TW201827060A - Use of zinc salts and zinc compounds for stimulating erythropoiesis characterized in that the amount of red blood cells in the organism can be effectively increased, and the diseases related to the deficiency of red blood cells can be treated or prevented - Google Patents

Abstract

The present invention provides the use of a zinc salts and zinc compounds for preparing a pharmaceutical composition capable of stimulating erythropeiesis. The pharmaceutical composition provided by the present invention includes an effectively therapeutic amount of zinc salts and zinc compounds, a pharmaceutically acceptable carrier and excipient. By providing the present pharmaceutical composition containing the zinc salts and zinc compounds, the amount of red blood cells in the organism can be effectively increased, thereby being applied to treat and/or prevent the diseases related to the deficiency of red blood cells.

Description

   Application of zinc salt and zinc compound for promoting red blood cell formation   

The invention relates to a new use of a zinc salt and a zinc compound for a pharmaceutical composition, and more particularly to a use of the zinc salt and a zinc compound for the preparation of a pharmaceutical composition for promoting red blood cell formation.

Anemia is a blood disease that generally refers to the state where the number of red blood cells (RBC), Hemoglobin (Hb), or mean corpuscular volume (MCV) in the blood has fallen below a reference value. It is most directly related to the ability of the blood to carry oxygen, so it is most common to use the hemoglobin content as the criterion for judging anemia. In general, if every 100 ml of blood, male hemoglobin is less than 14 grams or female hemoglobin is less than 12 grams, It is considered to have symptoms of anemia. And because prolonged anemia can lead to memory loss, heart failure and other conditions, and it may be a potential sign of some major diseases, although the damage caused is not immediate, it is still a very important disease.

There are many causes of anemia, including chronic diseases, acute bleeding, hereditary causes (such as thalassemia), anemia caused by insufficient nutrients (such as lack of nutrients such as folic acid, vitamin B12, or iron), or organ function decline Anemia caused by aging, such as the decline of the liver, thyroid or kidney, or anemia caused by related diseases. If the anemia is classified according to the etiology and pathogenesis, the anemia can be divided into reduced red blood cell anemia, excessive red blood cell destruction anemia, and hemorrhagic anemia. Among them, the cause of reduced red blood cell production includes the lack of hematopoietic materials and hematopoietic Caused by cell abnormalities or abnormal hematopoiesis regulation, among which abnormalities in hematopoietic cells and abnormalities in the regulation of hematopoietic processes are more likely to cause chronic and long-term anemia. Therefore, in the current research on the treatment of anemia, such anemia is often targeted As a priority prevention or treatment direction.

Erythropoietin (EPO) is a glycoprotein hormone used to control the production of red blood cells in the body. It is mainly synthesized and secreted in the liver and kidneys of the human body. After adulthood, it is mainly synthesized by the kidneys into the Lord. Under normal physiological conditions, when anemia occurs, healthy kidneys will feel the pressure of lack of oxygen in the blood circulation, and then generate more erythropoietin to stimulate hematopoietic stem cells in the bone marrow, create more red blood cells, and increase oxygen. supply. However, some patients with anemia, such as those with chronic renal failure, gradually lose their kidney function. When they feel the pressure of lack of oxygen in the blood circulation, they cannot produce enough red blood cells to stimulate bone marrow hematopoiesis to relieve the anemia. Symptoms that lead to their anemia cannot be improved or become worse, which is called renal anemia.

In general, there are many causes of renal anemia, including factors such as reduced red blood cell production, increased red blood cell destruction, increased bleeding opportunities, and lack of important nutrients. Among them, the decrease in red blood cell production is the main reason. At present, in response to the above-mentioned causes of renal anemia, the treatment methods can be divided into injections of artificially reconstituted erythropoietin, iron intake, dialysis therapy, androgen, blood transfusion, and other nutrients (such as vitamins) C, folic acid or vitamin B12), etc., in addition to the above-mentioned treatment methods, in recent years, biological agents other than erythropoietin have been continuously developed as erythrocyte stimulating agents (ESAs), including receptors for erythropoietin Peptide drugs with high affinity (eg, Peginesatide), Hypoxia-Inducible Factor Stabilizer or GATA-2 inhibitors that directly regulate the mechanism of red blood cell production.

However, some of the above-mentioned therapies are biologic drugs of recombinant proteins or peptides, which are complicated to prepare and have high doubts about the cost of treatment. In addition, other nutrients are supplemented, or blood components such as hemodialysis or blood transfusion are directly replaced. There are doubts about the treatment method that can only cure the symptoms but not the root cause. Therefore, how to find a more effective but relatively low-cost treatment method has become an urgent problem in related fields.

