Hemoglobin-based oxygen carrier and preparation method therefor

Abstract

The present invention relates to a hemoglobin-based oxygen carrier comprising a complex formed by chemically binding hemoglobin, PEG, and albumin, and a preparation method therefor. Furthermore, the present invention relates to an artificial blood composition comprising the hemoglobin-based oxygen carrier as an active ingredient. The hemoglobin-based oxygen carrier according to the present invention overcomes issues of low oxygen saturation and CO binding, and exhibits excellent biocompatibility and stability, thereby demonstrating oxygen transport capacity and oxidative stability comparable to or superior to those of natural blood.

Description

Hemoglobin-based oxygen carrier and method for producing the same

[0001] The present invention relates to a hemoglobin-based oxygen carrier comprising a complex formed by chemically combining hemoglobin, PEG, and albumin, and a method for producing the same.

[0002] Furthermore, the present invention relates to an artificial blood composition comprising the hemoglobin-based oxygen carrier as an active ingredient.

[0003] Globally, blood for transfusions is experiencing a chronic shortage. Demand for blood surges, particularly during emergencies such as epidemics, wars, and natural disasters, necessitating effective responses. Furthermore, declining birth rates and a rapidly aging population are expected to lead to a long-term decline in the number of blood donors, making the development of alternative technologies increasingly crucial. Against this backdrop, "artificial blood" technology, capable of transporting oxygen or acting as a blood substitute like real human blood, is attracting attention.

[0004] Artificial blood products can be broadly categorized into oxygen carriers and blood volume expanders. Among these, oxygen carrier artificial blood products are being developed to mimic the primary function of red blood cells: transporting oxygen. Hemoglobin-based oxygen carriers (HBOCs), in particular, are being actively researched.

[0005] Since the 1980s, various types of hemoglobin-based oxygen carriers (HBOCs) have been developed as red blood cell (RBC) substitutes. These include various technologies such as intramolecularly cross-linked Hb, polymerized Hb, PEGylated Hb, and hemoglobin nanocapsules. Because hemoglobin is a crucial component of blood that transports oxygen, ongoing efforts have been made to utilize hemoglobin extracted from red blood cells.

[0006] However, hemoglobin extracted from red blood cells is a tricky molecule that can be toxic when directly exposed to tissues and blood vessels. Consequently, technological improvements have been made to offset hemoglobin's toxicity while maintaining its oxygen-carrying capacity. While some products have undergone clinical trials, these technologies face technical limitations, including delays in FDA approval due to adverse effects (e.g., increased blood pressure and cardiac toxicity) and low efficacy.

[0007] The main limitations of existing HBOC technology include the fact that hemoglobin alone is difficult to recirculate and can be rapidly eliminated from the body; pure hemoglobin can cause renal toxicity and vasoconstriction; it has side effects such as increased blood pressure and cardiac toxicity; and the oxygen affinity of hemoglobin may not match the physiological conditions in the body, which may result in low oxygen transfer efficiency.

[0008] Based on the above problems, the present invention aims to provide a novel hemoglobin-based oxygen carrier and a method for producing the same, which can transport oxygen and replace blood, and can be included in an artificial blood composition for replacing red blood cells and used for the prevention or treatment of oxygen deficiency diseases.

[0009] Specifically, the present invention implements a form in which PEG is bound to a specific position of hemoglobin and then albumin is secondarily added to the end of the PEG chain by introducing a bi-functional or multi-functional PEG (e.g., Mal-PEG-SC) as a cross-linking agent between hemoglobin and albumin. This minimizes the influence on the oxygen transport capacity of hemoglobin, while increasing the physical size of the manufactured hemoglobin group, preventing it from easily escaping between lipid membranes and enabling recycling, thereby reducing toxicity in the body and increasing blood pressure can be expected.

[0010] [1] In one aspect of the present invention, the present invention relates to a hemoglobin-based oxygen carrier comprising a complex of the following [general formula 1] formed by chemically combining hemoglobin (Hb), PEG (polyethylene glycol), and albumin:

[0011] [General Formula 1]

[0012] Hb-PEG-Albumin.

[0013] [2] In the above [1], the PEG may include a bi-functional or multi-functional PEG.

