reticulum and auto-oxidation in air (37 ℃)
In a sterile atmosphere, a high-purity stroma-free hemoglobin solution (36 g / dL) obtained by purification from human erythrocytes derived from donated blood was added to pyridoxal 5 ′ phosphate (PLP, [PLP] / [Hb] = 2.5) and L-tyrosine were added at concentrations of 50, 100, 250, and 500 μM, or they were prepared without addition. The mixture was filtered through an FM microfilter (manufactured by Fuji Film) having a pore size of 0.22 μm using Remolino ^™ (manufactured by Millipore Japan) to obtain a charged hemoglobin solution. Add mixed lipid powder (phosphatidylcholine / cholesterol / DPEA mixture, manufactured by Nippon Seika Co., Ltd.) in small portions so that the lipid concentration is 4.5 wt%, and stir at 4 ° C for 12 hours to obtain hemoglobin-containing multilamellar vesicles It was. The particle size and the number of coating layers were controlled by the extrusion method using Remolino. FM microfilters were used in the order of pore sizes 3, 0.8, 0.65, 0.45, 0.3, 0.22 μm. The obtained hemoglobin vesicle dispersion was diluted with physiological saline, and after ultracentrifugation (50,000 g, 40 min), the supernatant hemoglobin solution was removed by suction. Polyoxyethylene-binding lipid dispersed in physiological saline [N- (monomethoxypolyethyleneglycol-carbamyl) distearoylphosphatidylethanolamine; N- (monomethoxypolyethyleneglyclycol-carbamyl) distearoylphosphatidylethanolamine, polyethyleneglycol molecular weight is 5300] Then, 0.3 mol% of lipid on the outer surface of the endoplasmic reticulum was added and stirred at 25 ° C. for 2 hours, and then the surface of the hemoglobin endoplasmic reticulum was modified with polyethylene glycol. The hemoglobin concentration was adjusted to 10 g / dL, and filtration was performed with a Dismic-25: 0.45 μm filter (ADVANTEC) to obtain polyethylene glycol-modified hemoglobin vesicles.

  L-tyrosine-containing hemoglobin endoplasmic reticulum ([L-tyrosine] = 50, 100, 250, 500 μM) or hemoglobin endoplasmic reticulum dispersion was shaken at 37 ° C in the atmosphere. The metation rate was calculated from the absorbance ratio of the vesicle dispersion at 405 nm and 430 nm. As a result, the L-tyrosine-encapsulated hemoglobin endoplasmic reticulum suppression rate was suppressed as the concentration of L-tyrosine added increased. When T1 / 2 is defined as the time at which the metation rate is 50%, T1 / 2 is 18 hours for L-tyrosine-unencapsulated hemoglobin vesicles, whereas L-tyrosine concentration is 50, 100, 250. In the 500 μM L-tyrosine-encapsulated hemoglobin endoplasmic reticulum, it was 20, 24, 27, and 30 hours, respectively, and T1 / 2 was significantly prolonged.

Auto-oxidation of L-tyrosine-encapsulated hemoglobin endoplasmic reticulum at an oxygen partial pressure of 40 Torr (37 ℃)
The L-tyrosine-encapsulated hemoglobin vesicle prepared in Example 1 ([L-tyrosine] = 50, 100, 250, 500 μM) or the hemoglobin vesicle dispersion was shaken at 37 ° C. at an oxygen partial pressure of 40 Torr. After collecting each sample, the metation rate was calculated from the absorbance ratio. As a result, the L-tyrosine-encapsulated hemoglobin endoplasmic reticulum suppression rate was suppressed as the concentration of L-tyrosine added increased. The time T1 / 2 at which the metation rate reaches 50% is 12.5 hours for L-tyrosine-unencapsulated hemoglobin vesicles, whereas L-tyrosine-encapsulated hemoglobin with L-tyrosine concentrations of 50, 100, 250, and 500 μM In the endoplasmic reticulum, it was 14, 15, 16, and 18.5 hours, respectively, and T1 / 2 was significantly prolonged.

Auto-oxidation of L-tyrosine-containing hemoglobin endoplasmic reticulum in the atmosphere (4 ℃)
The L-tyrosine-encapsulated hemoglobin endoplasmic reticulum ([L-tyrosine] = 1 mM) or hemoglobin endoplasmic reticulum dispersion prepared in Example 1 was stored at 4 ° C. in the atmosphere, and each sample was collected over time. The metation rate was calculated. As a result, the L-tyrosine-encapsulated hemoglobin endoplasmic reticulum suppression rate was suppressed as the concentration of L-tyrosine added increased. L-tyrosine-encapsulated hemoglobin endoplasmic reticulum and unencapsulated hemoglobin endoplasmic reticulum are 3.0% at the time of preparation, respectively, 4.4, 9.3% in one month, 10.2, 24.3% in three months, In the L-tyrosine-encapsulated hemoglobin endoplasmic reticulum, a significant reduction in the methation rate was observed.

