WO2020125464A1 - Use of composition containing ferrous amino acid chelate particles for preparing pharmaceutical product for treating or slowing autoimmunity-related diseases - Google Patents
Use of composition containing ferrous amino acid chelate particles for preparing pharmaceutical product for treating or slowing autoimmunity-related diseases Download PDFInfo
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- WO2020125464A1 WO2020125464A1 PCT/CN2019/123878 CN2019123878W WO2020125464A1 WO 2020125464 A1 WO2020125464 A1 WO 2020125464A1 CN 2019123878 W CN2019123878 W CN 2019123878W WO 2020125464 A1 WO2020125464 A1 WO 2020125464A1
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- amino acid
- composition
- ferrous
- acid chelate
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
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- A61K31/28—Compounds containing heavy metals
- A61K31/295—Iron group metal compounds
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Definitions
- the present invention relates to the use of a composition containing ferrous amino acid chelate particles sintered by a ferrous amino acid chelate compound, in particular for the treatment or alleviation of autoimmune-related diseases.
- autoimmune diseases are when the body's immune system functions out of balance, that is, the immune system attacks the tissues or organs in the body as foreign substances or pathogens, often causing chronic inflammation or even loss of function of the attacked tissues and organs.
- Common autoimmune diseases include: rheumatoid arthritis, systemic lupus erythematosus, and atopic dermatitis.
- MMP-9 matrix metalloproteinase family 2.
- MMP-9 is considered to be closely related to rheumatoid arthritis because it can decompose the important components of articular cartilage such as gelatin, type I, type IV, type V and type IX, and integrin.
- Symptoms of atopic dermatitis include itching, redness, and cracking of the skin, and they are prone to repeated attacks and are difficult to cure.
- environmental, psychological factors and oxidative stress such as free radicals
- oxidative stress can cause inflammation of the skin of people with genetic predisposition of immune disorders and cause clinical symptoms of atopic dermatitis.
- autoimmune diseases are systemic and difficult to diagnose, the treatment of autoimmune diseases is more laborious and lengthy than general diseases, so looking for a drug that effectively treats or slows down autoimmune-related diseases is an urgent problem that needs to be solved. .
- the present invention provides the use of a composition for the preparation of a medicament for the treatment or alleviation of autoimmune-related diseases, wherein the composition contains ferrous iron sintered by a ferrous amino acid chelate Amino acid chelate particles, and the average particle diameter of the ferrous amino acid chelate particles is 500 nm to 2600 nm, and the average molecular weight is 1,500 Dalton to 600,000 Dalton.
- the pharmaceutical product contains an effective dose of the composition and a pharmaceutically acceptable carrier.
- the average molecular weight of the ferrous amino acid chelate particles is 1,500 Daltons to 15,000 Daltons; in another embodiment, preferably, the The ferrous amino acid chelate particles have an average molecular weight of 400,000 Daltons to 550,000 Daltons, preferably, the ferrous amino acid chelate particles have an average molecular weight of 1,500 Daltons to 550,000 channels Dalton, more preferably, the average molecular weight of the ferrous amino acid chelate particles is 525,538 Daltons.
- the weight ratio of ferrous iron to amino acid of the ferrous amino acid chelate in the composition is between 1:1 and 1:4.
- the weight ratio of ferrous iron to amino acid of the ferrous amino acid chelate in the composition is between 1:1.5 and 1:2.5.
- the ferrous amino acid chelate in the composition is a ferric amino acid chelate prepared by mixing an iron compound and an amino acid and heating at 60°C to 90°C for 8 hours to 48 hours Among them, the iron compound is preferably an inorganic iron compound, more preferably an inorganic ferrous compound.
- the weight ratio of iron compound to amino acid is between 1:1.2 and 1:1.5.
- the iron compound is ferrous sulfate, ferrous chloride, ferrous pyrophosphate, or a combination thereof; the amino acid is glycine.
- the present invention further provides the use of a composition for preparing a medicament for treating or slowing down autoimmune-related diseases, wherein the composition is a ferrous amino acid chelate sintered from a ferrous amino acid chelate Composed of particles, and the average particle diameter of the ferrous amino acid chelate particles is 500 nanometers to 2,600 nanometers, the average molecular weight is 1,500 daltons to 600,000 daltons, and the pharmaceutical product is composed of an effective dose Consisting of the composition and a pharmaceutically acceptable carrier.
- the present invention further provides a composition for treating or alleviating autoimmune-related diseases, wherein the composition contains ferrous amino acid chelate particles sintered from ferrous amino acid chelate, and The ferrous amino acid chelate particles have an average particle size of 500 nm to 2,600 nm, and an average molecular weight of 1,500 Daltons to 600,000 Daltons.
- the present invention further provides a composition for treating or alleviating autoimmune-related diseases, wherein the composition is composed of ferrous amino acid chelate particles sintered from ferrous amino acid chelate, and The ferrous amino acid chelate particles have an average particle size of 500 nm to 2,600 nm, and an average molecular weight of 1,500 Daltons to 600,000 Daltons.
- the present invention further provides a method for treating or slowing down autoimmune-related diseases, which comprises administering an effective dose of a composition to a recipient and a pharmaceutically acceptable carrier, wherein the composition contains a ferric amino acid Ferrous amino acid chelate particles formed by sintering the chelate compound, and the average particle diameter of the ferric amino acid chelate particles is 500 nm to 2,600 nm, and the average molecular weight is 1,500 Daltons to 600,000 Daltons.
- the present invention additionally provides a method for treating or slowing down autoimmune-related diseases, which consists of administering an effective dose of a composition to a recipient and a pharmaceutically acceptable carrier, wherein the composition is composed of ferroamino acid
- the chelated ferrous amino acid chelate particles are sintered, and the average particle diameter of the ferric amino acid chelate particles is 500 nm to 2600 nm, and the average molecular weight is 1,500 dal Dayton to 600,000 Daltons.
- the "effective dose” described in the present invention refers to the dose required to achieve the effect of treating or slowing down autoimmune-related diseases; according to some embodiments of the present invention, it refers to the administration of a specific range of amounts containing ferrous amino acid
- the composition of ferrous amino acid chelate particles sintered by the chelate compound can reduce nitric oxide free radicals produced by macrophages, reduce the degranulation effect of basophilic spheres, inhibit the secretion of matrix metalloproteinases, Inhibit the migration of monocytes, inhibit the NF- ⁇ B and IKK- ⁇ pathways, reduce the incidence of lameness in rheumatoid arthritis, the degree of inflammation, slow the itching caused by atopic dermatitis, the severity of disease symptoms, and accelerate skin repair And increase the free radical scavenging rate.
- the effective dose is calculated based on the concentration of the composition of the present invention in the experimental examples of this specification. According to the initial estimation method announced by the US Food and Drug Administration, the dose is converted on the basis of an adult of 60 kg. It is calculated by converting 3.1 times the recommended daily intake per kg body weight (/kg bw/d) of the human body into 1 time the dose of the rabbit.
- the subject of administration of the composition of the present invention may be a human, and the effective dose of the composition may be 2.5 mg/kg to 10 mg/kg.
- the effective dose is calculated based on the concentration of the composition of the present invention in the experimental examples of this specification. According to the initial estimation method announced by the US Food and Drug Administration, the dose is converted on the basis of an adult of 60 kg. It is calculated by taking 1.8 times the recommended daily intake per kg of body weight (/kg bw/d) as the dog's 1 dose.
- the subject of administration of the composition of the present invention may be a human, and the effective dose of the composition may be 1 mg/kg to 10 mg/kg.
- the effective dose of the composition may be 3 mg/kg to 7 mg/kg, more preferably, the effective dose of the composition may be 5.5 mg/kg.
- the "pharmaceutically acceptable carrier” described in the present invention includes, but is not limited to, reducing agent, solvent, emulsifier, suspending agent, decomposer, Binder (binding agent), excipient (excipient), stabilizer (stabilizing agent), diluent (diluent), gelling agent (gelling agent), preservative (preservative), lubricant (lubricant), surfactant (surfactant), and other similar or applicable carriers of the invention.
- the reducing agent includes, but is not limited to, ascorbic acid, citric acid, acetic acid, propionic acid, butyric acid, and lactic acid ), hydroxysuccinic acid (malic acid), sulfonic acid (sulfonic acid), succinic acid (succinic acid) or a combination thereof.
- the "pharmaceutical products” described in the present invention may exist in various forms, including, but not limited to, liquid, semi-solid, and solid pharmaceutical forms, such as solutions, emulsions, suspensions, and powders (powder), tablet (tablet), pill (pill), lozenge (lozenge), tablet (troche), chewing gum (chewing gum), capsule (capsule), liposome, suppository and other similar or
- the dosage form of the present invention is suitable.
- the pharmaceutical product is an enteral or parenteral dosage form.
- the enteral dosage form is an oral dosage form
- the oral dosage form is a solution, an emulsion, a suspension, a powder, a lozenge, a pill, a lozenge, a tablet, a chewing gum, or a capsule.
- the treatment or alleviation of autoimmune related diseases is through the degranulation effect including suppression of immune cells.
- the aforementioned immune cells are mast cells.
- the aforementioned degranulation effect of suppressing immune cells can reduce the amount of histamine released.
- the autoimmune-related diseases include, but are not limited to rheumatoid arthritis.
- the autoimmune-related diseases include, but are not limited to atopic dermatitis.
- the composition of the present invention has the effects of reducing nitric oxide free radicals produced by macrophages, reducing the degranulation of basophiles, inhibiting the secretion of matrix metalloproteinases, inhibiting the migration of mononuclear cells, inhibiting NF- ⁇ B and IKK- Beta pathway and increase free radical scavenging rate and other effects, and it has been shown in animal experiments that it can reduce the incidence of lameness and inflammation in rheumatoid arthritis and can reduce the itching, disease severity and acceleration of skin caused by atopic dermatitis Repair, etc. Therefore, the composition of the present invention can effectively treat or slow down autoimmune-related diseases, especially rheumatoid arthritis and atopic dermatitis.
- Figure 1 The relative proportions of the effects of 0, 25, 50, 100 ⁇ g/mL treatment of macrophages Raw264.7 on cell growth.
- Figure 2 The relative proportions of cell growth influenced by the treatment of mononuclear cell THP-1 with 0, 50, 100, 250 ⁇ g/mL of the composition of the present invention.
- Figure 3 The relative proportion of nitric oxide production concentration after treatment of inflammation-induced macrophages Raw264.7 with 0, 25, 50, 100 ⁇ g/mL of the composition of the present invention.
- Fig. 4 Relative proportion of the amount of ⁇ -hexosaminidase released by the degranulation of basophilic cells RBL-2H3 treated with the composition of the present invention at 0, 10, 50 and 100 ⁇ g/mL.
- FIG. 5 shows the relative proportion of cell growth effects caused by treatment of synovial sarcoma cells SW982 stimulated with TNF- ⁇ protein with the composition of the present invention at 0, 50, 100, 250, and 500 ⁇ g/mL.
- FIG. 6 is an enzyme map of treatment of synovial sarcoma cells SW982 stimulated with TNF- ⁇ protein with the composition of the present invention at 0, 50, 100, 250, and 500 ⁇ g/mL.
- Figure 7 The relative proportion of the number of abnormal cell migration caused by the treatment of synovial sarcoma cells SW982 stimulated with TNF- ⁇ protein with 0, 50, 100, 250, 500 ⁇ g/mL synovial sarcoma cells to mononuclear THP-1 cells.
- Figure 8 The amount of RelA (NF- ⁇ B) in the nucleus of synovial sarcoma cells SW982 stimulated with TNF- ⁇ protein was treated with the composition of the present invention at 0, 50, 100, 250, 500 ⁇ g/mL.
- Fig. 9 The veterinarian checked the itchiness during the first to sixth visits.
- Figure 10 The veterinarian checked the severity of the symptoms at the first to sixth visits.
- Figure 11 Percent free radical scavenging of the compositions of the present invention at 0.25, 0.5, 1, 1.5, 2, 4 mg/mL.
- the composition containing ferrous amino acid chelate particles of the present invention is made by Taiwan Ligand Co., Ltd. of China (batch number: F171001; manufacturing date: October 5, 2017), and the composition is The freeze-dried powder was prepared in the following manner. First, ferrous sulfate and glycine acid (purity 98% or more) are mixed in a weight ratio of 1:1.3 and heated at 60°C to 90°C for 8 hours to 48 hours to obtain a ferrous amino acid chelate, in which The chelating ratio of ferrous iron to amino acid chelate is between 1:1 and 1:4, then the ferrous amino acid chelate is sintered at 200-240°C to Ferrous amino acid chelate particles were obtained.