[0014] [3] In the above [1], the PEG comprises a reactive functional group capable of chemically bonding with hemoglobin and albumin, such as Mal-PEG-SC (Maleimide-PEG-Succinimidyl Carbonate), Mal-PEG-NHS (Maleimide-PEG-N-Hydroxysuccinimide), Mal-PEG-NPC (Maleimide-PEG-Nitrophenyl carbonate), Mal-PEG-Ald (Maleimide-PEG-Aldehyde), SC-PEG-NHS (Succinimidyl Carbonate-PEG-N-Hydroxysuccinimide), SC-PEG-NPC (Succinimidyl Carbonate-PEG-Nitrophenyl carbonate), SC-PEG-Ald (Succinimidyl Carbonate-PEG-Aldehyde), NHS-PEG-NPC (N-Hydroxysuccinimide-PEG-Nitrophenyl carbonate), It may be characterized by being selected from the group consisting of NHS-PEG-Ald (N-Hydroxysuccinimide-PEG-Aldehyde), NPC-PEG-Ald (Nitrophenyl carbonate-PEG-Aldehyde), SC-PEG-SC (Succinimidyl Carbonate-PEG-Succinimidyl Carbonate), SP-PEG-SP (Succinimidyl Propionate-PEG-Succinimidyl Propionate), SSA-PEG-SSA (Succinamide-PEG-Succinamide), SCM-PEG-SCM (Succinimidyl Carboxymethyl-PEG-Succinimidyl Carboxymethyl), HO-PEG-SCM (Hydroxyl-PEG-Succinimidyl Carboxymethyl), and SC-PEG-CH2COOH (Succinimidyl Carbonate-PEG-Carboxylic Acid).

[0015] [4] In the above [1], the PEG may be characterized by having a molecular weight in the range of 1,000 to 50,000 Da.

[0016] [5] In the above [1], the albumin may be characterized by having a reactive functional group capable of binding to the chain terminal of the PEG.

[0017] [6] In the above [1], the complex is one in which PEG is primarily bound to hemoglobin and albumin is secondarily bound to the other chain terminal of the PEG; or

[0018] It can be characterized in that PEG is primarily bound to albumin, and hemoglobin is secondarily bound to the other chain terminal of the PEG.

[0019] [7] In one aspect of the present invention, the present invention relates to a method for producing a hemoglobin-based oxygen carrier, comprising a step of chemically bonding hemoglobin (Hb), PEG, and albumin to form a complex of Hb-PEG-Albumin.

[0020] [8] In one aspect of the present invention, the present invention relates to an artificial blood composition comprising a hemoglobin-based oxygen carrier according to any one of [1] to [6] as an active ingredient.

[0021] [9] In the above [8], the artificial blood composition may be characterized as being used as a substitute for red blood cells (RBC).

[0022]

[0010] In the above [8], the artificial blood composition may be characterized as being used for the prevention or treatment of oxygen deficiency disease.

[0023]

[0011] In the above

[0010] , the oxygen deficiency disease may be characterized as hypoxemic hypoxia, circulatory hypoxia (stasis / ischemic), anemic hypoxia, or histotoxic hypoxia.

[0024]

[0012] In one aspect of the present invention, the present invention relates to a method for preventing or treating an oxygen deficiency disease, comprising administering to a subject a therapeutically effective amount of the hemoglobin-based oxygen carrier according to any one of [1] to [6].

[0025]

[0013] In one aspect of the present invention, the present invention relates to the use of a therapeutically effective amount of the hemoglobin-based oxygen carrier according to any one of the above [1] to [6] for the prevention or treatment of an oxygen deficiency disease.

[0026]

[0014] In one aspect of the present invention, the present invention relates to the use of a therapeutically effective amount of the hemoglobin-based oxygen carrier according to any one of the above [1] to [6] for the preparation of a medicament for the prevention or treatment of an oxygen deficiency disease.

[0027] The hemoglobin-based oxygen carrier according to the present invention solves the problems of low oxygen saturation and CO binding of existing hemoglobin-based oxygen carriers (HBOCs) through the Hb-PEG-Albumin complex structure, and exhibits excellent biocompatibility and stability, demonstrating oxygen transport and oxidative stability similar to or superior to those of normal blood. In particular, it exhibits excellent oxygen transportability, low toxicity, and stability, thereby mimicking the gas exchange capacity of blood and enabling precise control through dosage adjustment. This is expected to increase its applicability in various clinical settings.

[0028] Figures 1a and 1b show the results of analyzing each sample using gel permeation chromatography (GPC) and high-performance liquid chromatography (HPLC) to verify the chemical binding of the hemoglobin-based oxygen carrier according to the present invention.

[0029] Figure 2 shows the results of SDS-PAGE performed to confirm whether the hemoglobin-based oxygen carrier according to the present invention forms a complex through chemical covalent bonding.

[0030] Figure 3 shows the results of measuring the change in absorbance while stepwise injecting oxygen or carbon dioxide to evaluate the reactivity of a hemoglobin-based oxygen carrier according to the present invention in an oxygen and carbon dioxide environment (red or gray bars - when carbon dioxide is injected; blue bars - when oxygen is injected).

[0031] Figures 4a and 4b show electrolyte (Na) stability in various temperature (4°C, 25°C, 40°C) and gas (air, O2, CO2) environments to evaluate the electrolyte stability of the hemoglobin-based oxygen carrier according to the present invention. + , K + , Cl - ) shows the results of evaluating stability and acid-base index changes.

[0032] Hereinafter, the present invention will be described in more detail.

[0033] Each description and embodiment disclosed in this invention can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this invention fall within the scope of this invention. Furthermore, the scope of this invention is not limited by the specific descriptions described below.

[0034] Furthermore, those skilled in the art will recognize or be able to ascertain, through routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Furthermore, such equivalents are intended to be encompassed by the present invention.

