Pharmaceutical composition for artificial oxygen carrier comprising plant exosomes containing hemoglobin or derivative thereof, and preparation method therefor

Abstract

The present invention relates to a pharmaceutical composition for an artificial oxygen carrier comprising plant exosomes containing hemoglobin or a derivative thereof, and a preparation method therefor and, more specifically, to: a pharmaceutical composition having an oxygen carrier function by preparing exosomes from plants containing antioxidant enzymes, removing enzyme and protein components to 10% or less in the prepared exosomes to improve safety when injected into the body, and encapsulating hemoglobin or a derivative thereof; and a preparation method therefor.

Description

Pharmaceutical composition for artificial oxygen carrier comprising plant exosome containing hemoglobin or derivative thereof and method for preparing same

[0001] The present invention relates to a pharmaceutical composition for an artificial oxygen carrier comprising a plant exosome containing hemoglobin or a derivative thereof and a method for producing the same.

[0002] Red blood cells are essential components of blood, supplying oxygen to the body's tissues and organs and removing carbon dioxide. When severe bleeding occurs due to accidents, trauma, surgery, or acute coagulation disorders, leading to hemorrhagic shock, death can occur if a new blood supply is not available. The oldest, most common, and most effective method for addressing these situations is allogeneic blood transfusion.

[0003] An artificial oxygen carrier is a general term for a substitute substance (such as recombinant / purified hemoglobin) that transports oxygen to each tissue of the body through the blood vessels in place of human red blood cells. The scientific understanding of blood began in 1611 when Harvey observed blood circulation, and in 1900, Landsteiner confirmed the possibility of transfusion by discovering the ABO blood type. An artificial oxygen carrier pursues oxygen transport capacity, maintenance of a constant colloidal osmotic pressure, long-term retention time in the blood, low toxicity and ease of excretion of material components, and ease of long-term preservation, and can be transfused regardless of blood type, making it very important for national social health and security.

[0004] To address the global blood shortage, advanced biotech countries are competitively developing blood substitutes. However, the development of effective artificial blood remains stagnant. Fundamentally, research and development is underway to mass-produce oxygen carriers that can replace red blood cells, such as cell-free and chemically modified hemoglobin and oxygen-affinity polymers. However, no successful examples have yet been reported. Developed countries, including the US, UK, and Japan, have attempted to develop blood substitutes to address the blood supply shortage. Various artificial blood products based on existing hemoglobin-based artificial red blood cell products are currently under development, but progress remains stagnant.

[0005] Cross-linked hemoglobin is a form that improves the stability of free hemoglobin through intramolecular cross-linking (main products: HemAssist, Optro). All cross-linked hemoglobins have failed clinically, and the limitation of this formulation is that, despite the presence of intramolecular cross-linking, the overall structure is similar to that of a single molecule of native hemoglobin, and its oxygen affinity curve is similar to that of normal red blood cells. It has a relatively short vascular residence time and low viscosity. The main side effect is increased blood pressure due to peripheral vasoconstriction, which has been reported to be caused by NO scavenging of free hemoglobin and decreased NO synthesis in vascular endothelial cells due to low viscosity.

[0006] Polymerized hemoglobin is a form that clusters several hemoglobin molecules through intermolecular cross-linking to improve stability and suppress nephrotoxicity (main products: Polyheme, HemoLink, Hemopure). Among polymerized hemoglobins, Hemopure is available for individual use in the United States for Jehovah's Witnesses who refuse blood transfusions, and was approved for use in surgical procedures for adult anemic patients in South Africa and in the treatment of acute anemia in Russia, but was withdrawn from the market due to safety issues.