- the average particle size of the ferroamino acid chelate particles was measured to be 1465.90 ⁇ 132.29 nanometers by dynamic light scattering in water through a laser particle size analyzer (Beckman Coulter, N5, Submicron Particle Size Analyzer). Using Waters Alliance 2695 System to perform gel penetration chromatography (GPC) to determine the number average molecular weight (Mn), weight average molecular weight (Mw), peak molecular weight (MP) and polydispersity (PDI), respectively, 68188 Dalton, 525,538 Dalton, 286,426 Dalton and 7.707205.
- GPC gel penetration chromatography
- Macrophages Raw264.7 (BCRC, Taiwan, 60001) were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium (Gibco 11965092) containing 10% heat-inactivated fetal bovine serum (FBS) at 37°C In the incubator of 5% carbon dioxide, it is about eight points full.
- DMEM Dulbecco's Modified Eagle Medium
- FBS heat-inactivated fetal bovine serum
- the basophilic cells RBL-2H3 (BCRC, Taiwan, 60310, China) were cultured in a minimum essential medium (MEM) medium (Gibco 11095-080) in a 37°C, 5% carbon dioxide incubator until about eight percent full.
- MEM minimum essential medium
- Synovial sarcoma cells SW982 (BCRC, Taiwan, 60535, China) were cultured in an incubator at 37°C and 0% carbon dioxide with L15 medium (Gibco, 11415-064) containing 10% FBS to about eight times full.
- Mononuclear THP-1 cells (BCRC, Taiwan, 60430) were cultured in RPMI1640 medium (Gibco, A10491-01) containing 10% heat-inactivated fetal bovine serum (fetal bovine serum, FBS) at 37°C, 5 % Carbon dioxide in the incubator to about eight full.
- RPMI1640 medium Gibco, A10491-01
- FBS heat-inactivated fetal bovine serum
- BNCS Boron neutron capture synovectomy
- Example 1 The effect of the composition of the present invention on the growth of macrophages Raw 264.7
- the enzyme of the mitochondria of living cells can be used to reduce the MTT (Thiazolyl Blue Tetrazolium Bromide) reagent to formazan crystals, and the absorbance value after dissolution is measured to reflect the survival rate of the cells.
- MTT Thiazolyl Blue Tetrazolium Bromide
- composition of the present invention is first dissolved in 0.1 M citric acid to prepare a solution (pH 3.5) with a concentration of 250 mg/mL, and then further diluted to a concentration of 25, 50, or 100 ⁇ g/mL.
- step (3) Treat the macrophage Raw264.7 of step (2) with different concentrations of the composition of the present invention in step (1), and use the composition of the present invention of 0 ⁇ g/mL as a control group, each condition being the same Three replicates were performed in each of the two sets of trials.
- the composition of the present invention is not toxic to immune cells (macrophages), and even shows that it can slightly promote the growth of immune cells compared to the control group.
- Example 2 Effect of the composition of the present invention on the growth of mononuclear THP-1 cells
- This example is the same as the method of Example 1, but the experiment was performed using THP-1 mononuclear cells (the cell density was 3 ⁇ 10 5 cells per well) of Preparation Example 5, and the concentration of the composition of the present invention was 50 , 100, 250 ⁇ g/mL).
- the enzyme of living cell mitochondria can reduce MTT (Thiazolyl Blue Tetrazolium Bromide) reagent to formazan crystals, and measure the absorbance value after dissolution to reflect the survival rate of cells.
- MTT Thiazolyl Blue Tetrazolium Bromide
- composition of the present invention is not toxic to immune cells (monocytes), and even shows that it can slightly promote the growth of immune cells compared to the control group.
- Example 3 The effect of the composition of the present invention on the production of nitric oxide by macrophage Raw264.7 in an inflammation model
- composition of the present invention is first dissolved in 0.1 M citric acid to prepare a solution (pH 3.5) with a concentration of 100 mg/mL, and then further diluted to a concentration of 25 ⁇ g/mL, 50 ⁇ g/mL, and 100 ⁇ g/mL.
- step (3) Treat the macrophages Raw264.7 with different concentrations of the composition of step (1) in step (1) for two hours, and take 0 ⁇ g/mL as the control group, each condition is tested in the same two groups Repeat three times in each of the steps, then add 1 ⁇ g/mL lipopolysaccharide (Sigma, L4391) to the group treated with the composition of the present invention, and use Raw264.7, a macrophage that only added 1 ⁇ g/mL lipopolysaccharide, as a positive control Group, continue to cultivate for 20 hours.
- 1 ⁇ g/mL lipopolysaccharide Sigma, L4391
- the composition of the present invention can inhibit the production of nitric oxide by macrophages during the inflammatory reaction induced by lipopolysaccharide and inhibit the inflammatory reaction.
- Example 4 ⁇ -hexosaminidase release amount of degranulation of basophils RBL-2H3 treated with the composition of the present invention
- basophils and mast cells in the body The function of basophils and mast cells in the body is mainly to perform degranulation when they are stimulated by allergens to release ⁇ -hexosaminidase, tryptase, histamine and other allergic mediators to cause immune reactions. . Because the properties of basophil RBL-2H3 are similar to mast cells, this experiment uses basophil RBL-2H3 to simulate the ⁇ -hexosaminidase in the allergic medium released by mast cells To evaluate the occurrence of de-granulation, the test method is as follows:
- composition of the present invention is first dissolved in 0.1 M citric acid to prepare a solution (pH 3.5) with a concentration of 100 mg/mL, and then further diluted to a concentration of 10 ⁇ g/mL, 50 ⁇ g/mL, and 100 ⁇ g/mL.
- Example 5 Effect of the composition of the present invention on the growth of synovial sarcoma cell SW982
- TNF- ⁇ tumor necrosis factor- ⁇
- MTT Thiazolyl Blue Tetrazolium Bromide
- composition of the present invention is first dissolved in 0.1 M citric acid to prepare a solution (pH 3.5) with a concentration of 250 mg/mL, and then further diluted to a concentration of 50, 100, 250, and 500 ⁇ g/mL.
- step (3) Treat the synovial sarcoma cell SW982 of step (3) with different concentrations of the composition of the invention in step (1), and use the composition of the invention of 0 ⁇ g/mL as a control group, each condition being the same Three replicates were performed in each of the two sets of trials.
- Matrix metalloproteinases decompose important components of articular cartilage, so they are considered to be closely related to rheumatoid arthritis. Matrix metalloproteinases are generally expressed in the form of zymogen (pro-MMP), and become MMP after secretion and activation. In this experiment, the amount of Pro-MMP-9 and MMP-9 in the culture solution of synovial sarcoma cell SW982 was tested by enzyme map. The experimental steps are as follows:
- composition of the present invention is first dissolved in 0.1 M citric acid to prepare a solution with a concentration of 250 mg/mL, and then further diluted to a concentration of 50, 100, 250, and 500 ⁇ g/mL.
- step (2) Treat the synovial sarcoma cells SW982 of step (2) for 48 hours with different concentrations of the composition of the invention and 0.1 M citric acid (ie, 0 ⁇ g/mL of the composition of the invention) in step (1).
- the colloid is washed once with dd H 2 O, then placed in a reaction buffer (40 mM Tris-HCl, pH 8.0, 10 mM CaCl 2 , 0.01% NaN 3 ), and shaken at 37° C. for 12-16 hours.
- a reaction buffer 40 mM Tris-HCl, pH 8.0, 10 mM CaCl 2 , 0.01% NaN 3
- the results are shown in Fig. 6.
- the control group had significantly less Pro-MMP-9 than the group treated with the composition of the present invention, while the MMP-9 matrix metal secreted by the synovial sarcoma cells SW982 treated with the composition of the present invention
- the protease is obviously higher than the control group, and it can be seen that the Pro-MMP-9 of the control group is modified into activated MMP-9 in a large amount compared to the group treated with the composition of the present invention.
- the higher the treatment concentration of the composition of the present invention the more it can inhibit the secretion of Pro-MMP-9 and MMP-9 matrix metalloproteinase from the synovial sarcoma cell SW982.
- the composition of the present invention can inhibit the decomposition of important components of articular cartilage by inhibiting the secretion of matrix metalloproteinases to achieve the effect of treating or slowing rheumatoid arthritis.
- Example 7 Effect of the composition of the present invention on the aggregation of inflammatory cells caused by rheumatoid arthritis
- the occurrence of an inflammatory reaction will release histamine from the damaged tissues, increase the permeability of blood vessels, and cause neutrophils and monocytes to aggregate and differentiate to produce an immune response.
- the synovial sarcoma cells SW982 of Preparation Example 4 were taken, planted in a 6-well cell culture plate at a density of 3 ⁇ 10 5 cells per well, and cultured in a 37°C incubator for 24 hours.
- the cells on the surface of the upper well plate were stained with 1/10 diluted Giemsa solution (Fluka, 48900) for one hour, and then wiped clean with a cotton swab above the upper well plate.
- the results are shown in Figure 7.
- the group stimulated with TNF- ⁇ protein alone significantly increased the number of migrating mononuclear cells THP-1, while the group treated with the composition of the present invention had an inhibitory mononuclear
- Example 8 Effect of the composition of the present invention on the IKK- ⁇ and RelA (NF- ⁇ B) pathways of TNF- ⁇ protein-stimulated synovial sarcoma cells SW982
- NF- ⁇ B transcription factor RelA
- IKK When subjected to external stimuli such as TNF- ⁇ , IKK will also be phosphorylated to further decompose IKK- ⁇ and render NF- ⁇ B activated. Activated NF- ⁇ B will enter the nucleus and trigger the expression and production of specific genes A large number of inflammatory factors cause a subsequent inflammatory response. Therefore, this experiment tested the occurrence of abnormal inflammation by measuring the amount of IKK- ⁇ in the cytoplasm and NF- ⁇ B in the nucleus after synovial sarcoma cells SW982 stimulated by TNF- ⁇ protein.
- composition of the present invention is first dissolved in 0.1 M citric acid to prepare a solution (pH 3.5) with a concentration of 250 mg/mL, and then further diluted to a concentration of 50, 100, 250, and 500 ⁇ g/mL.
- step (1) Treat cells with different concentrations of the composition of the present invention in step (1), and use 0.1M citric acid solution as a control group, place them in a 37°C incubator for 24 hours, and collect the cells.
- NC membrane nitrocellulose membrane
- the results of nuclear protein are shown in Figure 8.
- the amount of nuclear RelA (NF- ⁇ B) in the group treated with the composition of the present invention was lower than that of the control group, and the higher the concentration of the composition of the present invention was The lower the amount of RelA (NF- ⁇ B) in the nucleus of the membranous sarcoma cell SW982, the composition of the present invention can suppress the abnormal inflammatory response of cells and slow down the degree of inflammation of rheumatoid arthritis.
- Rheumatoid arthritis is a systemic chronic inflammatory disease characterized by hyperplasia of synovial membrane cells accompanied by infiltration of inflammatory cells, articular surface pannus (pannus formation) and joint damage.
- Experimental animals induced arthritis with antigens, the course of disease is similar to rheumatoid arthritis, so as in the literature Trivillin et al. (2014).
- the rabbits prepared in Preparation Example 6 were divided into a control group (without administration of the composition of the present invention) and an experimental group.
- the experimental group was administered the composition of the present invention for 60 days.
- the clinical symptoms that is, palpation during palpation or lameness, began to administer.
- the experimental group is further divided into a high-dose group and a low-dose group, wherein the high-dose group is administered orally 24 mg/kg once a day, and the low-dose group is administered orally 8 mg/kg once a day.
- the composition of the present invention is further divided into a high-dose group and a low-dose group, wherein the high-dose group is administered orally 24 mg/kg once a day, and the low-dose group is administered orally 8 mg/kg once a day.
- the rabbits of the experimental group and the control group are sacrificed, and the knee joint synovial tissue (synovial tissue) on both sides is collected and fixed with 4% paraformaldehyde; then embedded in paraffin and cut into Thin sections of 3-5 ⁇ m, stained with hematoxylin and eosin, observed with a microscope and real-time imaging system, and performed histopathological examination and basis Et al. (1997) to assess the severity of inflammation (Grade 0 is normal, Grade 3 is the most severe).
- rabbits gradually developed claudication symptoms, causing a total of 70% of rabbits to exhibit clinical symptoms of pain and claudication (of which 64% of rabbits presented before the composition of the invention was administered Clinical symptoms, the other 36% of the rabbits did not develop lameness symptoms until the composition of the present invention was administered).