[0035] definition

[0036] As used herein, the term “consisting of” means that the proportion of a particular component(s) totals 100%. The components or features listed below the term “consisting of” may be essential or mandatory.

[0037] As used herein, the term "comprising" means the presence of a feature, step, or component described below, and does not exclude the presence or addition of one or more features, steps, or components. Components or features described below "comprising" in this specification may be essential or mandatory, but some embodiments may further include other optional or non-essential components or features.

[0038] In this specification, the term “comprising” may, in some implementations, be modified to refer to “consisting essentially of” or “consisting of.”

[0039] As used herein, the term "hemoglobin" refers to one of the major proteins contained within red blood cells, responsible for transporting oxygen from the lungs to tissues and carbon dioxide from the tissues to the lungs. The functional unit of hemoglobin is a heterotetramer (α2β2). When exposed outside of red blood cells, hemoglobin rapidly dissociates into dimers and monomers, and this structural instability can lead to toxicity.

[0040] As used herein, the term “PEG” refers to a polymer consisting of repeating ethylene glycol units [-(CH2CH2O) n] refers to a polyether backbone polymer composed of PEG. In addition, “PEG” in the present specification is understood to include PEG derivatives, and may include Bi-functional PEG or Multi-functional PEG.

[0041] As used herein, the term "Bi-functional PEG" refers to a PEG derivative having two reactive functional groups at both ends of the PEG chain, and may include a Homobifunctional PEG having the same reactive functional group at both ends or a Heterobifunctional PEG having different reactive functional groups at both ends.

[0042] As used herein, the term "Multi-functional PEG" refers to a PEG derivative in which the PEG molecule has three or more reactive functional groups, which are mainly found in multi-arm or star-shaped PEG structures rather than linear PEG.

[0043] As used herein, the term "albumin" refers to a single-chain protein consisting of 585 amino acids, one of the most abundant protein components in mammalian serum. It can perform a variety of roles, including transport, pH regulation, osmotic pressure maintenance, and antioxidant activity.

[0044] As used herein, the term "oxygen carrier" refers to a substance designed to bind and release oxygen, primarily functioning to transport oxygen from the lungs to peripheral tissues and to facilitate the transport of carbon dioxide from the tissues to the lungs. Specifically, the oxygen carrier may include, but is not limited to, a hemoglobin-based oxygen carrier or a perfluorocarbon-based oxygen carrier. Furthermore, the term "oxygen carrier" in the present invention may be understood to encompass "artificial blood."

[0045] As used herein, the term “hemoglobin-based oxygen carrier (HBOC)” refers to an acellular system derived from natural hemoglobin that has been processed and modified through various chemical, genetic, or encapsulation strategies. Methods for preparing hemoglobin-based oxygen carriers may include, but are not limited to, methods utilizing chemically modified acellular suspensions (e.g., macromolecular bioconjugation (PEGylation), intramolecular cross-linking, polymerization, or enzymatic conjugation), encapsulation, recombinant production, or natural polymerization.

[0046] As used herein, the term “reactive functional group” refers to a specific chemical moiety that can form a covalent bond between two or more molecules (at least one of which is a biomolecule), which can establish a mechanism for labeling, cross-linking, or modifying the biomolecules.

[0047] As used herein, the term "oxygen-deficient disease" refers to a condition in which the level of oxygen in the body tissues is low, also known as hypoxia. The hypoxia may include, but is not limited to, hypoxemic hypoxia, circulatory hypoxia (stasis / ischemic hypoxia), anemic hypoxia, or histotoxic hypoxia. In addition, diseases that may cause hypoxia may include, but are not limited to, asthma, bronchitis, or chronic obstructive pulmonary disease (COPD).

[0048] In this specification, the term “prevention” means any act of suppressing the onset of clinical symptoms of a disease by administering a pharmaceutical composition according to the present invention.

[0049] In this specification, the term “treatment” means any action by which the clinical symptoms of a disease are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

[0050] Hemoglobin-based oxygen carrier and method for producing the same

[0051] In one aspect of the present invention, the present invention relates to a hemoglobin-based oxygen carrier comprising a complex of the following [general formula 1] formed by chemically bonding hemoglobin (Hb), PEG (polyethylene glycol), and albumin:

[0052] [General Formula 1]

[0053] Hb-PEG-Albumin.

[0054] In another aspect of the present invention, the PEG may include a bi-functional or multi-functional PEG.

[0055] Specifically, the Bi-functional PEG may include, but is not limited to, Homobifunctional PEG, for example, SC-PEG-SC (Succinimidyl Carbonate-PEG-Succinimidyl Carbonate), SP-PEG-SP (Succinimidyl Propionate-PEG-Succinimidyl Propionate), SSA-PEG-SSA (Succinamide-PEG-Succinamide), or SCM-PEG-SCM (Succinimidyl Carboxymethyl-PEG-Succinimidyl Carboxymethyl).