Measurement of methation time using endoplasmic reticulum containing tyrosine derivatives, antioxidants and phenol derivatives Preparation of hemoglobin endoplasmic reticulum dispersion containing tyrosine derivatives, various antioxidants and various phenol derivatives at a predetermined concentration The metation rate was measured in the same manner as in Example 1.

  The results are shown in Table 1. Table 1 below shows the initial methation rate and 50% methemolysis time of tyrosine derivatives, antioxidants and phenol derivatives. L-tyrosine was the most effective at inhibiting methemoglobin formation in the hemoglobin endoplasmic reticulum.

Methination inhibition test using L-tyrosine and D-tyrosine
A hemoglobin endoplasmic reticulum dispersion containing an oligopeptide containing D-tyrosine or L-tyrosine was prepared, and the 50% metation rate was measured in the same manner as in Example 1.

  As a result, it was confirmed that L-tyrosine was effective in suppressing methation (FIG. 1).

Methination suppression test when enzymes are used together (1)
Prepare hemoglobin endoplasmic reticulum dispersion containing 0.25 mL L-tyrosine, hemoglobin endoplasmic reticulum dispersion containing 50000 unit / mL catalase, and hemoglobin endoplasmic reticulum dispersion containing 0.25 mL L-tyrosine and 50000 unit / mL catalase. In the same manner as in Example 1, 50% metrification time was measured.

  The results are shown in Table 2.

  Table 2 above shows the methation-inhibiting effect by Tyr and the meth-ation-inhibiting effect by catalase. L-tyrosine significantly suppressed the metation rate compared to the control (without addition of L-tyrosine and catalase). Addition of catalase further enhanced the suppression of the metation rate.

Methination suppression test when enzymes are used together (2)
0.5 wt% methemoglobin / 1 mM L-tyrosine was added to a 5 g / dL oxyhemoglobin solution ([hemoglobin] = 775 μM). To this solution, 310 μM hydrogen peroxide (same concentration as methemoglobin heme) was added every 60 minutes under atmospheric conditions at 37 ° C. and stirring until 60 minutes. Catalase 20 μL (5000 units) was immediately added to each 300 μL sample collected immediately before each hydrogen peroxide addition to eliminate hydrogen peroxide, and then the methetation rate was calculated by the cyanomethemoglobin method.
The results are shown in FIG. In the system in which hydrogen peroxide was added to the oxyhemoglobin solution or oxyhemoglobin / L-tyrosine mixed solution, the metation rate increased linearly with the increase in the number of additions, reaching about 80% in 60 minutes. In addition, in the oxyhemoglobin solution / methemoglobin mixed solution, acceleration of methemoglobin due to denaturation of methemoglobin by the added hydrogen peroxide was observed, and oxyhemoglobin became 100% methemoglobin in 60 minutes. On the other hand, in the oxyhemoglobin / methemoglobin / L-tyrosine mixed solution, the increase in the methemoglobin rate was very slow, and after 40 minutes, it was 40%, and the methemoglobin oxyhemoglobin remained at 30%. From these results, it was confirmed that methemoglobin stably erased hydrogen peroxide like catalase in the presence of L-tyrosine and suppressed methemoglobin methemoglobin.

Preparation of high-concentration L-tyrosine, methemoglobin-encapsulated hemoglobin endoplasmic reticulum In a sterile atmosphere, pyridoxal 5 is an allosteric factor in a high-purity stroma-free hemoglobin solution (36 g / dL) obtained by purification from donated human erythrocytes 'Phosphoric acid (PLP, [PLP] / [Hb] = 2.5) was added. Furthermore, the methemoglobin solution obtained by methemoglobinization using potassium ferricyanide and the potassium ferricyanide removed by gel permeation chromatography is concentrated to 36 wt% by ultrafiltration so that the final concentration is 4 wt%. Added to. Furthermore, L-tyrosine was added to a concentration of 8.5 mM or prepared without addition. The mixture was filtered through an FM microfilter (manufactured by Fuji Film) having a pore size of 0.22 μm using Remolino ^™ (manufactured by Millipore Japan) to obtain a charged hemoglobin solution. The mixed lipid powder is a mixture of dipalmitoylphosphatidylcholine / cholesterol / 1,5-O-dihexadecyl-N-succinyl-L-glutamate / N- (monomethoxypolyethyleneglycol-carbamyl) distearoylphosphatidylethanolamine (Nippon Seika) The molecular weight of the polyethylene glycol chain is 5300. The mixed lipid powder was added in small portions so that the lipid concentration was 4.5 wt%, and stirred at 4 ° C. for 12 hours to obtain hemoglobin-encapsulating multilamellar vesicles. The particle size and the number of coating layers were controlled by the extrusion method using Remolino ^™ . FM microfilters were used in the order of pore sizes 3, 0.8, 0.65, 0.45, 0.3, 0.22 μm. The obtained hemoglobin endoplasmic reticulum dispersion was diluted with physiological saline, ultracentrifugated (50,000 g, 40 min), and the supernatant hemoglobin solution was removed by suction. The hemoglobin concentration was 10 g / dL, and the Dismic-25: 0.45 μm filter (ADVANTEC ) To obtain polyethylene glycol-modified hemoglobin vesicles.