- the rabbits in the control group had macroscopic lesions and more severe inflammation
- the rabbits in the high-dose group had macroscopic lesions (all accompanied by lameness symptoms) and a lower proportion of inflammation
- the lower dose group is between the two groups mentioned above, so the composition of the present invention has the effect of delaying or improving the course of disease, reducing the incidence of claudication and the degree of inflammatory response in rabbits. Therefore, the composition of the present invention has an effect of treating or slowing rheumatoid arthritis.
- the experimental animals of Preparation Example 7 were divided into two groups.
- 12 animals (A01-A12) received dermatological drug treatment, and the composition of the present invention was also administered once a day at a dose of 100 mg/10 kg.
- the control group had 2 animals (B01, B02) were also treated with skin medication, but were additionally given steroids (B01) or Oclacitinib (B02), respectively.
- Dermatological treatment medications were given according to the animal's condition, including antifungal drugs (itrconazole), antibiotics, and anti-tissue Amines, peripheral vasodilator drugs (pentoxifylline), antioxidants (Vitamin) E, etc., all experimental animals after the use of the composition of the present invention, every two weeks for a follow-up evaluation, evaluation tools include veterinarian examination records, skin bacteria And mold culture.
- pruritus visual analog (PVAS) as an evaluation tool (Olivry, T., Marsella, R., Iwasaki, T., Mueller, R., & International Task Force On On Canine Atopic Dermatitis. (2007).Validation of CADESI-03, a scale for clinical trials, enrolling dogs with a topic dermatitis. Veterinary dermatology, 18 (2), 78-86.), with 0 points for not itchy, and 10 points for extremely itchy (extremely itchy).
- the experimental group administered with the composition of the present invention has been examined by a veterinarian and the results are shown in Fig. 9. The degree of itching is alleviated; the control group is those who receive Oclacitinib (B02), the degree of itching relief and acceptance
- the composition of the present invention has similar animals.
- the composition of the present invention has the effect of treating or alleviating atopic dermatitis.
- Example 11 free radical scavenging ability of the composition of the present invention
- DPPH 1,1-diphenyl-2-picrylhydrazyl
- composition of the present invention is first dissolved in 0.1 M citric acid to prepare a solution (pH 3.5) with a concentration of 250 mg/mL, and then diluted to a concentration of 0.25, 0.5, 1, 1.5, 2, 4 mg/mL.
- step (1) Add 10 ⁇ L of step (1) the composition of the present invention at different concentrations, and use 0 mg/mL as a control group.
- the DPPH radical scavenging ability of the group to which the composition of the present invention is added is higher than that of the control group, and as the concentration of the composition of the present invention increases, the radical scavenging ability also increases, so the free The possibility of causing skin inflammation and related symptoms.
- the composition of the present invention can not only reduce nitric oxide free radicals produced by macrophages, reduce the degranulation of basophiles, inhibit the secretion of matrix metalloproteinases, inhibit the migration and inhibition of mononuclear cells Inflammation-related NF- ⁇ B and IKK- ⁇ pathways and increase free radical scavenging rate, etc., and animal experiments have shown that it can reduce the incidence of rheumatoid arthritis lameness, the degree of inflammation and can slow down the atopic dermatitis caused Itching, the severity of disease symptoms, and accelerated skin repair, etc. Therefore, the composition of the present invention can effectively treat or slow down autoimmune-related diseases, especially rheumatoid arthritis and atopic dermatitis.
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Abstract
The present invention provides a use of a composition for preparing a medicament for treating or relieving a autoimmunity-related disease, wherein the composition contains ferrous amino acid chelate particles sintered from a ferrous amino acid chelate, and the ferrous amino acid chelate particles have an average particle size of 500 nm to 2600 nm and an average molecular weight of 1,500 to 600,000 Daltons, and the medicament contains an effective dose of the composition and a pharmaceutically acceptable carrier. The composition of the present invention has the effect of treating or relieving a autoimmunity-related disease, especially rheumatoid arthritis and ectopic dermatitis.
Description
本发明涉及含有亚铁胺基酸螯合物烧结而成的亚铁胺基酸螯合物粒子的组合物的用途，特别是用于治疗或减缓自体免疫相关疾病。The present invention relates to the use of a composition containing ferrous amino acid chelate particles sintered by a ferrous amino acid chelate compound, in particular for the treatment or alleviation of autoimmune-related diseases.
自体免疫相关疾病是当身体的免疫系统功能失衡时，也就是免疫系统将体内的组织或器官视为外来的物质或病原体而攻击，常使受攻击的组织、器官慢性发炎甚或失去功能。常见的自体免疫疾病包括：类风湿性关节炎、全身性红斑性狼疮、异位性皮肤炎等。Autoimmune diseases are when the body's immune system functions out of balance, that is, the immune system attacks the tissues or organs in the body as foreign substances or pathogens, often causing chronic inflammation or even loss of function of the attacked tissues and organs. Common autoimmune diseases include: rheumatoid arthritis, systemic lupus erythematosus, and atopic dermatitis.
类风湿性关节炎常因发炎导致细胞浸润(cellular infiltration)、滑膜增生(synovial hyperplasia)，并进一步造成骨质侵蚀(bone erosion)，而基质金属蛋白酶(matrix metalloproteinase，MMP)家族中的MMP-2、MMP-9因可分解明胶、I型、IV型、V型和IX型胶原及黏合素(integrin)等关节软骨的重要成分，因而被认为与类风湿性关节炎密切相关。Rheumatoid arthritis often causes cellular infiltration, synovial hyperplasia, and further bone erosion due to inflammation, and MMP- in the matrix metalloproteinase (MMP) family 2. MMP-9 is considered to be closely related to rheumatoid arthritis because it can decompose the important components of articular cartilage such as gelatin, type I, type IV, type V and type IX, and integrin.
异位性皮肤炎的症状有发痒、红肿、皮肤龟裂，且容易反复发作难以根治。目前已知环境、心理因素及氧化压力(如：自由基)会引起免疫失调的遗传预先倾向性(genetic predisposition)者的皮肤的发炎变化并造成异位性皮肤炎的临床症状。Symptoms of atopic dermatitis include itching, redness, and cracking of the skin, and they are prone to repeated attacks and are difficult to cure. At present, it is known that environmental, psychological factors and oxidative stress (such as free radicals) can cause inflammation of the skin of people with genetic predisposition of immune disorders and cause clinical symptoms of atopic dermatitis.
由于自体免疫疾病是全身性的问题且确诊困难，自体免疫疾病的治疗相较于一般疾病更为辛苦且漫长，因此寻找一种有效治疗或减缓自体免疫相关疾病的药物是目前亟需解决的课题。Since autoimmune diseases are systemic and difficult to diagnose, the treatment of autoimmune diseases is more laborious and lengthy than general diseases, so looking for a drug that effectively treats or slows down autoimmune-related diseases is an urgent problem that needs to be solved. .
发明内容Summary of the invention
为达上述目的，本发明提供一种组合物用于制备治疗或减缓自体免疫相关疾病的医药品的用途，其中所述组合物中含有由亚铁胺基酸螯合物烧结而成的亚铁胺基酸螯合物粒子，且所述亚铁胺基酸螯合物粒子的平均粒径为500奈米至2600奈米、平均分子量为1,500道尔顿(Dalton)至600,000道尔顿，所述医药品含有有效剂量的所述组合物及 药学上可接受的载剂。To achieve the above object, the present invention provides the use of a composition for the preparation of a medicament for the treatment or alleviation of autoimmune-related diseases, wherein the composition contains ferrous iron sintered by a ferrous amino acid chelate Amino acid chelate particles, and the average particle diameter of the ferrous amino acid chelate particles is 500 nm to 2600 nm, and the average molecular weight is 1,500 Dalton to 600,000 Dalton. The pharmaceutical product contains an effective dose of the composition and a pharmaceutically acceptable carrier.
于其中一实施态样中，较佳地，所述亚铁胺基酸螯合物粒子的平均分子量为1,500道尔顿至15,000道尔顿；于另一实施态样中，较佳地，所述亚铁胺基酸螯合物粒子的平均分子量为400,000道尔顿至550,000道尔顿，较佳地，所述亚铁胺基酸螯合物粒子的平均分子量为1,500道尔顿至550,000道尔顿，更佳地，所述亚铁胺基酸螯合物粒子的平均分子量为525,538道尔顿。In one embodiment, preferably, the average molecular weight of the ferrous amino acid chelate particles is 1,500 Daltons to 15,000 Daltons; in another embodiment, preferably, the The ferrous amino acid chelate particles have an average molecular weight of 400,000 Daltons to 550,000 Daltons, preferably, the ferrous amino acid chelate particles have an average molecular weight of 1,500 Daltons to 550,000 channels Dalton, more preferably, the average molecular weight of the ferrous amino acid chelate particles is 525,538 Daltons.
较佳地，所述组合物中的亚铁胺基酸螯合物的亚铁与胺基酸的重量比是介于1：1至1：4之间。Preferably, the weight ratio of ferrous iron to amino acid of the ferrous amino acid chelate in the composition is between 1:1 and 1:4.
较佳地，所述组合物中的亚铁胺基酸螯合物的亚铁与胺基酸的重量比是介于1：1.5至1：2.5之间。Preferably, the weight ratio of ferrous iron to amino acid of the ferrous amino acid chelate in the composition is between 1:1.5 and 1:2.5.
较佳地，所述组合物中的亚铁胺基酸螯合物是由铁化合物与胺基酸混合并历经60℃至90℃加热8小时至48小时所制得的亚铁胺基酸螯合物，其中，铁化合物较佳为无机铁化合物，更佳为无机亚铁化合物。其中铁化合物与胺基酸的重量比例是介于1：1.2至1：1.5之间。Preferably, the ferrous amino acid chelate in the composition is a ferric amino acid chelate prepared by mixing an iron compound and an amino acid and heating at 60°C to 90°C for 8 hours to 48 hours Among them, the iron compound is preferably an inorganic iron compound, more preferably an inorganic ferrous compound. The weight ratio of iron compound to amino acid is between 1:1.2 and 1:1.5.
更佳地，所述铁化合物是硫酸亚铁、氯化亚铁、焦磷酸亚铁或其组合；所述胺基酸是甘胺酸。More preferably, the iron compound is ferrous sulfate, ferrous chloride, ferrous pyrophosphate, or a combination thereof; the amino acid is glycine.
本发明另外提供一种组合物用于制备治疗或减缓自体免疫相关疾病的医药品的用途，其中所述组合物由亚铁胺基酸螯合物烧结而成的亚铁胺基酸螯合物粒子所组成，且所述亚铁胺基酸螯合物粒子的平均粒径为500奈米至2,600奈米、平均分子量为1,500道尔顿至600,000道尔顿，且所述医药品由有效剂量的所述组合物及药学上可接受的载剂所组成。The present invention further provides the use of a composition for preparing a medicament for treating or slowing down autoimmune-related diseases, wherein the composition is a ferrous amino acid chelate sintered from a ferrous amino acid chelate Composed of particles, and the average particle diameter of the ferrous amino acid chelate particles is 500 nanometers to 2,600 nanometers, the average molecular weight is 1,500 daltons to 600,000 daltons, and the pharmaceutical product is composed of an effective dose Consisting of the composition and a pharmaceutically acceptable carrier.
本发明另外提供一种用于治疗或减缓自体免疫相关疾病的组合物，其中所述组合物中含有由亚铁胺基酸螯合物烧结而成的亚铁胺基酸螯合物粒子，且所述亚铁胺基酸螯合物粒子的平均粒径为500奈米至2,600奈米、平均分子量为1,500道尔顿至600,000道尔顿。The present invention further provides a composition for treating or alleviating autoimmune-related diseases, wherein the composition contains ferrous amino acid chelate particles sintered from ferrous amino acid chelate, and The ferrous amino acid chelate particles have an average particle size of 500 nm to 2,600 nm, and an average molecular weight of 1,500 Daltons to 600,000 Daltons.
本发明另外提供一种用于治疗或减缓自体免疫相关疾病的组合物，其中所述组合物由亚铁胺基酸螯合物烧结而成的亚铁胺基酸螯合物粒子所组成，且所述亚铁胺基酸螯合物粒子的平均粒径为500奈米至2,600奈米、平均分子量为1,500道尔顿至600,000道尔顿。The present invention further provides a composition for treating or alleviating autoimmune-related diseases, wherein the composition is composed of ferrous amino acid chelate particles sintered from ferrous amino acid chelate, and The ferrous amino acid chelate particles have an average particle size of 500 nm to 2,600 nm, and an average molecular weight of 1,500 Daltons to 600,000 Daltons.