[0056] Additionally, the Bi-functional PEG may include, but is not limited to, Heterobifunctional PEG, for example, Mal-PEG-SC (Maleimide-PEG-Succinimidyl Carbonate), HO-PEG-SCM (Hydroxyl-PEG-Succinimidyl Carboxymethyl), or SC-PEG-CH2COOH (Succinimidyl Carbonate-PEG-Carboxylic Acid).

[0057] Furthermore, the multi-functional PEG may include, but is not limited to, 4-Arm PEG-SC (4-Arm PEG-Succinimidyl Carbonate) or 4-Arm PEG-Succinamide Acid (4-Arm PEG-SAA).

[0058] In another aspect of the present invention, the PEG comprises a reactive functional group capable of chemically bonding with hemoglobin and albumin, such as Mal-PEG-SC (Maleimide-PEG-Succinimidyl Carbonate), Mal-PEG-NHS (Maleimide-PEG-N-Hydroxysuccinimide), Mal-PEG-NPC (Maleimide-PEG-Nitrophenyl carbonate), Mal-PEG-Ald (Maleimide-PEG-Aldehyde), SC-PEG-NHS (Succinimidyl Carbonate-PEG-N-Hydroxysuccinimide), SC-PEG-NPC (Succinimidyl Carbonate-PEG-Nitrophenyl carbonate), SC-PEG-Ald (Succinimidyl Carbonate-PEG-Aldehyde), NHS-PEG-NPC (N-Hydroxysuccinimide-PEG-Nitrophenyl carbonate), It may be characterized by being selected from the group consisting of NHS-PEG-Ald (N-Hydroxysuccinimide-PEG-Aldehyde), NPC-PEG-Ald (Nitrophenyl carbonate-PEG-Aldehyde), SC-PEG-SC (Succinimidyl Carbonate-PEG-Succinimidyl Carbonate), SP-PEG-SP (Succinimidyl Propionate-PEG-Succinimidyl Propionate), SSA-PEG-SSA (Succinamide-PEG-Succinamide), SCM-PEG-SCM (Succinimidyl Carboxymethyl-PEG-Succinimidyl Carboxymethyl), HO-PEG-SCM (Hydroxyl-PEG-Succinimidyl Carboxymethyl), and SC-PEG-CH2COOH (Succinimidyl Carbonate-PEG-Carboxylic Acid).

[0059] In one embodiment of the present invention, Mal-PEG-SC (Maleimide-Polyethylene Glycol-Succinimidyl Carbonate), which is a specific example of the Bi-functional PEG, can be prepared through the following [Reaction Scheme 1], but can also be prepared through a reaction scheme using another method generally usable by a person skilled in the art to which the present invention pertains, and is not limited thereto.

[0060] [Reaction Formula 1]

[0061]

[0062] In another aspect of the present invention, the PEG may be characterized by a molecular weight ranging from 1,000 to 50,000 Da. It is preferable that the hemoglobin-based oxygen carrier according to the present invention satisfies the above molecular weight range in order to prevent it from easily escaping between lipid membranes.

[0063] Specifically, the PEG may have a molecular weight in the range of 1,000 to 50,000 Da, 2,000 to 40,000 Da, 3,000 to 30,000 Da, 4,000 to 20,000 Da, 5,000 to 10,000 Da, 2,000 to 20,000 Da, 3,000 to 15,000 Da, 4,000 to 10,000 Da, 4,000 to 9,000 Da, 4,000 to 8,000 Da, 4,000 to 7,000 Da, 4,000 to 6,000 Da, 4,500 to 5,500 Da, 5,000 to 9,000 Da.

[0064] In another aspect of the present invention, the hemoglobin may be, but is not limited to, Hb A, Hb A2 or Hb F having normal oxygen transport function.

[0065] In another aspect of the present invention, the albumin may be characterized by having a reactive functional group capable of binding to a chain terminal of the PEG.

[0066] Specifically, if the albumin has a reactive functional group that can bind to the chain terminal of the PEG, it may be bovine serum albumin (BSA) or recombinant human albumin (rHSA, e.g., Cellastim). ® S rHSA, Exbumin ® rHSA, Optibumin ® 20 Rhsa, Optibumin ® May include, but is not limited to, 25 rHSA).

[0067] More specifically, the reactive functional groups include amine (-NH2), thiol (-SH), hydroxyl (-OH), carboxyl (-COOH), aldehyde (-CHO), ketone (-C(=O)R2), ester (-COO-R'), amide (-CONH2), and phosphate (-PO4 2- ), or ether (-O-), but is not limited thereto.

[0068] In another aspect of the present invention, the complex may be characterized in that PEG is primarily bound to hemoglobin and albumin is secondarily bound to the other chain terminal of the PEG; or PEG is primarily bound to albumin and hemoglobin is secondarily bound to the other chain terminal of the PEG. That is, the order in which hemoglobin and albumin bind to PEG in the complex is not limited.