The main object of the present invention is to provide a pharmaceutical composition containing zinc salts and zinc compounds to promote red blood cell production and use thereof. By providing such a pharmaceutical composition containing zinc salts and zinc compounds, it is possible to effectively improve the biological composition of organisms. The number of red blood cells is then used to treat diseases associated with lack of red blood cells.

Yet another object of the present invention is to provide a use of a zinc salt and a zinc compound for the preparation of a medicinal composition that promotes the formation of red blood cells. Using such a medicinal composition with a zinc salt and a zinc compound can provide a lower cost Choose to increase the number of red blood cells in the organism.

In order to achieve the above object, the present invention discloses the use of a zinc salt and a zinc compound for preparing a pharmaceutical composition for promoting the formation of red blood cells, wherein the pharmaceutical composition comprises an effective therapeutic amount of the zinc salt and the zinc compound and a pharmaceutically acceptable An excipient or vehicle.

In one embodiment of the present invention, the zinc salt and zinc compound include (but are not limited to) zinc sulfate, hydrohalide, nitrate, carbonate, citrate, etc. One of the group consisting of zinc sulfate, zinc nitrate, zinc chloride, zinc carbonate, zinc amino acid, and biological zinc, or any combination thereof.

In some embodiments of the present invention, it is disclosed that the medicinal composition described above promotes the formation of red blood cells by promoting the autocrine phenomenon of erythropoietin in specific cells.

In other embodiments of the present invention, it is also disclosed that the medicinal composition is used to improve the symptoms of red blood cell deficiency in animals.

In some embodiments of the present invention, the excipient is one selected from the group consisting of an adjuvant, a diluent, and a carrier, or any combination thereof.

In other embodiments of the present invention, the pharmaceutical composition is made into a dosage form, the dosage form is selected from a lozenge, a capsule, a powder, a powder, a solution, a suspension, One of the group consisting of an emulsion, a granule, a drop of pills, a pill, and an injection, or any combination thereof.

In other embodiments of the present invention, it is disclosed that the pharmaceutical composition is administered to a desired animal, and the animal may be a mammal, a bird or a fish.

The color scheme provided by the present invention is hereby stated in order to clearly commend the technical connotation and spirit of the invention provided by the present invention.

Figure 1: This is a comparison diagram of the number of cells in the head kidney cells of four fish species according to a preferred embodiment of the present invention. Figure 4 shows the relationship between the cell culture broth at different zinc salt addition concentrations and cell proliferation ratio in four types of fish according to a preferred embodiment of the present invention. Figure 3: This is a catfish head according to a preferred embodiment of the present invention. Renal cells, the microscopic field view of the cell culture solution after being cultured in the presence or absence of added zinc salt, and stained with cells; Figure 4: This is a grass carp, catfish and Wu Guo according to a preferred embodiment of the present invention Fish head kidney cells, whose cell culture solution is cultured with or without the addition of zinc salt, and the microscopic field view of the cells after staining; Figure 5: These are four types of fish according to a preferred embodiment of the present invention. After extracting zinc-binding protein from its cell culture solution, the components of protein electrophoresis and LC-MS / MS were performed; Figure 6: This is a Wu Guoyu, which is a preferred embodiment of the present invention, and its blood loss model in vivo Number of red blood cells, blood cell volume in blood at different time points And hemoglobin changes; Figure 7: This is a preferred embodiment of the Wu Guoyu, a blood sample collected at different time points in a living blood loss model after injection of saline or zinc sulfate solution. Microscopic view of the cells after staining; Figure 8: This is a catfish, a preferred embodiment of the present invention, the zinc-stimulated erythropoiesis reaction, and the addition of soluble erythropoietin receptors, or human recombinant erythropoietin Figure 9 shows the effect of red blood cells on antibody formation. Figure 9: This is a rat bone marrow cell of a preferred embodiment of the present invention. Relationship between the number of cells; Figure 10: This is a relationship between the number of cells and the number of cells in the culture medium of a rat bone marrow cell with / without the addition of zinc salts in a preferred embodiment of the present invention Figure 11: This is a rat bone marrow cell according to a preferred embodiment of the present invention. After the cell culture medium is cultured with or without the addition of zinc salt, it is subjected to CD71 transferrin receptor fluorescence staining method and membrane. protein Microscopic view of TER119 erythrocyte-specific blood group glycoprotein A staining; and Fig. 12: This is a rat bone marrow cell of a preferred embodiment of the present invention. The zinc salt promotes the red blood cell formation reaction and the addition of soluble red blood cell formation. Effect of serotonin receptors or rat erythropoietin neutralizing antibodies on erythrocyte production.