Autooxidation of high concentration L-tyrosine-encapsulated hemoglobin endoplasmic reticulum at an oxygen partial pressure of 40 Torr (37 ℃)
The L-tyrosine / methemoglobin-encapsulated hemoglobin vesicle ([L-tyrosine] = 8.5 mM) or hemoglobin vesicle dispersion prepared in Example 8 was shaken at an oxygen partial pressure of 40 Torr at 37 ° C. After collecting the sample, the metation rate was calculated from the absorbance ratio. As a result, the metation rate reached 20% after about 18 hours and 50% after 60 hours. In the case of L-tyrosine / methemoglobin-unencapsulated hemoglobin vesicles, the methation rate becomes 50% after about 13 hours under the same conditions. Therefore, high-concentration L-tyrosine / methemoglobin-encapsulated hemoglobin vesicles greatly increase the methemolysis rate. Suppressive effect was recognized.

High-concentration L-tyrosine-encapsulated hemoglobin vesicles continuously added with hydrogen peroxide L-tyrosine / methemoglobin-encapsulated oxyhemoglobin vesicles ([L-tyrosine] = 8.5 mM) or oxyhemoglobin vesicle dispersion 5 wt. Hydrogen peroxide (310 μM, same concentration as encapsulated methemoglobin) was added every 10 minutes (37 ° C. under air).

The measurement results are shown in FIG. As shown in FIG. 3, when hydrogen peroxide was added to a 5 wt% dispersion of oxyhemoglobin vesicles every 10 minutes, the metation rate reached 50% in 30 minutes ((◯ in FIG. 3). )). In contrast, 4 wt% of the 40 wt% hemoglobin encapsulated in the endoplasmic reticulum was converted to methemoglobin, and peroxidized in a 5 wt% dispersion of hemoglobin endoplasmic reticulum encapsulating 1 mM L-tyrosine. When hydrogen was added every 10 minutes, the metation rate reached 50% in 60 minutes ((■) in FIG. 3), indicating an approximately 2-fold extension effect. Furthermore, when 8.5 mM L-tyrosine was encapsulated, the metation rate reached only 40% even 420 minutes after addition of hydrogen peroxide ((●) in FIG. 3). This is a result showing a marked increase in the effect of inclusion of high concentration of L-tyrosine.

  Dispersions containing hemoglobin vesicles can be widely applied in medicine and pharmacy, and are used as blood substitutes in clinical medicine with the addition of various additives.

The figure which shows the comparison of the methation suppression effect of Hb by L-Tyr and D-Tyr. L-Tyr (◯), D-Tyr (●). Only L-Tyr suppresses hemoglobin methation, while D-tyrosine has no effect. This is a result showing that hemoglobin and L-Tyr interact specifically. The figure which shows the experimental result which produced | generated methemoglobin by the frequent addition of hydrogen peroxide with respect to the oxyhemoglobin solution which coexisted methemoglobin and L-tyrosine beforehand. In the system in which methemoglobin and L-tyrosine coexisted with oxyhemoglobin (●), the increase in the methemoglobin rate was significantly suppressed compared to the control oxyhemoglobin alone (◯). The system in which only L-tyrosine was added to oxyhemoglobin (□) showed almost the same increase in the metation rate as the control.In the system in which only methemoglobin was added to oxyhemoglobin (■), Methelation was promoted by side reactions (Fenton reaction, etc.) caused by iron ion release by hemoglobin denaturation. These results indicate that hydrogen peroxide is eliminated by peroxidase activity using methemoglobin L-tyrosine as a substrate. The figure which shows hydrogen peroxide continuous addition to the high concentration methemoglobin / L-tyrosine inclusion hemoglobin endoplasmic reticulum.

Claims (4)

  An anti-methotide containing tyrosine.   The anti-methetization agent according to claim 1, wherein tyrosine is L-tyrosine.   The anti-methetization agent according to claim 2, wherein the concentration of L-tyrosine is 0.01 mM to 20 mM. Furthermore, the methemoglobin prevention agent of any one of Claims 1-3 containing methemoglobin.

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