[0069] The hemoglobin-based oxygen carrier according to the present invention has a structure in which PEG is bound to a specific position of hemoglobin and albumin is added to the other chain terminal of the PEG, thereby minimizing the impact on the oxygen transport capacity of hemoglobin. Furthermore, the combined introduction of albumin and PEG, which have excellent biocompatibility, can reduce the human toxicity of hemoglobin. Furthermore, the combination of hemoglobin and albumin can improve circulation in the body, particularly oxygen circulation, when performing its function as artificial blood.

[0070] In one aspect of the present invention, the present invention relates to a method for producing a hemoglobin-based oxygen carrier as described above, comprising a step of chemically bonding hemoglobin (Hb), PEG, and albumin to form a complex of Hb-PEG-Albumin.

[0071] In another aspect of the present invention, there are various methods for chemically bonding PEG to hemoglobin (i.e., producing PEG-hemoglobin), but the type of covalent bond formed between PEG and hemoglobin changes depending on the use of different types of PEG derivatives, and the resulting three-dimensional structure also changes. For example, U.S. Patent No. 4,670,417 discloses reacting polyethylene glycol succinimidyl succinate-PEG (SS-PEG) with hemoglobin, and U.S. Patent No. 5,234,903 discloses reacting polyethylene glycol succinimidyl carbonate-PEG (SC-PEG) with hemoglobin to form PEG-hemoglobin, all of which are incorporated by reference in the present invention.

[0072] In one embodiment of the present invention, the method for producing a hemoglobin-based oxygen carrier may include any method capable of chemically binding PEG to hemoglobin or albumin, including a macromolecular bioconjugation (PEGylation) method, an intramolecular cross-linking method, a polymerization reaction, or an enzymatic conjugation method. Specifically, a macromolecular bioconjugation (PEGylation) method may be used, but is not limited thereto.

[0073] Artificial blood composition, pharmaceutical composition, preventive or therapeutic method, and use

[0074] In one aspect of the present invention, the present invention relates to an artificial blood composition comprising the hemoglobin-based oxygen carrier described above as an active ingredient.

[0075] In another aspect of the present invention, the artificial blood composition may be characterized as being used as a substitute for red blood cells (RBCs).

[0076] In another aspect of the present invention, the artificial blood composition may be characterized as being used for the prevention or treatment of oxygen deficiency diseases.

[0077] In another aspect of the present invention, the present invention relates to a pharmaceutical composition for preventing or treating an oxygen deficiency disease, comprising the hemoglobin-based oxygen carrier as an active ingredient.

[0078] Specifically, the above oxygen-deficient disease is also referred to as hypoxia and may include, but is not limited to, hypoxemic hypoxia, circulatory hypoxia (stasis / ischemic), anemic hypoxia, or histotoxic hypoxia.

[0079] In another aspect of the present invention, the present invention relates to a method for preventing or treating an oxygen deficiency disease, comprising administering to a subject a therapeutically effective amount of the hemoglobin-based oxygen carrier.

[0080] In another aspect of the present invention, the present invention relates to the use of a therapeutically effective amount of the hemoglobin-based oxygen carrier for the prevention or treatment of oxygen deficiency diseases.

[0081] In another aspect of the present invention, the present invention relates to the use of a therapeutically effective amount of the hemoglobin-based oxygen carrier for the preparation of a medicament for the prevention or treatment of an oxygen deficiency disease.

[0082] Among the terms or elements mentioned in the artificial blood composition, pharmaceutical composition, preventive or therapeutic method, and use, the same terms or elements as those described in the hemoglobin-based oxygen carrier and the method for producing the same are understood to be as mentioned in the description of the hemoglobin-based oxygen carrier and the method for producing the same.

[0083] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the content of the present invention and are not intended to limit the scope of the present invention to the following examples.

[0084]

[0085] Example

[0086] Example 1. Preparation of hemoglobin-based oxygen carrier

[0087] Maleimide (Mal), polyethylene glycol (PEG), succinimidyl carbonate (SC) for manufacturing hemoglobin-based oxygen carriers and the reagents used in the examples below were all purchased from Sigma Aldrich.

[0088] A DMSO solution of Mal-PEG-SC (50 mM, 9.6 mL, 5 kDa) was added dropwise to a PBS solution of hemoglobin (1 mM, 96 mL) (10 mM phosphate, 137 mM NaCl, pH 7.4), and the mixture was stirred at 4°C in a CO atmosphere for 90 min. Subsequently, unreacted PEG was removed using a gel filtration chromatography (GFC) column using PBS as the eluent, and a Mal-PEG-Hb PBS solution (288 mL ([Hb] = 0.33 mM)) was prepared.

[0089] A PB solution (38.8 mL, 58.3 mM sodium phosphate, 435.7 mM NaCl, 13.4 mM KCl, pH 7.2) containing 25% albumin (3.76 mM, 153.2 mL) was prepared, and the albumin solution (3 mM, 192 mL) was added to Mal-PEG-Hb. The reaction mixture (total 480 mL) was then stirred at 4°C under CO purge and dark conditions for 72 h.