In order for your reviewers to have a better understanding and understanding of the features of the present invention and the effects achieved, I would like to refer to the preferred embodiments and detailed explanations as follows: In the present invention, the current recombinant protein or Peptide biological preparations are complicated in preparation and costly, and treatment methods such as hemodialysis or blood transfusion can only treat the symptoms but not the root cause, and provide a novel medicinal composition that promotes the formation of red blood cells and uses thereof. By promoting the formation of red blood cells, the pharmaceutical composition of the present invention can effectively increase the amount of red blood cells in the body of the user, thereby achieving the purpose of controlling the amount of red blood cells in the body. At the same time, the preparation cost of the used medical composition components is relatively low, and Without excessively complicated preparation technology, it can effectively reduce the financial burden of patients during treatment.

Therefore, the present invention provides a use of a zinc salt and a zinc compound for preparing a pharmaceutical composition that promotes the formation of red blood cells, wherein the pharmaceutical composition comprises an effective therapeutic amount of the zinc salt and the zinc compound and a pharmaceutically acceptable carrier or agent. Shape agent. The pharmaceutical composition mainly uses zinc salts and zinc compounds to effectively increase the concentration of Erythropoietin (EPO) in the body, thereby achieving the purpose of promoting the formation of red blood cells in the body. On the other hand, the pharmaceutical composition can allow zinc salts and zinc compounds to enter the body in a variety of forms through the action of adjuvants, providing the delivery of the pharmaceutical composition components in a manner most suitable for the needs of patients.

In the present invention, the effective therapeutic amount refers to the dose or amount of a drug that achieves a therapeutic response or desired effect after a subject takes the pharmaceutical composition, which is usually between the minimum effective dose (minimum effective dose, The range between MED) and the maximum tolerated dose (MTD), which is usually higher than one of the average values that can cause the drug's efficacy, and it needs to be statistically higher than that given to users by placebo. Effectiveness of treatment.

In the present invention, the zinc salt and zinc compound used are a compound formed by combining zinc metal ion (Zn ²⁺ ) with acid ion or non-metal ion. Although zinc ion is an ion capable of forming a compound with all anions, However, in order to allow the zinc salt to dissociate into zinc ions for pharmacological effects after delivery to the body, in the present invention, only the zinc salt that can be dissolved in water or a similar solvent is provided for the medicine provided by the present invention Preparation of composition. The preparation of zinc salts and zinc compounds and the acquisition of their material sources are generally not complicated. For example, after reacting zinc metal with a source of halogen gas or solid, a zinc halide compound (such as zinc chloride or zinc bromide, etc.) can be obtained, which is a fairly common source of zinc salt. Based on the above principles, the zinc salts and zinc compounds provided by the present invention are, for example, salts formed by zinc ions and sulfuric acid, hydrohalic acid, nitric acid, citric acid, and the like. For example, the zinc salt according to the present invention may be selected from one of the group consisting of zinc sulfate, zinc nitrate and zinc chloride, or any combination thereof. However, the types and sources of zinc salts and zinc compounds provided by the present invention are not limited thereto, and any zinc salt and zinc compound that conforms to the spirit of the foregoing invention should be the object of the protection scope of the present invention.

Among them, the excipient used in the present invention is a compound used in a pharmaceutical composition to enhance the effect of an active ingredient in a drug, or to promote dissolution and absorption in a final dosage form of a drug to enhance the display of the active ingredient. In addition, excipients can also stabilize the physical and chemical properties of the active ingredients during the pharmaceutical manufacturing process, avoiding the denaturation of the pharmaceutical composition and stabilizing its quality. In addition to the factors mentioned above, the choice of excipients is still It depends on other factors such as the route of administration, dosage form or active ingredients of the pharmaceutical composition after preparation. Based on the above principles, the excipient provided by the present invention is selected from one of the group consisting of an adjuvant, a diluent, and a carrier, or any combination thereof.

Among them, the medicinal composition provided by the present invention is mainly administered by oral administration or injection. However, the administration route provided by the present invention is not limited to this. And the way of administration of zinc compounds to the body to achieve a sufficient drug blood concentration should be the subject of the scope of the present invention. Based on the foregoing principles, the pharmaceutical composition provided by the present invention is made into a dosage form for the delivery of zinc salts and zinc compounds, wherein the dosage form is selected from a lozenge, a capsule, a powder, a powder, a solution Agent, a suspension agent, an emulsion, a granule, a drop, a pill and an injection, or any combination thereof, however, the dosage form of the pharmaceutical composition provided by the present invention is not based on This is limited.