[0007] On the other hand, Polyheme and HemoLink, which have similar structures, failed clinical trials due to cardiovascular adverse effects (hypertension and myocardial infarction). While hypertension was also reported with Hemopure, the level was lower compared to the two products mentioned above. The likely cause is that larger hemoglobin polymers reduce leakage into the interstitial space, thereby reducing vasoconstrictive effects. In other words, while Polyheme and HemoLink contain a portion of monomolecular hemoglobin, Hemopure's optimized process minimizes the content of monomolecular hemoglobin, which is believed to be the primary reason for its clinical efficacy and safety.

[0008] PEGylated hemoglobin has the property of alleviating hemoglobin's vasoactivity by modifying it with the biocompatible molecule polyethylene glycol (PEG) (Main products: PHP, Hemospan, Sanguinate). Hemospan completed Phase 3 clinical trials in Europe and Phase 2 in the US for use as an artificial blood product. However, despite the efficacy and safety (no significant cardiovascular side effects), development was halted due to the manufacturer's bankruptcy. Sanguinate is currently in Phase 2 clinical trials for sickle cell anemia, and adverse safety concerns have been reported in the clinical trials.

[0009] A comprehensive review of products attempted to be developed to date shows that there is a decrease in efficacy and nephrotoxicity due to rapid renal excretion by free hemoglobin, cardiovascular side effects due to rapid extravasation and nitric oxide depletion by free hemoglobin, reflex hypertension due to off-target delivery of oxygen when administering hemoglobin formulations with low oxygen binding capacity, and instability (MetHb formation) occurs during storage.

[0010] These problems stem from the use of hemoglobin or its derivatives without an artificial membrane, which generates large amounts of free oxygen radicals and causes adverse side effects. To overcome this, there is an urgent need to develop a new paradigm for artificial blood that transcends existing limitations. Artificial blood coated with hemoglobin or other components within an artificial cell membrane is being proposed as an alternative. While attempts using lipid or polymer membranes have been made, none have yet been successful.

[0011] Accordingly, the purpose of the present invention is to develop an oxygen carrier having a form similar to red blood cells by introducing a plant exosome with an artificial cell membrane concept and encapsulating hemoglobin therein.

[0012] The present invention provides a pharmaceutical composition for an artificial oxygen carrier comprising a plant exosome and hemoglobin or a derivative thereof encapsulated within the exosome.

[0013] In addition, the present invention provides artificial blood comprising the pharmaceutical composition for the artificial oxygen carrier.

[0014] In addition, the present invention provides a method for producing a pharmaceutical composition for the artificial oxygen carrier.

[0015] The present invention relates to a pharmaceutical composition for an artificial oxygen carrier comprising a plant exosome containing hemoglobin or a derivative thereof and a method for producing the same. More specifically, the present invention produces an exosome from a plant containing an antioxidant enzyme, removes enzyme and protein components from the produced exosome to 10% or less, thereby improving safety when administered into the body, and encapsulates hemoglobin or a derivative thereof therein to provide a pharmaceutical composition having the function of an oxygen carrier, so that the composition can be utilized as a new concept artificial blood substitute without concerns about the side effects of existing artificial blood products.

[0016] Figure 1 is a schematic diagram showing a pharmaceutical composition for an artificial oxygen carrier according to the present invention.

[0017] Figure 2 shows the results of measuring the particle size of plant exosomes containing hemoglobin.

[0018] Hereinafter, the present invention will be described in more detail.

[0019]

[0020] The present invention provides a pharmaceutical composition for an artificial oxygen carrier comprising a plant exosome and hemoglobin or a derivative thereof encapsulated within the exosome.

[0021] The above pharmaceutical composition may contain 0.1 to 10 parts by weight of hemoglobin or a derivative thereof based on 100 parts by weight of plant exosomes. In this case, if the content of hemoglobin or a derivative thereof exceeds the above range, the encapsulation rate of hemoglobin and derivatives may be low, and a problem may arise in which unencapsulated hemoglobin accounts for more than 50%.

[0022] The above plant may be a plant containing an antioxidant enzyme, and may be one or more plants selected from the group consisting of, for example, beet, ginseng, edelweiss, aloe, desert rose, purslane, and watercress, but is not limited thereto.