[0090] Example 2. Chemical bond verification

[0091] To confirm the chemical binding of hemoglobin-based oxygen carriers, each sample was analyzed by gel permeation chromatography (GPC) and high-performance liquid chromatography (HPLC) to confirm the peak positions of each component, and the distributions when each component exists alone and when it is a mixture after binding were compared.

[0092] In gel permeation chromatography, for sample 1, a unique peak appeared at the position corresponding to the green box, as a single PEG component.

[0093] For sample 2, a peak appeared at the location indicated by the red box, as it was a single component of hemoglobin (Hb).

[0094] For sample 3, PEG and hemoglobin were mixed, and the two components did not chemically combine, so each peak appeared separately.

[0095] For sample 4, the peak appeared at the location corresponding to the blue box, as it was the only component of albumin.

[0096] In the case of sample 5, PEG and albumin were mixed, and in this case too, the two components did not chemically combine, so each peak appeared separately.

[0097] For sample 6, albumin was reacted after the PEG / hemoglobin reaction, for sample 7, hemoglobin was reacted after the PEG / albumin reaction, and for sample 8, PEG and albumin / hemoglobin were reacted simultaneously, and a new peak with a higher molecular weight than the single component of sample 1, 2, or 4, and the mixed component of sample 3 or 5 appeared. This confirmed the formation of a hemoglobin-based oxygen carrier of Hb-PEG-Albumin.

[0098] In particular, this new peak was observed at the combined position of the individual peaks of PEG, hemoglobin, and albumin, thereby confirming that PEG, hemoglobin, and albumin were chemically combined to form a single hemoglobin-based oxygen carrier (Fig. 1a).

[0099] HPLC also showed the same results as GPC, and the complex peak was confirmed at the same location, confirming that the binding of Hb-PEG-Albumin was stable and reproducible (Fig. 1b).

[0100] Example 3. Confirmation of protein binding (SDS-PAGE)

[0101] To confirm that the hemoglobin-based oxygen carrier of Hb-PEG-Albumin was formed into a single complex through chemical covalent bonding rather than simple mixing, SDS-PAGE was performed.

[0102] The red dotted line in the SDS-PAGE experimental results represents a hemoglobin-based oxygen carrier formed by chemical bonding of PEG, hemoglobin, and albumin.

[0103] As can be seen in Fig. 2, it was confirmed that the hemoglobin-based oxygen carrier of Hb-PEG-Albumin formed a complex structure by forming a chemically stable bond.

[0104] Example 4. Evaluation of oxygen transport function

[0105] In order to evaluate the oxygen transport and oxidation stability of the hemoglobin-based oxygen carrier of Hb-PEG-Albumin, whole blood and the hemoglobin-based oxygen carrier according to the present invention (Hepharmin-16, sample #1) were compared using a CO-Oximeter, and the results were as shown in Table 1 below.

[0106] Item Description Whole Blood Hempharmin-16 (#1) Interpretation tHb Total hemoglobin concentration 16.1 g / dL 13.8 g / dL Slightly low but sufficient for clinical function FO2Hb Oxygenated hemoglobin ratio 96.6% 97.1% Higher oxygen binding capacity FCOHb CO2 bound Hb ratio 2.5% 2.3% Low CO2 binding ratio, less interference with oxygen transport FMetHb Oxidized inactive Hb ratio 0.3% 0.3% Same, secured oxidation stability FHHb Oxygen-free Hb ratio 0.3% 0.3% Same, excellent oxygen retention

[0107] As confirmed in the results in Table 1, the hemoglobin-based oxygen carrier of Hb-PEG-Albumin stably delivered oxygen to the tissues with a FO2Hb value of 97.1%, which was higher than that of normal blood (96.6%). In addition, the FCOHb value was 2.3%, which was lower than that of normal blood, confirming that there was less interference in oxygen transport. Furthermore, the FmetHb value was 0.3%, which was the same as that of normal blood, confirming that the function was maintained even in the oxidative environment of the body. The oxygen retention capacity was also confirmed to stably maintain oxygen with a FHHb value of 0.3%.

[0108] Example 5. Evaluation of oxygen and carbon dioxide reactivity

[0109] To evaluate the reactivity of the hemoglobin-based oxygen carrier Hb-PEG-Albumin in oxygen and carbon dioxide environments, oxygen or carbon dioxide was injected stepwise and absorbance changes were measured. An increase in signal intensity indicates gas binding or reaction.

[0110] The red or gray bars in Figure 3 indicate that the hemoglobin-based oxygen carrier of Hb-PEG-Albumin is sensitive to carbon dioxide and capable of responding to respiratory gas exchange reactions, as evidenced by a gradual increase in response intensity upon carbon dioxide injection. This demonstrates its applicability to patients with respiratory diseases and oxygen-deficient conditions.

[0111] Additionally, the blue bars indicate that the response signal increases rapidly when oxygen is injected, indicating that the cells function as oxygen carriers, demonstrating the potential for oxygen delivery to tissues.