Among them, the zinc salt and zinc compound provided by the present invention are used for the preparation for promoting the formation of red blood cells, by utilizing the phenomenon of promoting the autocrine phenomenon of erythropoietin in specific cells to achieve the purpose of promoting the formation of red blood cells. The specific cell line Contains head kidney cells of fish animals or bone marrow cells of mammals. By promoting the autocrine phenomenon of erythropoietin in specific cells, erythropoietin will further stimulate the differentiation and production of red blood cells, thereby improving the lack of red blood cells in the body, and then treating various diseases caused by the lack of red blood cells in the body. The basis of novel pharmaceutical composition uses.

In the following, specific implementation examples are used for the description of the organizational technical content, features, and results of this invention, and can be implemented accordingly, but the scope of protection of the present invention is not limited thereto.

【Materials and Methods】

[Experimental animal]

The adult male rats (Sprague-Dawley breed) used in the experiments provided by the present invention weigh 210 ± 20 grams and are housed in indoor animal facilities at 22 ± 3 ° C and 55 ± 5% relative humidity. Light-dark cycle with replacement every 12 hours. The use of animals has been reviewed and approved by the Institutional Animal Care and Use Committee of National Ocean University of Taiwan.

There are four types of fish used in the experiments provided by the present invention: crucian carp, grass carp, silver carp, and tilapia. These fish were obtained from the farm before the experiment. After fingerlings, they are kept in polyethylene tanks for two months to prepare. The average data of experimental fish species are as follows: the total length of catfish is 22 ± 3 cm and weighs about 207 ± 69 g (n = 44); the total length of grass carp is 41 ± 2 cm and weighs about 680 ± 101 g (n = 6); The total length is 39 ± 3 cm and weighs about 556 ± 73 grams (n = 6); the 27 ± 1 fish has a total length of 27 ± 1 cm and weighs about 362 ± 43 grams (n = 15). All animals are used by National Taiwan Review and approval by Institutional Animal Care and Use Committee of Ocean University.

[Sample preparation of head kidney cells of each species]

In the experiment provided by the present invention, the head kidney cell line used was obtained from the head kidney of each of the aforementioned fish species. The fish's head and kidneys were sieved through a sieve with a pore size of 26 microns to obtain cells, and then washed with DMEM cell culture solution (Dulbecco's Modified Eagle's medium, DMEM), and then repeated centrifugal washing to remove impurities, and then used the cell culture solution The remaining cells are resuspended in the culture medium to complete the preparation of the fish head and kidney cell suspension.

[Sample preparation of rat bone marrow cells]

In the experiment provided by the present invention, the bone marrow cell line used was isolated from the femur of a 12-week-old rat. It was rinsed with DMEM cell culture solution (Dulbecco's Modified Eagle's medium, DMEM) and collected from the femur. After the bone marrow contents are repeatedly washed by centrifugation to remove impurities, the remaining cells are resuspended in the culture solution with the cell culture solution to complete the preparation of the rat bone marrow cell suspension.

[Example 1]

[ Effects of zinc salts and zinc compounds on erythrocyte production of head kidney cells in four fish species]

In order to observe the effect of zinc salts and zinc compounds on the erythropoiesis function of the head kidney cells of the four fish species, the present invention uses the head kidney cells of the four fish species in cell culture fluid (10% of fish serum, corresponding to the head kidney cells). Source) The culture was carried out in suspension culture for one day, and each group was added with a zinc chloride ranging from 0.01 mM to 2 mM as an experimental group for comparison. The comparison item is the number of the first type of cells obtained after the cell culture fluid is subjected to Percoll density chromatography. As mentioned earlier, the first type of cell line is an immature red blood cell with a density of 1.020 g / mL. .

From the results, it can be found that after the kidney cells of the four fish species were cultured for one day with a cell culture solution containing different concentrations of zinc chloride, the total number of cells in the cell culture solution significantly changed (as shown in Figure 1). However, an increase in the number of cells of the first type and a decrease in the number of the cells of the second type were observed in the four types of fish at the same time (as shown in Fig. 1). After the significant increase on the first day, it entered the plain period and no longer had significant changes. The proportion of cells increased was three times that of the control group without added zinc chloride, indicating that the added zinc salt stimulated the immature Proliferation of red blood cells, and the number of cells increased by three times.