本发明另外提供一种治疗或减缓自体免疫相关疾病的方法，其包含对受体施予有效剂量的组合物以及药学上可接受的载剂，其中所述组合物中含有由亚铁胺基酸螯合物烧结而成的亚铁胺基酸螯合物粒子，且所述亚铁胺基酸螯合物粒子的平均粒径为500奈米至2,600奈米、平均分子量为1,500道尔顿至600,000道尔顿。The present invention further provides a method for treating or slowing down autoimmune-related diseases, which comprises administering an effective dose of a composition to a recipient and a pharmaceutically acceptable carrier, wherein the composition contains a ferric amino acid Ferrous amino acid chelate particles formed by sintering the chelate compound, and the average particle diameter of the ferric amino acid chelate particles is 500 nm to 2,600 nm, and the average molecular weight is 1,500 Daltons to 600,000 Daltons.
本发明另外提供一种治疗或减缓自体免疫相关疾病的方法，其由对受体施予有效剂量的组合物以及药学上可接受的载剂所组成，其中所述组合物由亚铁胺基酸螯合物烧结而成的亚铁胺基酸螯合物粒子所组成，且所述亚铁胺基酸螯合物粒子的平均粒径为500奈米至2600奈米、平均分子量为1,500道尔顿至600,000道尔顿。The present invention additionally provides a method for treating or slowing down autoimmune-related diseases, which consists of administering an effective dose of a composition to a recipient and a pharmaceutically acceptable carrier, wherein the composition is composed of ferroamino acid The chelated ferrous amino acid chelate particles are sintered, and the average particle diameter of the ferric amino acid chelate particles is 500 nm to 2600 nm, and the average molecular weight is 1,500 dal Dayton to 600,000 Daltons.
本发明所述的「有效剂量」是指达成治疗或减缓自体免疫相关疾病的效果的所需剂量；依据本发明的一些实施例，是指透过施予特定范围量的含有亚铁胺基酸螯合物烧结而成的亚铁胺基酸螯合物粒子的组合物，能够降低巨噬细胞产生的一氧化氮自由基、降低嗜碱性球的去颗粒作用、抑制基质金属蛋白酶的分泌、抑制单核球细胞的迁移、抑制NF-κB及IKK-β途径、降低类风湿性关节炎跛行发生率、炎症程度、减缓异位性皮肤炎造成的搔痒、病兆严重程度、加速皮肤的修复及增加自由基清除率等。The "effective dose" described in the present invention refers to the dose required to achieve the effect of treating or slowing down autoimmune-related diseases; according to some embodiments of the present invention, it refers to the administration of a specific range of amounts containing ferrous amino acid The composition of ferrous amino acid chelate particles sintered by the chelate compound can reduce nitric oxide free radicals produced by macrophages, reduce the degranulation effect of basophilic spheres, inhibit the secretion of matrix metalloproteinases, Inhibit the migration of monocytes, inhibit the NF-κB and IKK-β pathways, reduce the incidence of lameness in rheumatoid arthritis, the degree of inflammation, slow the itching caused by atopic dermatitis, the severity of disease symptoms, and accelerate skin repair And increase the free radical scavenging rate.
依据本发明，有效剂量的计算方式是以本说明书的实验例中本发明的组合物的浓度为基准，根据美国食品药物管理局公告的实验初期估算方法，以60公斤的成人为基准，剂量换算以人体每日每公斤体重之建议摄取量(/kg b.w./d)的3.1倍为兔子的1倍剂量换算而得。According to the present invention, the effective dose is calculated based on the concentration of the composition of the present invention in the experimental examples of this specification. According to the initial estimation method announced by the US Food and Drug Administration, the dose is converted on the basis of an adult of 60 kg. It is calculated by converting 3.1 times the recommended daily intake per kg body weight (/kg bw/d) of the human body into 1 time the dose of the rabbit.
较佳地，本发明的组合物的给药对象可为人类，所述组合物的有效剂量可为2.5mg/kg至10mg/kg。Preferably, the subject of administration of the composition of the present invention may be a human, and the effective dose of the composition may be 2.5 mg/kg to 10 mg/kg.
依据本发明，有效剂量的计算方式是以本说明书的实验例中本发明的组合物的浓度为基准，根据美国食品药物管理局公告的实验初期估算方法，以60公斤的成人为基准，剂量换算以人体每日每公斤体重之建议摄取量(/kg b.w./d)的1.8倍为狗的1倍剂量换算而得。According to the present invention, the effective dose is calculated based on the concentration of the composition of the present invention in the experimental examples of this specification. According to the initial estimation method announced by the US Food and Drug Administration, the dose is converted on the basis of an adult of 60 kg. It is calculated by taking 1.8 times the recommended daily intake per kg of body weight (/kg bw/d) as the dog's 1 dose.
较佳地，本发明的组合物的给药对象可为人类，所述组合物的有效剂量可为1mg/kg至10mg/kg。较佳地，所述组合物的有效剂量可为3mg/kg至7mg/kg，更佳地，所述组合物的有效剂量可为5.5mg/kg。Preferably, the subject of administration of the composition of the present invention may be a human, and the effective dose of the composition may be 1 mg/kg to 10 mg/kg. Preferably, the effective dose of the composition may be 3 mg/kg to 7 mg/kg, more preferably, the effective dose of the composition may be 5.5 mg/kg.
本发明所述之「药学上可接受的载剂」包含，但不限于还原剂(reducing agent)、溶剂(solvent)、乳化剂(emulsifier)、悬浮剂(suspending agent)、分解剂(decomposer)、黏结剂(binding agent)、赋形剂(excipient)、安定剂(stabilizing agent)、稀释剂(diluent)、胶凝剂(gelling agent)、防腐剂(preservative)、润滑剂(lubricant)、表面活性剂(surfactant)，及其他类似或适用本发明的载剂。The "pharmaceutically acceptable carrier" described in the present invention includes, but is not limited to, reducing agent, solvent, emulsifier, suspending agent, decomposer, Binder (binding agent), excipient (excipient), stabilizer (stabilizing agent), diluent (diluent), gelling agent (gelling agent), preservative (preservative), lubricant (lubricant), surfactant (surfactant), and other similar or applicable carriers of the invention.
较佳地，所述还原剂包含，但不限于抗坏血酸(ascorbic acid)、柠檬酸(citric acid)、乙酸(acetic acid)、丙酸(propionic acid)、丁酸(butyric acid)、乳酸(lactic acid)、羟琥珀酸(malic acid)、磺酸(sulfonic acid)、丁二酸(succinic acid)或其组合。Preferably, the reducing agent includes, but is not limited to, ascorbic acid, citric acid, acetic acid, propionic acid, butyric acid, and lactic acid ), hydroxysuccinic acid (malic acid), sulfonic acid (sulfonic acid), succinic acid (succinic acid) or a combination thereof.
本发明所述的「医药品」可以多种形式存在，该等形式包含，但不限于液体、半固体及固体药剂形式，诸如溶液(solution)、乳剂(emulsion)、悬浮液(suspension)、粉末(powder)、锭剂(tablet)、丸剂(pill)、口含锭(lozenge)、片剂(troche)、口嚼胶(chewing gum)、胶囊(capsule)、脂质体、栓剂以及其他类似或适用本发明的剂型。The "pharmaceutical products" described in the present invention may exist in various forms, including, but not limited to, liquid, semi-solid, and solid pharmaceutical forms, such as solutions, emulsions, suspensions, and powders (powder), tablet (tablet), pill (pill), lozenge (lozenge), tablet (troche), chewing gum (chewing gum), capsule (capsule), liposome, suppository and other similar or The dosage form of the present invention is suitable.
较佳地，所述医药品是经肠道的或非经肠道的剂型。Preferably, the pharmaceutical product is an enteral or parenteral dosage form.
更佳地，所述经肠道的剂型是口服剂型，其口服剂型是溶液、乳剂、悬浮液、粉末、锭剂、丸剂、口含锭、片剂、口嚼胶或胶囊。More preferably, the enteral dosage form is an oral dosage form, and the oral dosage form is a solution, an emulsion, a suspension, a powder, a lozenge, a pill, a lozenge, a tablet, a chewing gum, or a capsule.
较佳地，所述治疗或减缓自体免疫相关疾病是藉由包括抑制免疫细胞的去颗粒作用。Preferably, the treatment or alleviation of autoimmune related diseases is through the degranulation effect including suppression of immune cells.
更佳地，前述免疫细胞为肥大细胞。More preferably, the aforementioned immune cells are mast cells.
其中，前述抑制免疫细胞的去颗粒作用可降低组织胺的释出量。Among them, the aforementioned degranulation effect of suppressing immune cells can reduce the amount of histamine released.
较佳地，所述自体免疫相关疾病包括，但不限于类风湿性关节炎。Preferably, the autoimmune-related diseases include, but are not limited to rheumatoid arthritis.
较佳地，所述自体免疫相关疾病包括，但不限于异位性皮肤炎。Preferably, the autoimmune-related diseases include, but are not limited to atopic dermatitis.
本发明的组合物有降低巨噬细胞产生的一氧化氮自由基、降低嗜碱性球的去颗粒作用、抑制基质金属蛋白酶的分泌、抑制单核球细胞的迁移、抑制NF-κB及IKK-β途径及增加自由基清除率等功效，并在动物实验中显示，能降低类风湿性关节炎跛行发生率、炎症程度并能减缓异位性皮肤炎造成的搔痒、病兆严重程度及加速皮肤的修复等，因此本发明的组合物可有效治疗或减缓自体免疫相关疾病，特别是类风湿性关节炎及异位性皮肤炎。The composition of the present invention has the effects of reducing nitric oxide free radicals produced by macrophages, reducing the degranulation of basophiles, inhibiting the secretion of matrix metalloproteinases, inhibiting the migration of mononuclear cells, inhibiting NF-κB and IKK- Beta pathway and increase free radical scavenging rate and other effects, and it has been shown in animal experiments that it can reduce the incidence of lameness and inflammation in rheumatoid arthritis and can reduce the itching, disease severity and acceleration of skin caused by atopic dermatitis Repair, etc. Therefore, the composition of the present invention can effectively treat or slow down autoimmune-related diseases, especially rheumatoid arthritis and atopic dermatitis.
图1以0、25、50、100μg/mL本发明的组合物处理巨噬细胞Raw264.7对细胞生长造成影响的相对比例。Figure 1 The relative proportions of the effects of 0, 25, 50, 100 μg/mL treatment of macrophages Raw264.7 on cell growth.
图2以0、50、100、250μg/mL本发明的组合物处理单核球细胞THP-1对细胞生长造成影响的相对比例。Figure 2 The relative proportions of cell growth influenced by the treatment of mononuclear cell THP-1 with 0, 50, 100, 250 μg/mL of the composition of the present invention.
图3以0、25、50、100μg/mL本发明的组合物处理经发炎诱导的巨噬细胞Raw264.7后一氧化氮生成浓度的相对比例。Figure 3 The relative proportion of nitric oxide production concentration after treatment of inflammation-induced macrophages Raw264.7 with 0, 25, 50, 100 μg/mL of the composition of the present invention.
图4以0、10、50、100μg/mL本发明的组合物处理的嗜碱性球细胞RBL-2H3之去颗粒作用的β-己糖胺酶(β-hexosaminidase)释放量相对比例。Fig. 4 Relative proportion of the amount of β-hexosaminidase released by the degranulation of basophilic cells RBL-2H3 treated with the composition of the present invention at 0, 10, 50 and 100 μg/mL.
图5以0、50、100、250、500μg/mL本发明的组合物处理经TNF-α蛋白质刺激的滑膜肉瘤细胞SW982造成的细胞生长影响相对比例。FIG. 5 shows the relative proportion of cell growth effects caused by treatment of synovial sarcoma cells SW982 stimulated with TNF-α protein with the composition of the present invention at 0, 50, 100, 250, and 500 μg/mL.
图6以0、50、100、250、500μg/mL本发明的组合物处理经TNF-α蛋白质刺激的滑膜肉瘤细胞SW982的酵素图谱。FIG. 6 is an enzyme map of treatment of synovial sarcoma cells SW982 stimulated with TNF-α protein with the composition of the present invention at 0, 50, 100, 250, and 500 μg/mL.
图7以0、50、100、250、500μg/mL本发明的组合物处理经TNF-α蛋白质刺激的滑膜肉瘤细胞SW982对于单核球细胞THP-1造成的异常细胞迁移数量相对比例。Figure 7 The relative proportion of the number of abnormal cell migration caused by the treatment of synovial sarcoma cells SW982 stimulated with TNF-α protein with 0, 50, 100, 250, 500 μg/mL synovial sarcoma cells to mononuclear THP-1 cells.