[0112] As the reactivity increases linearly with the gas concentration of oxygen or carbon dioxide, it was confirmed that the effect can also be controlled by precisely controlling the concentration (Fig. 3).

[0113] Example 6. Evaluation of electrolyte stability and acid-base indices

[0114] To evaluate the electrolyte stability of hemoglobin-based oxygen carriers of Hb-PEG-Albumin, electrolyte (Na) was tested under various temperature (4°C, 25°C, 40°C) and gas (air, O2, CO2) environments. + , K + , Cl - ) stability and acid-base index changes were evaluated.

[0115] In the case of a CO2 environment, a gas of a constant flow rate (approximately 1 L / min) and concentration (5% CO2 / 95% N2 mixed gas) was continuously injected into a sealed container containing a hemoglobin-based oxygen carrier sample of Hb-PEG-Albumin manufactured in Example 1 to simulate the carbon dioxide partial pressure conditions at the venous blood level in the body.

[0116] In the case of the O2 environment, high-concentration (purity of approximately 95% or more) oxygen gas was injected into the container containing the sample to simulate the hyperoxic conditions at the level of arterial blood in the body, and the electrolyte stability and pH changes of the sample were observed under those conditions.

[0117] For air environments, samples were stored under external atmospheric conditions (maintaining temperature settings, relative humidity 40-60%) to evaluate stability under typical room temperature and atmospheric gas composition (approximately 21% O2, 0.04% CO₂, 78% N₂).

[0118] Electrolyte (Na + , K + , Cl - ) concentration and acid-base indicators (pH, pCO2, HCO3) - Changes in blood gas (etc.) were measured using a blood gas analyzer (BGA, model name: i-STAT1, manufacturer: Abbott). The samples were incubated for 24 hours under sealed conditions according to gas conditions (air, 5% CO2, high concentration O2) and temperature (4℃, 25℃, 40℃), and then the changes according to each condition were evaluated. Before measurement, the samples were stabilized at room temperature (approximately 20-25℃) for 10 minutes and then injected into the analyzer, and Na was electrochemically measured using an i-STAT dedicated electrolyte measurement cartridge (Electrolyte / chemistry cartridge). + , K + , Cl - , pH, pCO2, HCO3 - The figures were automatically analyzed.

[0119] Measurements were repeated three times (n=3) for each gas condition and temperature, and the measured values ​​were expressed as mean ± standard deviation (mean ± SD).

[0120] Additionally, the concentration ratio between electrolytes in the sample (Na + / Cl - , Na+ / K + etc.) and Anion Gap (Na + + K + ) - (Cl - + HCO3 - ) values ​​were calculated, and the possibility of abnormal acid-base equilibrium and ion imbalance in the sample was further analyzed.

[0121] In Fig. 4a, the electrolyte stability and acid-base index changes in various temperature environments were confirmed.

[0122] Na + It is known that Na is the main cation of extracellular fluid and plays an important role in regulating osmotic pressure and body fluid volume. Experimental results show that Na + It was confirmed that the concentration was stably maintained without change at all temperatures, which means that even if the hemoglobin-based oxygen carrier according to the present invention is administered into the body, it does not disturb the blood sodium concentration, so it may have fewer side effects on body fluid balance and osmotic pressure control.

[0123] K + It is known that K is the main cation in intracellular fluid and plays an important role in nerve signal transmission, muscle contraction, and maintenance of cell membrane potential. Experimental results show that K + It was confirmed that the concentration was stably maintained without change at all temperatures, and this means that even if the hemoglobin-based oxygen carrier according to the present invention is administered into the body, it does not disturb the blood potassium concentration, so that side effects on cell function, especially cardiac arrhythmia, can be reduced.

[0124] Cl - It is known that Cl is the main anion of extracellular fluid and plays an important role in body fluid balance, acid-base balance, and osmotic pressure regulation. Experimental results show that Cl - It was confirmed that the concentration was stable up to 25℃, but decreased at 40℃. This is because the material was denatured or new ions were generated in the high temperature environment of 40℃, resulting in Cl- It was confirmed that it is stable when stored at room temperature / refrigerated, but caution is needed when storing at high temperatures for a long period of time, as it may have affected the concentration.

[0125] Anion Gap is an indicator that reflects the concentration of unmeasured anions, is a representative indicator of acid-base imbalance, and is calculated using the following general formula 1.

[0126] [General Formula 1]

[0127] Anion Gap = (Na + + K + ) - (Cl - + TCO2 or HCO3 - )

[0128] As a result of the experiment, it was confirmed that the Anion Gap was stable up to 25℃, but decreased at 40℃. This indicates that there is a possibility of structural, chemical stability or metabolic changes in a high temperature environment of 40℃. Therefore, it was confirmed that it is stable when stored at room temperature / refrigerated, but caution is required when storing at high temperatures for a long period of time.