[Example 2]

[Effects of Different Zinc Salt Concentrations on the Red Blood Cell Production Function of Head Kidney Cells in Four Fishes]

In order to observe the effect of different zinc salt concentrations on the erythropoiesis function of the head kidney cells of the four fish species, the present invention uses the head kidney cells of the four fish species in cell culture fluid (10% fish serum, corresponding to the source of the head kidney cells) The suspension culture was performed for one day, and the group without adding zinc salt in the cell culture solution was used as the control group, and the group with 0.3 mM-1.2 mM zinc chloride in the cell culture solution was used as the experimental group. Comparison. The comparison items are divided into the total number of cells in the cell culture solution after four days of culture, and the total number of cells in the two major categories (fraction 1 and fraction 2) classified by density after the cell culture solution is subjected to Percoll density chromatography. The layered state of the cell culture fluid was selected for observation after Percoll density chromatography, because the maturity of red blood cells is closely related to the cell size in the bone cartilage. Therefore, the mature and immature red blood cells are distinguished by cell density chromatography. And observe the number of cells. The first type of cells are immature red blood cells with a density of 1.020 g / mL, and the second type of cells are mature red blood cells with a density of 1.070 g / mL. .

From the results, it can be found that after the kidney cells of the four types of fish were cultured for one day with a cell culture solution containing different concentrations of zinc chloride, the number of the first type of cells in the cell culture solution changed significantly (as shown in Figure 2). Among them, crucian carp has a significant immature red blood cell proliferation effect when the zinc chloride is added at a concentration greater than 0.6mM, and has the largest cell proliferation ratio (500%) when the zinc chloride is added at a concentration of 1.2mM. Grass carp (Grass carp) has significant immature red blood cell proliferation effect when zinc chloride is added at a concentration greater than 0.2mM, and has the largest cell proliferation ratio (520%) when zinc chloride is added at a concentration of 0.6mM. Carp (silver carp) has significant immature red blood cell proliferation effect when zinc chloride is added at a concentration greater than 0.3mM, and has the largest cell proliferation ratio (660%) when zinc chloride is added at a concentration of 0.3mM. However, tilapia has significant immature red blood cell proliferation effect when zinc chloride is added at a concentration greater than 1.2 mM, and has maximum cell proliferation when zinc chloride is added at a concentration of 1.5 mM. Example (290%).

[Example 3]

[Observation of erythrocytes of sturgeon head kidney cells by zinc salts and zinc compounds with microscope and cell staining ]

In order to observe the effect of zinc salt on the erythrocyte production function of the head kidney cells of the four fish species, the present invention is to culture the head kidney cells and the cell culture fluid of the four fish species in suspension culture for one day, The group without zinc salt was used as the control group. 1.2 mM, 0.6 mM, 0.3 mM, and 1.5 mM zinc chloride (Carassius auratus, catfish, and kokoku) were added to the cell culture medium depending on the species of fish. In the experimental group, the two groups were compared. The comparison items are divided into the composition of the cell particle size distribution of the cell culture medium after one day of culture, and observation with a microscope with Giemsa Stain and transferrin receptor immunofluorescence staining Observe.

Observing the state of the stained cells under a microscope, it can be found that most of the cells present in the uncultured cell culture medium are lymphocytes (a) (about 70%), neutrophils (b), and Basophilic cells (c) (as shown in Figure 3 (A)), and after a day of culture with 0.3 mM zinc chloride in the cell culture medium, cells in the culture medium began to stimulate the proliferation of red blood cells. (About 42%), including (1) lymphoid hemoblasts, (2) early erythroblasts, (3) polychromatophilic erythroblast, (4 ) Orthochromatic erythroblasts and (5) erythrocytes (as shown in Figure 3 (B)), only cells (1) ~ (3) of these cells will be transferred to iron Protein receptor staining, while cells (4) ~ (5) cannot be stained by transferrin receptor (as shown in Figure 3 (D)), indicating that immunofluorescent staining of transferrin receptor can be used to identify Mature red blood cells.

Based on this, we observed that the head kidney cells of grass carp, catfish, and kelp were cultured with zinc salt-added cell culture solution. Before the culture, no cells were immunofluorescently stained at the transferrin receptor. Medium color (as shown in Figure 4 (A) (C) (E)), and after one day of cell culture, it can be clearly observed that some cells show color in immunofluorescent staining of transferrin receptor, indicating that After the head kidney cells of grass carp, catfish, and pelagic fish were cultured in a cell culture solution supplemented with zinc salt for one day, they did have newly-born immature red blood cells. (As shown in Figure 4 (B) (D) (F))

[Example 4]

[Observe the composition of blood components in proliferating blood cells by protein electrophoresis and LC-MS / MS]

Generally speaking, transferrin is considered to be an active substance that carries zinc in fish serum to stimulate the head kidney to produce erythrocyte hyperplasia. Therefore, in order to confirm which component of blood in the present invention carries zinc, the present invention uses the aforementioned The proliferating cell sample obtained in the experiment was extracted with zinc-binding proteins in the sample by means of detergent and Immobilized Metal Affinity Chromatography (IMAC). Observe by protein electrophoresis and LC-MS / MS.