图8以0、50、100、250、500μg/mL本发明的组合物处理经TNF-α蛋白质刺激的滑膜肉瘤细胞SW982细胞核中的RelA(NF-κB)的量。Figure 8 The amount of RelA (NF-κB) in the nucleus of synovial sarcoma cells SW982 stimulated with TNF-α protein was treated with the composition of the present invention at 0, 50, 100, 250, 500 μg/mL.
图9第一次至第六次就诊时兽医师检查纪录搔痒程度。Fig. 9 The veterinarian checked the itchiness during the first to sixth visits.
图10第一次至第六次就诊时兽医师检查纪录病兆严重程度。Figure 10: The veterinarian checked the severity of the symptoms at the first to sixth visits.
图11 0.25、0.5、1、1.5、2、4mg/mL本发明的组合物的自由基清除百分比。Figure 11 Percent free radical scavenging of the compositions of the present invention at 0.25, 0.5, 1, 1.5, 2, 4 mg/mL.
发明将由下列的实施例做为进一步说明，这些实施例并不限制本发明前面所揭示的内容。熟习本发明的技艺者，可以做些许的改良与修饰，但不脱离本发明的范畴。The invention will be further illustrated by the following examples, which do not limit the previously disclosed content of the invention. Those skilled in the art of the present invention can make some improvements and modifications without departing from the scope of the present invention.
制备例1制备含有亚铁胺基酸粒子的组合物Preparation Example 1 Preparation of a composition containing ferrous amino acid particles
本发明的含有亚铁胺基酸螯合物粒子的组合物是由中国台湾配位体股份有限公司制作(批次号码：F171001；制造日期：2017年10月5日)，且该组合物为冷冻干燥的粉末，其是以下述方式制备。首先，将硫酸亚铁与甘胺酸(纯度98％以上)以重量比1：1.3混合并历经60℃至90℃加热8小时至48小时，以获得亚铁胺基酸螯合物，其中亚铁胺基酸螯合物的亚铁与胺基酸螯合比例是介于1：1至1：4之间，接着，亚铁胺基酸螯合物在200-240℃下进行烧结，以获得亚铁胺基酸螯合物粒子。经由雷射粒径分析仪(Beckman Coulter,N5,Submicron Particle Size Analyzer)在水中进行动态光散射测得亚铁胺基酸螯合物粒子的平均粒径为1465.90±132.29奈米。利用Waters Alliance 2695 System进行凝胶穿透层析仪(GPC)测定数目平均分子量(Mn)、重量平均分子量(Mw)、峰值分子量(MP)和多分散性(polydispersity，PDI)，分别为68188道尔顿、525,538道尔顿、286,426道尔顿及7.707205。The composition containing ferrous amino acid chelate particles of the present invention is made by Taiwan Ligand Co., Ltd. of China (batch number: F171001; manufacturing date: October 5, 2017), and the composition is The freeze-dried powder was prepared in the following manner. First, ferrous sulfate and glycine acid (purity 98% or more) are mixed in a weight ratio of 1:1.3 and heated at 60°C to 90°C for 8 hours to 48 hours to obtain a ferrous amino acid chelate, in which The chelating ratio of ferrous iron to amino acid chelate is between 1:1 and 1:4, then the ferrous amino acid chelate is sintered at 200-240°C to Ferrous amino acid chelate particles were obtained. The average particle size of the ferroamino acid chelate particles was measured to be 1465.90±132.29 nanometers by dynamic light scattering in water through a laser particle size analyzer (Beckman Coulter, N5, Submicron Particle Size Analyzer). Using Waters Alliance 2695 System to perform gel penetration chromatography (GPC) to determine the number average molecular weight (Mn), weight average molecular weight (Mw), peak molecular weight (MP) and polydispersity (PDI), respectively, 68188 Dalton, 525,538 Dalton, 286,426 Dalton and 7.707205.
制备例2培养巨噬细胞Raw264.7Preparation Example 2 Cultured macrophages Raw264.7
将巨噬细胞Raw264.7(中国台湾BCRC，60001)以含有10％热失活的胎牛血清(fetal bovine serum，FBS)的Dulbecco's Modified Eagle Medium(DMEM)培养基(Gibco 11965092)培养于37℃、5％二氧化碳的培养箱中，至约八分满。Macrophages Raw264.7 (BCRC, Taiwan, 60001) were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium (Gibco 11965092) containing 10% heat-inactivated fetal bovine serum (FBS) at 37°C In the incubator of 5% carbon dioxide, it is about eight points full.
制备例3培养嗜碱性球细胞RBL-2H3Preparation Example 3 Culture of basophilic sphere cells RBL-2H3
将嗜碱性球细胞RBL-2H3(中国台湾BCRC，60310)以Minimum Essential Medium(MEM)培养基(Gibco 11095-080)培养于37℃、5％二氧化碳的培养箱中，至约八分满。The basophilic cells RBL-2H3 (BCRC, Taiwan, 60310, China) were cultured in a minimum essential medium (MEM) medium (Gibco 11095-080) in a 37°C, 5% carbon dioxide incubator until about eight percent full.
制备例4培养滑膜肉瘤细胞SW982Preparation Example 4 Culture of synovial sarcoma cells SW982
将滑膜肉瘤细胞SW982(中国台湾BCRC，60535)以含有10％FBS的L15培养基(Gibco，11415-064)培养于37℃、0％二氧化碳的培养箱中，至约八分满。Synovial sarcoma cells SW982 (BCRC, Taiwan, 60535, China) were cultured in an incubator at 37°C and 0% carbon dioxide with L15 medium (Gibco, 11415-064) containing 10% FBS to about eight times full.
制备例5培养单核球细胞THP-1Preparation Example 5 Culture of mononuclear cell THP-1
将单核球细胞THP-1(中国台湾BCRC，60430)以含有10％热失活的胎牛血清(fetal bovine serum,FBS)的RPMI1640培养基(Gibco，A10491-01)培养于37℃、5％二氧化碳的培养箱中至约八分满。Mononuclear THP-1 cells (BCRC, Taiwan, 60430) were cultured in RPMI1640 medium (Gibco, A10491-01) containing 10% heat-inactivated fetal bovine serum (fetal bovine serum, FBS) at 37°C, 5 % Carbon dioxide in the incubator to about eight full.
制备例6类风湿性关节炎实验动物Preparation Example 6 Rheumatoid Arthritis Experimental Animal
新西兰大白兔，3月龄，体重约3公斤共6只。并参考Trivillin et al.(2014).Boron neutron capture synovectomy(BNCS)as a potential therapy for rheumatoid arthritis:boron biodistribution study in a model of antigen-induced arthritis in rabbits.Radiation and environmental biophysics,53(4),635-643进行抗原诱导关节炎(antigen-induced arthritis)模式，方法如下所述：New Zealand white rabbit, 3 months old, weighs about 3 kg, a total of 6. Also refer to Trivillin et al. (2014). Boron neutron capture synovectomy (BNCS) as potential healing for rhumatoid arthritis: boron biodistribution study model in anti-gengen-induced arthritis in rabbits. Radiation and bioenvironment 53 -643 carries on the antigen-induced arthritis model, the method is as follows:
(1)兔子接受2次皮内注射卵白蛋白乳剂(Ovoalbumin emulsion，OVA,Sigma Chemicals,USA,5mg/mL在0.9％NaCl中)，OVA先以1:1比例与完全弗氏佐剂(complete Freund’s adjuvant)(Difco,USA)混合制成乳剂。2次皮内注射间隔为15天。(1) Rabbits received 2 intradermal injections of ovalbumin emulsion (Ovoalbumin emulsion, OVA, Sigma Chemicals, USA, 5mg/mL in 0.9% NaCl), OVA first with complete Freund's adjuvant (complete Freund's) in a 1:1 ratio adjuvant) (Difco, USA) mixed to make an emulsion. The interval between 2 intradermal injections is 15 days.
(2)兔子于第2次皮内注射后的第10天及第20天，分别于右膝关节内注射1mL OVA(5mg/mL，在0.9％NaCl中)。(2) On the 10th and 20th days after the second intradermal injection, the rabbits were injected with 1 mL OVA (5 mg/mL in 0.9% NaCl) into the right knee joint, respectively.
(3)兔子于第1次关节内注射OVA后的第50-60天期间，评估抗原诱导关节炎的临床征状(关节肿胀、触诊时的疼痛反应)。(3) Rabbits were evaluated for clinical symptoms of antigen-induced arthritis (swollen joints, pain response at palpation) during the 50-60 days after the first intra-articular injection of OVA.
制备例7异位性皮肤炎实验动物Preparation Example 7 Atopic Dermatitis Experimental Animal
纳入本研究的动物共有15只患有异位性皮肤炎的动物，分别为14只犬、1只猫，均为1岁龄以上，有一只犬只在研究计划途中退出，因此总共有14只动物的研究结果资料呈现。A total of 15 animals with atopic dermatitis were included in this study, including 14 dogs and 1 cat, all of which are over 1 year old. One dog withdrew on the way of the research plan, so there were 14 in total The results of animal research are presented.
实施例1本发明的组合物对巨噬细胞Raw 264.7生长的影响Example 1 The effect of the composition of the present invention on the growth of macrophages Raw 264.7
本实施例利用活细胞粒线体的酵素能将MTT(Thiazolyl Blue Tetrazolium Bromide)试剂还原成formazan结晶，测量其溶解后的吸光值以反映细胞的存活率。MTT试验方法如下所述：In this embodiment, the enzyme of the mitochondria of living cells can be used to reduce the MTT (Thiazolyl Blue Tetrazolium Bromide) reagent to formazan crystals, and the absorbance value after dissolution is measured to reflect the survival rate of the cells. The MTT test method is as follows:
(1)将本发明的组合物先溶于0.1M柠檬酸配制成浓度250mg/mL的溶液(pH3.5)，再进一步稀释成25、50、100μg/mL的浓度。(1) The composition of the present invention is first dissolved in 0.1 M citric acid to prepare a solution (pH 3.5) with a concentration of 250 mg/mL, and then further diluted to a concentration of 25, 50, or 100 μg/mL.
(2)取制备例2的巨噬细胞Raw264.7，以每孔4×10
4细胞的密度种 植到24孔细胞培养盘中，并于37℃、5％CO
2培养箱中培养24小时。
(2) Raw264.7 macrophages from Preparation Example 2 were planted into a 24-well cell culture plate at a density of 4×10 4 cells per well, and cultured in a 37° C., 5% CO 2 incubator for 24 hours.
(3)以步骤(1)中的不同浓度本发明的组合物处理步骤(2)的巨噬细胞Raw264.7，并以0μg/mL的本发明的组合物为对照组，每一条件于相同的两组试验中各进行三重复。(3) Treat the macrophage Raw264.7 of step (2) with different concentrations of the composition of the present invention in step (1), and use the composition of the present invention of 0 μg/mL as a control group, each condition being the same Three replicates were performed in each of the two sets of trials.
(4)分别在0、24、48及72小时的时间点加入MTT，并观察细胞生长情形。亦即，在前述每一时间点将每一格细胞中的培养液置换成500μL的0.5mg/mL的MTT(Sigma，M2128)溶液，接着，于37℃、5％CO
2培养箱中培养4小时后，将MTT溶液移除。
(4) Add MTT at 0, 24, 48 and 72 hours respectively, and observe the growth of cells. That is, at each of the aforementioned time points, the culture solution in each cell was replaced with 500 μL of 0.5 mg/mL MTT (Sigma, M2128) solution, followed by incubation at 37°C in a 5% CO 2 incubator. 4 After hours, the MTT solution was removed.
(5)侦测formazan吸光值。具体而言，用200μL的100％乙醇与DMSO(体积比1：1)的溶液溶解formazan结晶后，在振荡器上震荡20分钟让formazan结晶完全溶解，吸取50μL溶液到96孔细胞培养盘中，用酵素免疫分析测读仪读取565nm的吸光值，并以对照组的吸光值为基准作图。(5) Detect the absorbance of formazan. Specifically, after dissolving formazan crystals with 200 μL of a solution of 100% ethanol and DMSO (volume ratio 1:1), shake on a shaker for 20 minutes to completely dissolve the formazan crystals, and draw 50 μL of the solution into a 96-well cell culture dish. Use the enzyme immunoassay reader to read the absorbance at 565nm, and plot the absorbance of the control group as a reference.
结果如图1所示，本发明的组合物对免疫细胞(巨噬细胞)不具毒性，甚至相较于对照组显示可些微促进免疫细胞生长。As a result, as shown in FIG. 1, the composition of the present invention is not toxic to immune cells (macrophages), and even shows that it can slightly promote the growth of immune cells compared to the control group.