[0023] The above hemoglobin means an oxygen-bonded (or CO-bonded), active polypeptide that is not chemically cross-linked through treatment with a chemical cross-linking agent, such as dialdehyde, and may contain less than 5%, less than 3%, or less than 1% cross-linked hemoglobin.

[0024] The above hemoglobin derivative may be selected from the group consisting of, but is not limited to, PEG cross-linked hemoglobin, thiol cross-linked nano-type hemoglobin derivative, nanoconjugate-type hemoglobin derivative and sugar cross-linked hemoglobin derivative.

[0025] The above pharmaceutical composition can treat or prevent hypoxia resulting from disease, injury and damage, blood loss due to acute bleeding, anemia, and shock by delivering oxygen to tissues.

[0026]

[0027] In addition, the present invention provides artificial blood comprising the pharmaceutical composition.

[0028] The artificial blood described above is useful for replacing blood in connection with trauma, stroke, ischemia / reperfusion injury, surgery, anemia, or other injuries, injuries, and diseases requiring transfusion, and refers to a hemoglobin formulation capable of transporting oxygen to tissues. The formulation is useful for injuries, injuries, and diseases in subjects having an appropriate blood volume but whose blood has an inadequate ability to transport and / or deliver oxygen to tissues. The artificial blood may be formulated in a hypotonic, isotonic, or hypertonic salt solution.

[0029]

[0030] In addition, the present invention provides a method for manufacturing a pharmaceutical composition for an artificial oxygen carrier, comprising the steps of: preparing a plant exosome (step 1); preparing a first solution containing ascorbic acid in the plant exosome prepared in the first step (step 2); preparing a second solution by dissolving hemoglobin or a derivative thereof in a solvent (step 3); placing a dialysis bag containing the first solution into a container containing the second solution and stirring and reacting the same (step 4); and removing unencapsulated hemoglobin or a derivative thereof (step 5).

[0031] The above plant exosome can be manufactured through the steps of 1) adding distilled water to a plant, centrifuging the separated supernatant, filtering under reduced pressure, and concentrating it, and 2) recovering the concentrated liquid, filtering under reduced pressure, and concentrating it.

[0032] The above plant exosomes can remove enzyme and protein components in an amount of 1 to 10 wt% or less.

[0033] The above plant may be a plant containing an antioxidant enzyme, and may be one or more plants selected from the group consisting of, for example, beet, ginseng, edelweiss, aloe, desert rose, purslane, and watercress, but is not limited thereto.

[0034] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0035]

[0036] <Example> Preparation of plant exosomes containing hemoglobin

[0037] 1. Preparation of plant exosomes

[0038] Beets were washed thoroughly with DW, weighed, and ground together in a blender at a ratio of 1:1 w / v % of beets: DW. 545.25 g of beets and 545 mL of DW were added together. Beet juice was placed in six 50 mL conical tubes and centrifuged at 2,000 xg for 20 minutes. The supernatant was collected and centrifuged at 4,000 xg for 20 minutes. The supernatant was collected and centrifuged at 8,000 xg for 30 minutes. The supernatant was collected and centrifuged at 10,000 xg for 1 hour. The supernatant was collected and filtered under reduced pressure with a 0.45 ㎛ nylon filter. After attaching a 100 kDa filter disc to an Amicon stirred cell, 200 mL of beet juice was added and the lid was closed. Filtration was performed by connecting to a nitrogen tank, applying 2 bar of pressure, and magnetic stirring at 100 rpm. After confirming that the mixture was concentrated to 20 mL, 80 mL of DW (4 times the volume) was added to make a total of 100 mL of the mixture. The mixture was concentrated to 20 mL. After a final three-step washing process, 20 mL of the concentrate was obtained. At this time, the concentrate was collected while making sure that the pipette did not touch the membrane. After attaching a 500 kDa filter disc to an Amicon stirred cell, 200 mL of beet juice was added, and the lid was closed. Filtration was performed by connecting to a nitrogen tank, applying 2 bar of pressure, and magnetic stirring at 100 rpm. After confirming that the mixture was concentrated to 20 mL, the final filtrate was treated with a 0.22 μm PES syringe filter to obtain plant exosomes.