From the results, it can be found that in the proliferating blood cell samples of the four fish, the performance was seen at the protein position of 72 kDa. In addition, the results of LC-MS / MS can also be used in the acetate buffer solution (acetate buffer). Under elution, a significant absorption value was obtained at a wavelength of 280 nm (as shown in Figure 5), which proves that the blood cell proliferation sample provided in this experiment does have transferrin as the zinc-carrying zinc in the serum. Active substance.

[Example 5]

[ Effects of zinc salts and zinc compounds on erythrocyte function in fish blood loss models ]

In order to observe the effect of zinc and its derivatives on the function of erythrocytes, the present invention will provide a simulation model of blood loss of Wu Guoyu. The blood loss model provides 54 Wu Guoyu for testing, of which 0.2 mL of Blood was used as the reference line for the experimental control, and then the remaining 48 tails of kelp were sampled with 3.0 mL of blood by tail vein puncture, and expressed as a state of blood loss of 20%. Then, 48 fins were divided into two groups, and 0.25 mL of saline solution (control group) and 0.25 mL of zinc sulfate solution (concentration of 1.47 mg / mL or 5.1 mM) were injected, and 2 hours (0 Day), 3rd day, 6th day and 12th day, 6 fish were selected from each experimental group for 0.2mL blood sampling, and the collected blood was used for blood analysis and microscope observation.

It can be found from the results that two hours after Wu Guoyu took blood as a simulated blood loss of 20%, the number of red blood cells, hematocrit, and hemoglobin in his body decreased to about the initial reference value. 55% -65% (as shown in Figure 6). In the control group injected with saline solution, there were no significant changes in the number of red blood cells, blood cell volume ratio, and hemoglobin protein in the body of Wu Guoyu from 2 hours (day 0) to day 12 after injection. On the contrary, when From the third day after the injection of zinc sulfate aqueous solution, Wu Guoyu, which simulates blood loss, has significantly improved both the number of red blood cells, the volume ratio of hemoglobin and hemoglobin, and by the 6th and 12th days, The trend of continuous increase shows that zinc salt in Wu Guoyu's blood loss model does have the ability to promote red blood cells.

In addition, by observing the sampled cells with Giemsa Stain and a microscope, it can be found that the group injected with saline solution (control group) and the group injected with zinc sulfate aqueous solution (experimental group) on day 0, The cell composition in the blood is mainly mature red blood cells (cells 5 shown in Figure 7 (A) (B)), and the samples taken on the 3rd and 6th days were injected with saline solution. The cell composition of the group (control group) is still dominated by mature red blood cells (such as cells 5 shown in Figure 7 (C) (E)) with a small number of orthochromatic erythroblasts (Cells 4 shown in Figure 7 (C)); and the cell composition of the group (experimental group) injected with a zinc sulfate aqueous solution on the 3rd day was mainly the newly proliferated lymphoid hemoblasts (lymphoid hemoblasts) , Early erythroblasts and polychromatophilic erythroblast (early erythroblasts) (cells 1, 2 and 3 as shown in Figure 7 (D)), and the group injected with zinc sulfate aqueous solution (Experimental group) On the 6th day, further positive staining was found Chromochromoblasts (cell 4 shown in Figure 7 (F)), prove that zinc salt can indeed induce erythropoiesis within three days in Wu Guoyu's blood loss model, and it begins to form after day 6 More mature red blood cells (positive staining red blood cells).

[Example 6]

[Zinc salts and zinc compounds affect the way red blood cells are produced in fish head and kidney cells]

In order to observe the effect of zinc salts and zinc compounds on the red blood cell formation of fish head and kidney cells, in this example, the carp head kidney cells, cell culture fluid (DMEM / F12 medium and 20% carp serum, FBS), and 1.2 mM chloride were used. Zinc mixed cell culture solution, after adding various concentrations of soluble erythropoietin receptor or recombinant human erythropoietin antibody, the cells were collected for one day after co-cultivation, and the cells were collected and centrifuged through Percoll density Isolate and calculate the number of cells in the first type of cells, compare with the number of cells at the beginning of the culture and calculate the ratio, and finally observe the results.

It can be found from the results that after adding different concentrations of soluble erythropoietin receptors or human recombinant erythropoietin antibodies to co-culture, the amount of erythropoiesis induced by zinc salts will increase with the added soluble The concentration of erythropoietin receptor, or human recombinant erythropoietin antibody increases and decreases, when the concentration of soluble erythropoietin receptor is 7.5 μg / mL, or when the concentration of human recombinant erythropoietin antibody is 200 μg / mL At about 80% of the Erythropoiesis effect was removed (as shown in Figure 8). In the case that the erythropoietin is not added in the culture system provided by the present invention, and the erythropoietin immune activity is not detected in the cell culture medium, it can be proved that the erythropoiesis induced by the zinc salt provided by the present invention is borrowed It is achieved by making fish head kidney cells autocrine erythropoietin.