实施例2本发明的组合物对单核球细胞THP-1生长的影响Example 2 Effect of the composition of the present invention on the growth of mononuclear THP-1 cells
本实施例同实施例1的方法，但改以制备例5的单核球细胞THP-1(细胞密度为每孔3×10
5细胞)进行实验，且本发明的组合物的使用浓度为50、100、250μg/mL)。利用活细胞粒线体的酵素能将MTT(Thiazolyl Blue Tetrazolium Bromide)试剂还原成formazan结晶，测量其溶解后的吸光值以反映细胞的存活率。
This example is the same as the method of Example 1, but the experiment was performed using THP-1 mononuclear cells (the cell density was 3×10 5 cells per well) of Preparation Example 5, and the concentration of the composition of the present invention was 50 , 100, 250 μg/mL). The enzyme of living cell mitochondria can reduce MTT (Thiazolyl Blue Tetrazolium Bromide) reagent to formazan crystals, and measure the absorbance value after dissolution to reflect the survival rate of cells.
结果如图2所示，本发明的组合物对免疫细胞(单核球细胞)不具毒性，甚至相较于对照组显示可些微促进免疫细胞生长。The results are shown in FIG. 2. The composition of the present invention is not toxic to immune cells (monocytes), and even shows that it can slightly promote the growth of immune cells compared to the control group.
实施例3本发明的组合物对发炎模型中巨噬细胞Raw264.7产生一氧化氮的影响Example 3 The effect of the composition of the present invention on the production of nitric oxide by macrophage Raw264.7 in an inflammation model
当巨噬细胞受到外来物刺激时，会增加诱发型一氧化氮合酶(inducible nitric oxide synthase，iNOS)的表现，产生大量一氧化氮，此为巨噬细胞参与免疫反应及发炎的重要机制。因此以格兰氏阴性菌产生的内毒素－脂多醣(lipopolysaccharide，LPS)刺激巨噬细胞生成一氧 化氮，并测试本发明组合物对一氧化氮生成浓度的影响，试验方法如下：When macrophages are stimulated by foreign substances, they will increase the performance of inducible nitric oxide synthase (iNOS), producing a large amount of nitric oxide, which is an important mechanism for macrophages to participate in immune reactions and inflammation. Therefore, endotoxin-lipopolysaccharide (LPS) produced by Gram-negative bacteria was used to stimulate macrophages to produce nitric oxide, and the effect of the composition of the present invention on the concentration of nitric oxide was tested. The test method is as follows:
(1)将本发明的组合物先溶于0.1M柠檬酸配制成浓度100mg/mL的溶液(pH3.5)，再进一步稀释成25μg/mL、50μg/mL、100μg/mL的浓度。(1) The composition of the present invention is first dissolved in 0.1 M citric acid to prepare a solution (pH 3.5) with a concentration of 100 mg/mL, and then further diluted to a concentration of 25 μg/mL, 50 μg/mL, and 100 μg/mL.
(2)取制备例2的巨噬细胞Raw264.7，以每孔1×10
6细胞的密度种植到24孔细胞培养盘中，并于37℃、5％CO
2培养箱中培养24小时。
(2) Raw264.7 macrophages from Preparation Example 2 were planted into a 24-well cell culture plate at a density of 1×10 6 cells per well, and cultured in a 37° C., 5% CO 2 incubator for 24 hours.
(3)以步骤(1)中的不同浓度本发明的组合物处理步骤(2)巨噬细胞Raw264.7两个小时，并以0μg/mL为对照组，每一条件于相同的两组试验中各进行三重复，接着经本发明的组合物处理的组别再加入1μg/mL的脂多醣(Sigma，L4391)，另以仅加入1μg/mL脂多醣的巨噬细胞Raw264.7为正对照组，继续培养20小时。(3) Treat the macrophages Raw264.7 with different concentrations of the composition of step (1) in step (1) for two hours, and take 0 μg/mL as the control group, each condition is tested in the same two groups Repeat three times in each of the steps, then add 1 μg/mL lipopolysaccharide (Sigma, L4391) to the group treated with the composition of the present invention, and use Raw264.7, a macrophage that only added 1 μg/mL lipopolysaccharide, as a positive control Group, continue to cultivate for 20 hours.
(4)收集细胞培养液，以Biovision K262试剂组并参照其使用手册进行一氧化氮浓度测定，并以对照组一氧化氮浓度为基准作图。(4) Collect the cell culture solution, use the Biovision K262 reagent group and refer to its instruction manual to measure the concentration of nitric oxide, and draw the graph based on the nitric oxide concentration of the control group.
结果如图3所示，本发明的组合物可以在脂多醣诱导之发炎反应中，抑制巨噬细胞生成一氧化氮，抑制发炎反应。As a result, as shown in FIG. 3, the composition of the present invention can inhibit the production of nitric oxide by macrophages during the inflammatory reaction induced by lipopolysaccharide and inhibit the inflammatory reaction.
实施例4经本发明组合物处理的嗜碱性球细胞RBL-2H3去颗粒作用(degranulation)的β-己糖胺酶(β-hexosaminidase)释放量Example 4 β-hexosaminidase release amount of degranulation of basophils RBL-2H3 treated with the composition of the present invention
嗜碱性球细胞和肥大细胞在体内的功用主要是在受到过敏原刺激时进行去颗粒作用释放β-己糖胺酶、类胰蛋白酶、组织胺(histamine)等过敏介质(mediators)引起免疫反应。由于嗜碱性球细胞RBL-2H3的性质与肥大细胞(mast cell)相似，因此本实验使用嗜碱性球细胞RBL-2H3模拟肥大细胞所释放出的过敏介质中的β-己糖胺酶以评估去颗粒作用的发生，试验方法如下所述：The function of basophils and mast cells in the body is mainly to perform degranulation when they are stimulated by allergens to release β-hexosaminidase, tryptase, histamine and other allergic mediators to cause immune reactions. . Because the properties of basophil RBL-2H3 are similar to mast cells, this experiment uses basophil RBL-2H3 to simulate the β-hexosaminidase in the allergic medium released by mast cells To evaluate the occurrence of de-granulation, the test method is as follows:
(1)将本发明的组合物先溶于0.1M柠檬酸配制成浓度100mg/mL的溶液(pH3.5)，再进一步稀释成10μg/mL、50μg/mL、100μg/mL的浓度。(1) The composition of the present invention is first dissolved in 0.1 M citric acid to prepare a solution (pH 3.5) with a concentration of 100 mg/mL, and then further diluted to a concentration of 10 μg/mL, 50 μg/mL, and 100 μg/mL.
(2)将制备例3的培养的嗜碱性球细胞RBL-2H3培养皿加入1mL胰蛋白酶(trypsin)裂解液静置于37℃、5％二氧化碳的培养箱中，加入 2mL MEM培养基制成细胞悬浮液后放入水平离心机以1000rpm离心3分钟后移除上清液以去除胰蛋白酶后，加入新鲜培养液重新悬浮细胞后取20μL置于细胞计数盘上计数细胞，将细胞以每孔4×10
4的细胞密度种植于96孔盘中。
(2) Put the cultured basophil RBL-2H3 culture dish of Preparation Example 3 into 1mL trypsin lysate and place it in a 37°C, 5% carbon dioxide incubator, add 2mL MEM medium Put the cell suspension in a horizontal centrifuge at 1000 rpm for 3 minutes, remove the supernatant to remove the trypsin, add fresh culture medium to resuspend the cells, take 20 μL and place the cells on the cell counting plate to count the cells. The cell density of 4×10 4 was planted in 96-well dishes.
(3)待细胞完全贴附后，加入anti-dinitrophenyl IgE(Sigma，D8406)，使每一孔的最终浓度达1μg/mL致敏18小时。(3) After the cells are completely attached, add anti-dinitrophenyl IgE (Sigma, D8406) to sensitize the final concentration of each well to 1 μg/mL for 18 hours.
(4)以PBS清洗3次完全去除anti-dinitrophenyl IgE后，加入100μL MEM培养基并于实验组分别加入步骤(1)中的不同浓度本发明的组合物及0.1M柠檬酸(即，0μg/mL本发明的组合物)作为对照组，处理时间为1小时，每一条件于相同的两组试验中各进行三重复。(4) After washing 3 times with PBS to completely remove anti-dinitrophenyl IgE, add 100 μL of MEM medium and add different concentrations of the composition of the present invention and 0.1 M citric acid (ie, 0 μg/ mL of the composition of the invention) as a control group, the treatment time is 1 hour, and each condition is repeated three times in the same two sets of tests.
(5)接着使用tyrode’s buffer(135mM NaCl,5mM KCl,1mM MgCl
2,1.8mM CaCl
2,20mM Hepes,pH 7.4，使用当天再加入0.04％BSA)清洗2次，接着每孔添加100μL tyrode’s buffer并加入DNP-BSA(Thermo fisher，A23018)使其最终浓度为0.5μg/mL，静置于37℃烘箱刺激30分钟。
(5) Then use tyrode's buffer (135 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 20 mM Hepes, pH 7.4, then add 0.04% BSA on the day of use) to wash twice, then add 100 μL tyrode's buffer to each well and add The final concentration of DNP-BSA (Thermo fisher, A23018) was 0.5 μg/mL, and it was allowed to stand in a 37°C oven for 30 minutes for stimulation.
(6)刺激期间配制pNAG(Merck，487052)3.5mg/mL柠檬酸缓冲液(citrate buffer)，分注到新的96孔盘，共两盘，一盘标注上清液(supernatant)、另一盘标注细胞液(lysate)。(6) Prepare pNAG (Merck, 487052) 3.5mg/mL citrate buffer during stimulation, and dispense into a new 96-well plate, a total of two plates, one plate is marked with supernatant and the other The dish is labeled with lysate.
(7)吸取50μL上清液置入于标有上清液的pNAG 96孔盘内，接着放入37℃烘箱90分钟。(7) Pipette 50 μL of supernatant into the pNAG 96-well dish marked with supernatant, and then put it in a 37°C oven for 90 minutes.
(8)对含有细胞的96孔盘每孔加入150μL 0.1％ Triton X-100，并用分注器上下吸取5次后放入37℃烘箱10分钟，接着吸取50μL置入标有细胞液的pNAG 96孔盘内，接着放入37℃烘箱90分钟。(8) Add 150 μL of 0.1% Triton X-100 to each well of a 96-well dish containing cells, pipette up and down 5 times with a dispenser, and place in a 37°C oven for 10 minutes, then draw 50 μL of pNAG labeled with cell solution Then place it in the orifice tray for 90 minutes at 37°C.
(9)中止反应，每孔加入100μL 400mM甘胺酸(glycine)溶液，当颜色转变为黄色时代表有β-己糖胺酶存在，使用酵素免疫测读仪侦测405nm下的吸光值。(9) To stop the reaction, add 100 μL of 400 mM glycine solution to each well. When the color changes to yellow, it indicates the presence of β-hexosaminidase. Use an enzyme immunoreader to detect the absorbance at 405 nm.
(10)去颗粒百分比为100×(上清液含量)/(上清液含量+细胞液含量)，但上清液只取了1/2、裂解液只取了1/4，且裂解液中亦包含一部份来自上清液的β-己糖胺酶，所以应改为％＝100×[2×(上清-培养盘blank)]/[1/2(上清-盘养盘blank)]+[4×(细胞液-盘养盘blank)]，并以对照组的去颗粒百分比为基准作图。(10) The percentage of degranulation is 100×(supernatant content)/(supernatant content + cell fluid content), but only 1/2 of the supernatant, 1/4 of the lysate, and the lysate It also contains a part of β-hexosaminidase from the supernatant, so it should be changed to %=100×[2×(supernatant-culture plate blank)]/[1/2(supernatant-plate culture plate blank)]+[4×(cell liquid-disk culture blank)], and plot based on the degranulation percentage of the control group.
结果如图4所示，经由本发明的组合物处理的组别β-己糖胺酶释放百分比相较于对照组低降，且随着处理本发明的组合物浓度的增加，β-己糖胺酶释放的量下降，显示本发明的组合物可以降低嗜碱性球细胞或肥大细胞的去颗粒作用、抑制免疫细胞的去颗粒作用、降低类风湿性关节炎的发炎反应。The results are shown in Figure 4. The percentage of β-hexosaminidase released by the group treated with the composition of the present invention was lower than that of the control group, and as the concentration of the treated composition of the present invention increased, β-hexose The decreased amount of aminase release shows that the composition of the present invention can reduce the degranulation of basophils or mast cells, suppress the degranulation of immune cells, and reduce the inflammatory response of rheumatoid arthritis.