[0129] TCO₂ is a major component of the bicarbonate-carbonate buffer system, the main buffering system in blood, and is an important indicator of acid-base balance. Experimental results showed that TCO₂ was stable up to 25°C but decreased at 40°C. This suggests that the acid-base buffering capacity may be reduced or destabilized in a high-temperature environment such as 40°C. Therefore, while TCO₂ is stable when stored at room temperature / refrigerated, caution is advised when storing at high temperatures for long periods.

[0130] In Fig. 4b, the electrolyte stability and acid-base index changes in various gaseous environments were confirmed.

[0131] In a CO2 environment, Na + and K +was stable, and the TCO2 decrease was reduced. Through this, it was confirmed that the hemoglobin-based oxygen carrier according to the present invention can respond to oxygen deficiency diseases by regulating the acid-base balance in response to changes in physiological CO2 levels.

[0132] In an O2 environment, Na + and K + was stable, Cl - , Anion Gap, and TCO₂ decreased. Through this, it was confirmed that the hemoglobin-based oxygen carrier according to the present invention is not destroyed by oxygen, but rather significantly reacts with oxygen, exhibits a gas exchange mechanism similar to blood, and maintains oxygen reactivity.

Claims

A hemoglobin-based oxygen carrier comprising a complex of the following [general formula 1] formed by chemically combining hemoglobin (Hb), PEG (polyethylene glycol), and albumin: [General Formula 1] Hb-PEG-Albumin. A hemoglobin-based oxygen carrier in claim 1, wherein the PEG comprises a bi-functional or multi-functional PEG. In claim 1, the PEG comprises a reactive functional group capable of chemically bonding with hemoglobin and albumin, such as Mal-PEG-SC (Maleimide-PEG-Succinimidyl Carbonate), Mal-PEG-NHS (Maleimide-PEG-N-Hydroxysuccinimide), Mal-PEG-NPC (Maleimide-PEG-Nitrophenyl carbonate), Mal-PEG-Ald (Maleimide-PEG-Aldehyde), SC-PEG-NHS (Succinimidyl Carbonate-PEG-N-Hydroxysuccinimide), SC-PEG-NPC (Succinimidyl Carbonate-PEG-Nitrophenyl carbonate), SC-PEG-Ald (Succinimidyl Carbonate-PEG-Aldehyde), NHS-PEG-NPC (N-Hydroxysuccinimide-PEG-Nitrophenyl carbonate), A hemoglobin-based oxygen carrier, characterized in that it is selected from the group consisting of NHS-PEG-Ald (N-Hydroxysuccinimide-PEG-Aldehyde), NPC-PEG-Ald (Nitrophenyl carbonate-PEG-Aldehyde), SC-PEG-SC (Succinimidyl Carbonate-PEG-Succinimidyl Carbonate), SP-PEG-SP (Succinimidyl Propionate-PEG-Succinimidyl Propionate), SSA-PEG-SSA (Succinamide-PEG-Succinamide), SCM-PEG-SCM (Succinimidyl Carboxymethyl-PEG-Succinimidyl Carboxymethyl), HO-PEG-SCM (Hydroxyl-PEG-Succinimidyl Carboxymethyl) and SC-PEG-CH2COOH (Succinimidyl Carbonate-PEG-Carboxylic Acid). A hemoglobin-based oxygen carrier, characterized in that the PEG in claim 1 has a molecular weight in the range of 1,000 to 50,000 Da. A hemoglobin-based oxygen carrier, characterized in that in claim 1, the albumin has a reactive functional group capable of binding to the chain terminal of the PEG. In the first paragraph, The above complex is one in which PEG is primarily bound to hemoglobin and albumin is secondarily bound to the other chain terminal of the PEG; or A hemoglobin-based oxygen carrier, characterized in that PEG is primarily bound to albumin and hemoglobin is secondarily bound to the other chain terminal of the PEG. A method for producing a hemoglobin-based oxygen carrier according to any one of claims 1 to 6, comprising a step of chemically combining hemoglobin (Hb), PEG, and albumin to form a complex of [general formula 1]. An artificial blood composition comprising a hemoglobin-based oxygen carrier according to any one of claims 1 to 6 as an active ingredient. An artificial blood composition according to claim 8, characterized in that the artificial blood composition is used as a substitute for red blood cells (RBC). An artificial blood composition according to claim 8, characterized in that the artificial blood composition is used for the prevention or treatment of oxygen deficiency diseases. In the 10th paragraph, the oxygen deficiency disease is, An artificial blood composition that is hypoxemic hypoxia, circulatory hypoxia (stasis / ischemic), anemic hypoxia, or histotoxic hypoxia. A method for preventing or treating an oxygen deficiency disease, comprising administering to a subject a therapeutically effective amount of the hemoglobin-based oxygen carrier according to any one of claims 1 to 6. Use of a therapeutically effective amount of a hemoglobin-based oxygen carrier according to any one of claims 1 to 6 for the prevention or treatment of oxygen deficiency diseases. Use of a therapeutically effective amount of a hemoglobin-based oxygen carrier according to any one of claims 1 to 6 for the preparation of a medicament for the prevention or treatment of oxygen deficiency diseases.

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