[Example 7]

[ Effects of zinc salts and zinc compounds on erythrocyte production of rat bone marrow cells]

In order to observe the effect of zinc salts and zinc compounds on the erythropoiesis function of rat bone marrow cells, in this example, rat bone marrow cells and cell culture fluid (DMEM / F12 medium and 10% fetal bovine serum, FBS) were cultured in suspension. The culture was performed for one day, and the group without adding zinc salt in the cell culture solution was used as the control group, and the group with 0.3 mM zinc chloride in the cell culture solution was used as the experimental group for comparison. The comparison items are divided into the composition of the cell particle size distribution of the cell culture solution after one day of culture, and observation with a microscope, Giemsa Stain, and O-dianisidine stain. ), CD71 transferrin receptor fluorescent staining method, membrane protein TER119 erythroid-specific glycophorin A (erythroid-specific glycophorin A) fluorescent staining method and DNA staining method, etc. The physiological state of cells in the culture medium was observed.

It can be seen from the results that, after the rat bone marrow cells were cultured for one day with 0.3 mM zinc chloride-containing cell culture solution, the number of cells with a particle size of 5.1 μm in the cell culture solution after one day of culture was larger than the same particle diameter before the culture. The number of large and small cells increased by 1.64 times (n = 11), and the number of cells with a particle size of 6.1 μm in the culture medium decreased by 55 days compared with the number of cells with the same particle size without culture. % (As shown in Figure 9). In addition, after observing under the microscope with Giemsa Stain and o-dianisidine stain, cells can be further found. The cell line with a particle size of 5.1 μm in the uncultured cell culture medium is a reticulocyte (the R cell shown in Figure 10 (A)), while the cell with a particle size of 6.1 μm Erythrocytes or part of Lymphocytes (E and L cells as shown in Figure 10 (A)); cells added after cultured with 0.3mM zinc chloride for one day Although the particle size of the cells is about 5.1 μm, It can be further judged that the appearance is a multi-lobed star shape, which is an immature reticular red blood cell (such as the x cell shown in Figure 10 (B) (D)); and observe the cell type and shape through a microscope By comparison, it can be found that the number of erythrocytes has decreased (as shown in Figure 10 (C) (D)), and this result is also consistent with the aforementioned cell count results.

In addition, during the process of red blood cell production, during the differentiation of preerythroblasts into orthochromatic erythroblasts and reticulocytes, the CD71 transferrin receptor and The membrane protein TER119 is highly expressed in erythroid-specific glycophorin A. Therefore, in the experiments provided by the present invention, the cells were cultured for one day without being cultured and cultured with 0.3 mM zinc chloride. All subsequent cell samples were further stained with CD71 transferrin receptor fluorescence staining, membrane protein TER119 red blood cell-specific blood group glycoprotein A (erythroid-specific glycophorin A) fluorescence staining, and DNA staining. The state of the cell.

It was found from the staining results that "Cell X" had a corresponding antibody response to CD71 transferrin receptor and membrane protein TER119 (as shown in Figure 11 (A) (B)), but no DNA staining reaction (as shown in Figure 11). Figures (C) and (D)), from which it is judged that these cells with a particle size of about 5 μm not only have the characteristic of staining with heme, but also have a multi-leafed appearance, and are associated with CD71 transferrin and The membrane protein TER119 has a corresponding antibody response, but no cell nucleus (no DNA staining reaction). The newly formed star “cell x” is confirmed to be immature reticulocytes; the last step in the production of red blood cells is to transform the positive Orthochromatic erythroblasts differentiate into a nucleated reticulocyte and another type of "pyrenocyte" containing a thin layer of cytoplasm. From (Figure 10 (B) (D) and Figure 10), it can be found that "cell y" is separated from "cell x" and cannot be stained by heme, but contains a nucleus with a cytoplasmic membrane coating. Therefore, it is determined that "cell y" is a "pyrenocyte" with the nucleus of red blood cells squeezed out.

Finally, in order to confirm that the addition of zinc chloride contributed to the increase in the number of cells of the aforementioned immature reticulocytes (the number of cells was 164% of the control group, n = 11), so in addition to the experimental conditions (DMEM / F12 medium, In addition to 10% FBS and 0.3 mM zinc chloride), we also tested zinc chloride and fetal bovine serum albumin (FBS) separately from the experimental group of cell culture to observe whether it affects the function of stimulating cell proliferation, and in order to Test whether other metal ions can achieve the same effect. Two experimental groups, ferric chloride and ferric chloride added fetal bovine serum protein (FBS), were tested to see if it affected the function of stimulating cell proliferation. The experimental results It was confirmed that the other groups had almost no effect on cell culture, and the immature reticulocytes in the experimental samples did not increase (data not provided), indicating that only the aforementioned experimental conditions (DMEM / F12 medium, 10% FBS and 0.3mM chlorine Zinc) can achieve the effect of promoting the increase of the number of immature reticulocytes, and the purpose of red blood cell formation.