实施例5本发明的组合物对滑膜肉瘤细胞SW982生长的影响Example 5 Effect of the composition of the present invention on the growth of synovial sarcoma cell SW982
类风湿性关节炎的关节软骨和骨的损伤主要是由于滑膜细胞的增生所引起，而滑膜细胞的增生可经由肿瘤坏死因子-α(Tumor necrosis factor-α，TNF-α)的刺激所引起。本实施例以TNF-α刺激滑膜肉瘤细胞并利用活细胞粒线体的酵素能将MTT(Thiazolyl Blue Tetrazolium Bromide)试剂还原成formazan结晶，测量其溶解后的吸光值以反映细胞的生长、存活率。本实验的步骤如下所述：The damage of articular cartilage and bone in rheumatoid arthritis is mainly caused by the proliferation of synovial cells, and the proliferation of synovial cells can be stimulated by tumor necrosis factor-α (Tumor necrosis factor-α, TNF-α) cause. In this example, TNF-α was used to stimulate synovial sarcoma cells and the enzyme of living cell mitochondria was used to reduce MTT (Thiazolyl Blue Tetrazolium Bromide) reagent to formazan crystals, and the absorbance value after dissolution was measured to reflect the growth and survival of cells rate. The steps of this experiment are as follows:
(1)将本发明的组合物先溶于0.1M柠檬酸配制成浓度250mg/mL的溶液(pH3.5)，再进一步稀释成50、100、250、500μg/mL的浓度。(1) The composition of the present invention is first dissolved in 0.1 M citric acid to prepare a solution (pH 3.5) with a concentration of 250 mg/mL, and then further diluted to a concentration of 50, 100, 250, and 500 μg/mL.
(2)取制备例4的滑膜肉瘤细胞SW982，以每孔2×10
4细胞的密度种植到24孔细胞培养盘中，并于37℃、0％CO
2培养箱中培养24小时。
(2) The synovial sarcoma cells SW982 of Preparation Example 4 were taken, planted in a 24-well cell culture dish at a density of 2×10 4 cells per well, and cultured in a 37° C., 0% CO 2 incubator for 24 hours.
(3)将细胞分别以0或10ng/mL的TNF-α蛋白质(R&D，210-TA)处理，并置于37℃培养箱培养24小时。(3) Treat cells with TNF-α protein (R&D, 210-TA) at 0 or 10 ng/mL, respectively, and place them in a 37°C incubator for 24 hours.
(4)以步骤(1)中的不同浓度本发明的组合物处理步骤(3)的滑膜肉瘤细胞SW982，并以0μg/mL的本发明的组合物为对照组，每一条件于相同的两组试验中各进行三重复。(4) Treat the synovial sarcoma cell SW982 of step (3) with different concentrations of the composition of the invention in step (1), and use the composition of the invention of 0 μg/mL as a control group, each condition being the same Three replicates were performed in each of the two sets of trials.
(5)分别在0、24、48及72小时的时间点加入MTT，并观察细胞生长情形。亦即，在前述每一时间点将每一格细胞中的培养液置换成500μL的1mg/mL的MTT(Sigma，M2128)溶液，接着，于37℃培养箱中培养3小时后，将MTT溶液移除。(5) Add MTT at the time points of 0, 24, 48 and 72 hours, and observe the growth of cells. That is, at each of the aforementioned time points, the culture solution in each cell was replaced with 500 μL of 1 mg/mL MTT (Sigma, M2128) solution, and then, after incubating in a 37°C incubator for 3 hours, the MTT solution Remove.
(6)侦测formazan吸光值。具体而言，用200μL的100％乙醇与DMSO(体积比1：1)的溶液溶解formazan结晶后，在振荡器上震荡20分钟让formazan结晶完全溶解，吸取50μL溶液到96孔细胞培养盘中，用酵素免疫分析测读仪读取565nm的吸光值，并以对照组的吸光值为基准作图。(6) Detect the absorbance of formazan. Specifically, after dissolving formazan crystals with 200 μL of a solution of 100% ethanol and DMSO (volume ratio 1:1), shake on a shaker for 20 minutes to completely dissolve the formazan crystals, and pipette 50 μL of the solution into a 96-well cell culture dish. Use the enzyme immunoassay reader to read the absorbance at 565nm, and plot the absorbance of the control group as a reference.
结果如图5所示，以TNF-α蛋白质处理的滑膜肉瘤细胞SW982相较于对照组细胞异常增生。而随着处理本发明的组合物浓度的增加，增加了滑膜肉瘤细胞增生的抑制。因此本发明的组合物能有效抑制滑膜肉瘤细胞SW982的增生而降低类风湿性关节炎的发生。The results are shown in Fig. 5. Synovial sarcoma cells SW982 treated with TNF-α protein were abnormally proliferated compared with the control group. As the concentration of the composition treated with the present invention increases, the inhibition of synovial sarcoma cell proliferation is increased. Therefore, the composition of the present invention can effectively inhibit the proliferation of synovial sarcoma cells SW982 and reduce the occurrence of rheumatoid arthritis.
实施例6基质金属蛋白酶酵素图谱(Zymography)Example 6 Zymography of matrix metalloproteinases
基质金属蛋白酶会分解关节软骨的重要成分，因此被认为与类风湿性关节炎密切相关，而基质金属蛋白酶一般以酶原(zymogen，pro-MMP)的形式表现，分泌、活化后成为MMP。本实验以酵素图谱测试滑膜肉瘤细胞SW982的培养液中Pro-MMP-9、MMP-9的量，实验步骤如下所述：Matrix metalloproteinases decompose important components of articular cartilage, so they are considered to be closely related to rheumatoid arthritis. Matrix metalloproteinases are generally expressed in the form of zymogen (pro-MMP), and become MMP after secretion and activation. In this experiment, the amount of Pro-MMP-9 and MMP-9 in the culture solution of synovial sarcoma cell SW982 was tested by enzyme map. The experimental steps are as follows:
(1)将本发明的组合物先溶于0.1M柠檬酸配制成浓度250mg/mL的溶液，再进一步稀释成50、100、250、500μg/mL的浓度。(1) The composition of the present invention is first dissolved in 0.1 M citric acid to prepare a solution with a concentration of 250 mg/mL, and then further diluted to a concentration of 50, 100, 250, and 500 μg/mL.
(2)取制备例4的滑膜肉瘤细胞SW982，以每孔3×10
5细胞的密度种植到6孔细胞培养盘中，并于37℃培养箱中培养24小时。
(2) The synovial sarcoma cells SW982 of Preparation Example 4 were taken and planted in a 6-well cell culture plate at a density of 3×10 5 cells per well, and cultured in a 37°C incubator for 24 hours.
(3)先用PBS清洗细胞一次，接着以1mL含有10ng/mL TNF-α蛋白质(R&D，210-TA)之无血清L15培养液处理每孔细胞，并置于37℃培养箱培养24小时。(3) Wash the cells with PBS once, then treat each cell with 1 mL of serum-free L15 medium containing 10 ng/mL TNF-α protein (R&D, 210-TA) and place in a 37°C incubator for 24 hours.
(4)以步骤(1)中的不同浓度本发明的组合物及0.1M柠檬酸(即，0μg/mL本发明的组合物)处理步骤(2)的滑膜肉瘤细胞SW982 48小时。(4) Treat the synovial sarcoma cells SW982 of step (2) for 48 hours with different concentrations of the composition of the invention and 0.1 M citric acid (ie, 0 μg/mL of the composition of the invention) in step (1).
(5)收集每孔的培养液，并加入1mM苯甲基磺酰氟(Phenylmethanesulfonyl Fluoride，PMSF)(Applichem，A0999.0005)，再以400xg离心5分钟，去除沉淀物后，置于冰上保冰。(5) Collect the culture solution in each well, add 1 mM Phenylmethanesulfonyl Fluoride (PMSF) (Applichem, A0999.0005), centrifuge at 400xg for 5 minutes, remove the precipitate, and store on ice. ice.
(6)取10μL培养液混合5x loading buffer(不含β-Mercaptoethanol或其他还原剂)后，注入0.1％Gelatin-7.5％SDS-PAGE进行电泳，并以含有Pro-MMP-9、MMP-9两蛋白质的肺腺癌细胞A549的培养液为对照组。(6) Mix 10xL of culture medium with 5x loading buffer (without β-Mercaptoethanol or other reducing agents), inject 0.1% Gelatin-7.5% SDS-PAGE for electrophoresis, and use Pro-MMP-9 and MMP-9. The culture medium of protein lung adenocarcinoma cell A549 was the control group.
(7)跑胶完毕后，取出胶体置入washing buffer(2.5％triton X-100)中，室温摇荡30分钟，重复两次。(7) After running the gel, remove the gel and place it in the washing buffer (2.5% triton X-100), shake it at room temperature for 30 minutes, and repeat twice.
(8)胶体用d.d H
2O清洗一次，接着置入reaction buffer(40mM Tris-HCl,pH 8.0,10mM CaCl
2,0.01％NaN
3)，在37℃下摇晃12-16小 时。
(8) The colloid is washed once with dd H 2 O, then placed in a reaction buffer (40 mM Tris-HCl, pH 8.0, 10 mM CaCl 2 , 0.01% NaN 3 ), and shaken at 37° C. for 12-16 hours.
(9)用d.d H
2O清洗两次胶后，以0.25％Coomassie blue R-250(Panreac，254932)染色20分钟。
(9) After washing the gel twice with dd H 2 O, stain with 0.25% Coomassie blue R-250 (Panreac, 254932) for 20 minutes.
(10)用退色液(50％methanol,7％acetic acid)退染、封胶。(10) Decolorize and seal with fading solution (50% methanol, 7% acetic acid).
结果如图6所示，对照组的Pro-MMP-9明显少于以本发明的组合物处理的组别，而以本发明的组合物处理的滑膜肉瘤细胞SW982分泌的MMP-9基质金属蛋白酶明显比对照组要高，可见得对照组的Pro-MMP-9相较于以本发明的组合物处理的组别大量修饰成为活化的MMP-9。且随着本发明的组合物处理浓度越高，越可以抑制滑膜肉瘤细胞SW982分泌Pro-MMP-9、MMP-9基质金属蛋白酶。本发明的组合物可通过抑制基质金属蛋白酶分泌来抑制关节软骨的重要成分分解，而达到治疗或减缓类风湿性关节炎的效果。The results are shown in Fig. 6. The control group had significantly less Pro-MMP-9 than the group treated with the composition of the present invention, while the MMP-9 matrix metal secreted by the synovial sarcoma cells SW982 treated with the composition of the present invention The protease is obviously higher than the control group, and it can be seen that the Pro-MMP-9 of the control group is modified into activated MMP-9 in a large amount compared to the group treated with the composition of the present invention. And the higher the treatment concentration of the composition of the present invention, the more it can inhibit the secretion of Pro-MMP-9 and MMP-9 matrix metalloproteinase from the synovial sarcoma cell SW982. The composition of the present invention can inhibit the decomposition of important components of articular cartilage by inhibiting the secretion of matrix metalloproteinases to achieve the effect of treating or slowing rheumatoid arthritis.
实施例7本发明的组合物对类风湿性关节炎造成的发炎细胞聚集的影响Example 7 Effect of the composition of the present invention on the aggregation of inflammatory cells caused by rheumatoid arthritis
发炎反应的发生会使破坏的组织释出组织胺，而增加血管的通透性，使嗜中性球、单核球聚集、进行分化产生免疫反应。The occurrence of an inflammatory reaction will release histamine from the damaged tissues, increase the permeability of blood vessels, and cause neutrophils and monocytes to aggregate and differentiate to produce an immune response.
(一)制备SW982条件培养基(conditioned medium)：(1) Preparation of SW982 conditioned medium (conditioned medium):
(1)取制备例4的滑膜肉瘤细胞SW982，以每孔3×10
5细胞的密度种植到6孔细胞培养盘中，并于37℃培养箱中培养24小时。
(1) The synovial sarcoma cells SW982 of Preparation Example 4 were taken, planted in a 6-well cell culture plate at a density of 3×10 5 cells per well, and cultured in a 37°C incubator for 24 hours.
(2)以含有0或10ng/mL TNF-α蛋白质(R&D，210-TA)之10％FBS L15培养基处理细胞，并在37℃培养箱中培养24小时。(2) Treat cells with 10% FBS L15 medium containing 0 or 10 ng/mL TNF-α protein (R&D, 210-TA), and incubate in a 37°C incubator for 24 hours.