[0039]

[0040] 2. Hemoglobin encapsulation

[0041] 20 mg of hemoglobin was weighed and added to 2 mL of the obtained exosomes (solution 1). The mixture was stirred at room temperature for 4 hours at 20 rpm. 20 mg of hemoglobin was weighed and added to 2 mL of the obtained exosomes. 17.613 mg of hemoglobin was weighed and added to 20 mL of deionized water (DW) (5 mM, solution 2). The solution 1 was placed in a dialysis bag, and the bag was tied. The solution 2 was placed in a beaker, and the mixture was stirred at room temperature for 4 hours at 200 rpm. Ice was placed in a sonicator, sonicated for 5 minutes, and vortexed for 1 minute. The above process was repeated three times. The mixture was frozen in a nitrogen solution and thawed in a 30°C water bath for 3 minutes. The freezing and thawing processes 2–3 were repeated 10 times. The sample was centrifuged at 20,000 xg for 30 min at 4°C. Unencapsulated hemoglobin was removed.

[0042]

[0043] 3. Exosome particle size analysis

[0044] The average size of the exosome particles manufactured above was measured using a dynamic light scattering device (Otsuka Electronics / Japan) by placing 2 mL of exosomes into a measurement cell and measuring the particle size and zeta potential. As a result of the measurement, the size of the exosomes before encapsulation was 101 nm, and the particle size of the manufactured hemoglobin-containing exosomes was 254.6 nm.

[0045]

[0046] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for an artificial oxygen carrier comprising a plant exosome and hemoglobin or a derivative thereof encapsulated within the exosome.

2. A pharmaceutical composition for an artificial oxygen carrier, characterized in that in paragraph 1, the pharmaceutical composition contains 0.1 to 10 parts by weight of hemoglobin or a derivative thereof with respect to 100 parts by weight of plant exosomes.

3. A pharmaceutical composition for an artificial oxygen carrier, characterized in that the plant in paragraph 1 is a plant containing an antioxidant enzyme.

4. A pharmaceutical composition for an artificial oxygen carrier, characterized in that in the third paragraph, the plant is at least one plant selected from the group consisting of beet, ginseng, edelweiss, aloe, desert rose, purslane, and watercress.

5. Artificial blood containing a pharmaceutical composition according to any one of clauses 1 to 4.

6. Step of preparing plant exosomes (Step 1); A step (step 2) of preparing a first solution containing ascorbic acid in the plant exosomes prepared in the above step 1; A step of preparing a second solution by dissolving hemoglobin or a derivative thereof in a solvent (step 3); Step 4: placing a dialysis bag containing the first solution into a container containing the second solution, stirring, and reacting; and A method for producing a pharmaceutical composition for an artificial oxygen carrier, comprising a step of removing unencapsulated hemoglobin or a derivative thereof (step 5).

7. In the 6th paragraph, the plant exosome is characterized in that the manufacturing method is manufactured through the steps of 1) adding distilled water to a plant, centrifuging, filtering under reduced pressure and concentrating the separated supernatant, and 2) recovering the concentrated solution, filtering under reduced pressure, and concentrating it.

8. A manufacturing method according to claim 6, characterized in that the plant exosome has enzyme and protein components removed to 1 to 10 wt% or less.

9. A manufacturing method according to claim 6, characterized in that the plant is a plant containing an antioxidant enzyme.

10. A manufacturing method according to claim 6, characterized in that the plant is at least one plant selected from the group consisting of beet, ginseng, edelweiss, aloe, desert rose, purslane, and watercress.

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