[Example 8]

[Zinc salts and zinc compounds affect red blood cell formation in rat bone marrow cells]

In order to observe the effect of zinc salts and zinc compounds on the erythrocyte formation of rat bone marrow cells, in this example, rat bone marrow cells, cell culture fluid (DMEM / F12 medium and 20% fetal bovine serum, FBS) and 0.6 mM chlorinated The mixed cell culture solution of zinc was added with various concentrations of soluble erythropoietin receptor or rat erythropoietin antibody, and the results were observed after co-culture for one day.

It can be found from the results that after adding different concentrations of soluble erythropoietin receptors or rat erythropoietin neutralizing antibodies and co-cultivation, the amount of erythropoiesis induced by zinc salts will increase as the cells are added. When the concentration of soluble erythropoietin receptor or rat erythropoietin antibody increases and decreases, when the concentration of soluble erythropoietin receptor is 30 μg / mL, about 60% of the effect of erythropoiesis (Erythropoiesis) is eliminated When the concentration of rat erythropoietin-neutralizing antibody is 70 μg / mL, the effect of erythrocyte production will be completely eliminated (as shown in Figure 12). Therefore, in the case that the erythropoietin is not added to the culture system provided by the present invention, and the erythropoietin immune activity is not detected in the cell culture solution, the erythropoiesis system induced by the zinc salt provided by the present invention can be proved This is achieved by causing bone marrow cells to secrete erythropoietin.

In summary, the present invention does provide a use of a zinc salt and a zinc compound for preparing a medicinal composition that promotes the formation of red blood cells. The zinc salt and the zinc compound provided by the present invention can promote the secretion of erythropoietin by specific cells. The phenomenon achieves the purpose of promoting the formation of red blood cells, and its ability to promote the production of reticulocytes has been verified on fish and mammals. By providing such a use, zinc salts and zinc compounds can be used to promote the autocrine of erythropoietin to accelerate the efficiency of erythrocyte production in animals, to improve the lack of red blood cells in animals, and to reduce related diseases caused by lack of red blood cells (such as :anemia). At the same time, because zinc salts and zinc compounds have relatively low production costs and ease of acquisition, the present invention does provide a more economical and economical choice of pharmaceutical compositions for the purpose of promoting the formation of red blood cells.

However, the above are only the preferred embodiments of this creation, and are not intended to limit the scope of implementation of this creation. For example, all changes and modifications based on the shapes, structures, features, and spirits described in the scope of this creation application patent Shall be included in the scope of the patent application for this creation.

Claims (10)

    A zinc salt and a zinc compound are used for preparing a medicinal composition that promotes the formation of red blood cells, wherein the medicinal composition comprises an effective therapeutic amount of a zinc salt and a pharmaceutically acceptable excipient.         The use according to item 1 of the scope of patent application, wherein the zinc salt and zinc compound are selected from the group consisting of zinc sulfate, zinc nitrate, zinc chloride, zinc carbonate, zinc amino acid, and biological zinc One of them or any combination thereof.         The use as described in item 1 of the scope of the patent application, wherein the pharmaceutical composition achieves the purpose of promoting red blood cell production by promoting the autocrine phenomenon of erythropoietin in specific cells.         The use according to item 1 of the scope of the patent application, wherein the pharmaceutical composition is used to improve the symptoms related to lack of red blood cells in animals.         The use according to item 1 of the scope of patent application, wherein the symptom associated with the lack of red blood cells is anemia.         The use according to item 1 of the scope of patent application, wherein the excipient is selected from one of the group consisting of an adjuvant, a diluent, and a carrier, or any combination thereof.         The use according to item 1 of the scope of patent application, wherein the pharmaceutical composition is made into a dosage form, the dosage form is selected from a lozenge, a capsule, a powder, a powder, a solution, and a suspension. , An emulsion, a granule, a drop, a pill, and an injection, or any combination thereof.         The use according to item 1 of the patent application scope, wherein the pharmaceutical composition is administered to a mammal or a fish animal as required.         The use according to item 3 of the patent application scope, wherein the specific cell line is a head kidney cell of a fish animal.         The use according to item 3 of the scope of patent application, wherein the specific cell is a bone marrow cell of a mammal.