(3)用PBS清洗细胞一次，将培养液换成含10ng/mL的TNF-α蛋白质及0、50、100、250或500μg/mL的本发明的组合物的无血清L15培养基，放置到37℃培养箱中培养24小时，对照组为0μg/mL的本发明的组合物且无TNF-α蛋白质处理。(3) Wash the cells once with PBS, change the culture medium to serum-free L15 medium containing 10 ng/mL of TNF-α protein and 0, 50, 100, 250 or 500 μg/mL of the composition of the present invention, and place Incubated for 24 hours in a 37°C incubator, the control group was 0 μg/mL of the composition of the present invention without TNF-α protein treatment.
(4)收集培养液，离心400xg五分钟，去除沉淀物后备用。(4) Collect the culture solution, centrifuge at 400xg for five minutes, and remove the precipitate before use.
(二)细胞迁移试验(Migration assay)(2) Cell migration test (Migration assay)
(1)收集制备例5的单核球细胞THP-1，用PBS清洗两次后，使细胞悬浮在1mL的0.5％FBS L15培养液，置入37℃培养箱里使细胞饥饿两小时。(1) Collect THP-1 mononuclear cells from Preparation Example 5, wash them twice with PBS, suspend the cells in 1 mL of 0.5% FBS L15 culture solution, and place them in a 37°C incubator to starve the cells for two hours.
(2)取300μl的THP-1细胞液(含有1×10
5细胞)种入上层孔盘(Corning，3421)里，接着将所述上层孔盘放入含600μL的0.5％FBS SW982条件培养基之24孔培养盘中。
(2) Take 300 μl of THP-1 cell solution (containing 1×10 5 cells) into the upper well plate (Corning, 3421), and then put the upper well plate into 600 μL of 0.5% FBS SW982 conditioned medium In a 24-well culture dish.
(3)将培养盘置入37℃培养箱里，使细胞爬行2小时。(3) Place the culture plate in a 37°C incubator and let the cells crawl for 2 hours.
(4)取出上层孔盘，并将培养液移除，接着将上层孔盘浸入甲醇中，固定8分钟后，取出上层孔盘、风干。(4) Take out the upper well plate and remove the culture solution, then immerse the upper well plate in methanol, fix it for 8 minutes, take out the upper well plate and air dry.
(5)用1/10稀释的Giemsa溶液(Fluka，48900)染上层孔盘表面的细胞一小时，接着用棉花棒将上层孔盘上方擦拭干净。(5) The cells on the surface of the upper well plate were stained with 1/10 diluted Giemsa solution (Fluka, 48900) for one hour, and then wiped clean with a cotton swab above the upper well plate.
(6)数细胞，并以对照组的细胞数为基准作图。(6) Count the cells, and plot based on the number of cells in the control group.
结果如图7所示，单纯经TNF-α蛋白质刺激组相较于对照组，迁移的单核球细胞THP-1数量明显增加，而处理有本发明的组合物的组别则具有抑制单核球细胞THP-1迁移的能力，且随浓度增加，抑制单核球细胞THP-1迁移的能力越明显。因此，本发明的组合物能有效抑制类风湿性关节炎引起之发炎细胞聚集、减缓类风湿性关节炎的发炎程度。The results are shown in Figure 7. Compared with the control group, the group stimulated with TNF-α protein alone significantly increased the number of migrating mononuclear cells THP-1, while the group treated with the composition of the present invention had an inhibitory mononuclear The ability of THP-1 to migrate from sphere cells, and as the concentration increases, the more obvious the ability to inhibit the migration of THP-1 from monocytes. Therefore, the composition of the present invention can effectively inhibit the aggregation of inflammatory cells caused by rheumatoid arthritis and slow down the degree of inflammation of rheumatoid arthritis.
实施例8本发明的组合物对TNF-α蛋白质刺激的滑膜肉瘤细胞SW982的IKK-β及RelA(NF-κB)途径的影响Example 8 Effect of the composition of the present invention on the IKK-β and RelA (NF-κB) pathways of TNF-α protein-stimulated synovial sarcoma cells SW982
转录因子RelA(NF-κB)的活化在免疫、发炎的反应中扮演重要的角色。在受到外来刺激如TNF-α时，IKK也会被磷酸化进一步使IKK-β分解，并使NF-κB呈现活化的状态，受活化的NF-κB会进入细胞核中引发特定基因的表现、产生大量的发炎因子造成后续的发炎反应。因此本实验透过测定TNF-α蛋白质刺激的滑膜肉瘤细胞SW982后细胞质中的IKK-β及细胞核的中NF-κB的量以测试异常发炎的发生。The activation of transcription factor RelA (NF-κB) plays an important role in immune and inflammatory responses. When subjected to external stimuli such as TNF-α, IKK will also be phosphorylated to further decompose IKK-β and render NF-κB activated. Activated NF-κB will enter the nucleus and trigger the expression and production of specific genes A large number of inflammatory factors cause a subsequent inflammatory response. Therefore, this experiment tested the occurrence of abnormal inflammation by measuring the amount of IKK-β in the cytoplasm and NF-κB in the nucleus after synovial sarcoma cells SW982 stimulated by TNF-α protein.
(一)蛋白质萃取(1) Protein extraction
(1)将本发明的组合物先溶于0.1M柠檬酸配制成浓度250mg/mL的溶液(pH3.5)，再进一步稀释成50、100、250、500μg/mL的浓度。(1) The composition of the present invention is first dissolved in 0.1 M citric acid to prepare a solution (pH 3.5) with a concentration of 250 mg/mL, and then further diluted to a concentration of 50, 100, 250, and 500 μg/mL.
(2)取制备例4的滑膜肉瘤细胞SW982以每孔3×10
5细胞的密度种植到6孔细胞培养盘中，并于37℃培养箱中培养24小时。
(2) The synovial sarcoma cells SW982 of Preparation Example 4 were planted in a 6-well cell culture dish at a density of 3×10 5 cells per well, and cultured in a 37°C incubator for 24 hours.
(3)将细胞分别处理0或10ng/mL的TNF-α蛋白质(R&D，210-TA)，并置于37℃培养箱培养24小时。(3) Treat cells with 0 or 10 ng/mL of TNF-α protein (R&D, 210-TA) and place them in a 37°C incubator for 24 hours.
(4)以步骤(1)中的不同浓度本发明的组合物处理细胞，并以0.1M柠檬酸溶液为对照组，放置到37℃培养箱培养24小时后，收集细胞。(4) Treat cells with different concentrations of the composition of the present invention in step (1), and use 0.1M citric acid solution as a control group, place them in a 37°C incubator for 24 hours, and collect the cells.
(5)萃取蛋白质：用PBS清洗细胞2次后，将培养盘置于冰上，加入含有蛋白酶抑制剂(protease inhibitor)(Thermo Pierce，87786)的100μL RIPA lysis buffer(Sigma，R0278)，用刮勺将细胞刮下来并收集到离心管中，再置于冰上20分钟。接着以12,000rpm离心10分钟，分别收集细胞质上清液蛋白质及细胞核蛋白质。利用BCA试剂组(Thermo Pierce，23227)定量蛋白质浓度，分配等量的蛋白质与sample buffer(Sigma，S3401)混匀，沸水煮10分钟。(5) Protein extraction: After washing the cells twice with PBS, place the culture plate on ice, add 100 μL of RIPA Analysis buffer (Sigma, R0278) containing protease inhibitor (Thermo Pierce, 87786), and scrape Scoop down the cells and collect them in a centrifuge tube, then place on ice for 20 minutes. Then centrifuged at 12,000 rpm for 10 minutes to collect cytoplasmic supernatant protein and nuclear protein. Use the BCA reagent set (Thermo Pierce, 23227) to quantify the protein concentration, dispense an equal amount of protein and mix with the sample buffer (Sigma, S3401), and boil for 10 minutes in boiling water.
(二)西方墨点法：(2) Western blot method:
(1)配制9％SDS-PAGE，取30μg的前述步骤(一)之(5)的蛋白质检体进行蛋白质电泳，接着转印一小时。(1) Prepare 9% SDS-PAGE, take 30 μg of the protein sample of the previous step (a) (5) and perform protein electrophoresis, followed by transfer for one hour.
(2)取出转印好的硝酸纤维素膜(nitrocellulose membrane，NC membrane)，浸入3％脱脂乳中进行blocking一小时。(2) Take out the transferred nitrocellulose membrane (NC membrane), immerse in 3% skim milk and block for one hour.
(3)将硝酸纤维素膜置换到一级抗体中，室温作用4小时。(β-Actin：Novus，NB600-501；RelA(NF-κB)Novus，NBP1-96139)，并以β-Actin及Lamin B1(Novus，NB100-56403)分别作为细胞质和细胞核的loading control蛋白质。(3) Replace the nitrocellulose membrane with the primary antibody and let it work at room temperature for 4 hours. (β-Actin: Novus, NB600-501; RelA (NF-κB) Novus, NBP1-96139), and β-Actin and Lamin B1 (Novus, NB100-56403) were used as loading control proteins of cytoplasm and nucleus, respectively.
(4)使用3％脱脂乳清洗硝酸纤维素膜三次。(4) Wash the nitrocellulose membrane three times with 3% skim milk.
(5)将硝酸纤维素膜置换到二级抗体中，室温作用2小时。(Goat anti rabbit IgG(H+L)HRP JacksonImmunoResearch，111-035-003、Goat anti mouse IgG(H+L)HRP JacksonImmunoResearch，115-035-003)。(5) Replace the nitrocellulose membrane with the secondary antibody, and act at room temperature for 2 hours. (Goat anti-rabbit IgG(H+L)HRP JacksonImmunoResearch, 111-035-003, Goat antimouse IgG(H+L)HRP JacksonImmunoResearch, 115-035-003).
(6)TBS buffer清洗硝酸纤维素膜三次后以定影剂、显影剂(Kodak，1901875)显影。(6) After cleaning the nitrocellulose membrane three times with TBS buffer, it was developed with a fixer and developer (Kodak, 1901875).
细胞核蛋白质结果如图8所示，以本发明的组合物处理的组别中细胞核的RelA(NF-κB)的量均较对照组低，且随处理本发明的组合物的浓度越高，滑膜肉瘤细胞SW982细胞核中的RelA(NF-κB)量越低，因此本发明的组合物可以抑制细胞异常发炎反应、减缓类风湿性关节炎的发炎程度。The results of nuclear protein are shown in Figure 8. The amount of nuclear RelA (NF-κB) in the group treated with the composition of the present invention was lower than that of the control group, and the higher the concentration of the composition of the present invention was The lower the amount of RelA (NF-κB) in the nucleus of the membranous sarcoma cell SW982, the composition of the present invention can suppress the abnormal inflammatory response of cells and slow down the degree of inflammation of rheumatoid arthritis.
实施例9类风湿性关节炎动物实验Example 9 Animal Experiment of Rheumatoid Arthritis
类风湿性关节炎为一种全身性慢性炎症疾病，滑液膜细胞增生伴随炎症细胞浸润、关节面血管翳(pannus formation)及关节损伤为其特征。实验动物以抗原诱导关节炎，其病程相似于类风湿性关节炎，因而如文献Trivillin et al.(2014).Boron neutron capture synovectomy(BNCS)as a potential therapy for rheumatoid arthritis:boron biodistribution study in a model of antigen-induced arthritis in rabbits.Radiation and environmental biophysics,53(4),635-643所述被长期应用于研究人类类风湿性关节炎的致病机制和治疗药物研发。Rheumatoid arthritis is a systemic chronic inflammatory disease characterized by hyperplasia of synovial membrane cells accompanied by infiltration of inflammatory cells, articular surface pannus (pannus formation) and joint damage. Experimental animals induced arthritis with antigens, the course of disease is similar to rheumatoid arthritis, so as in the literature Trivillin et al. (2014). Boron neutron capture synovectomy (BNCS) as potential treatment for rheumatoid arthritis:boron biodistribution study in a model Antigen-induced arthritis in rabbits. Radiation and environmental biophysics, 53(4), 635-643 has been used for a long time to study the pathogenesis of human rheumatoid arthritis and the development of therapeutic drugs.
取制备例6的兔子，分为对照组(不投予本发明的组合物)及实验组，实验组投予本发明的组合物60天，投予本发明组合物的时间点为兔子产生明显临床征状时，亦即触诊时有疼痛反应或跛行时即开始投予。实验组又分为高剂量组及低剂量组，其中高剂量组为每日以口服方式投予一次24mg/kg本发明的组合物、低剂量组为每日以口服方式投予一次8mg/kg本发明的组合物。并于本发明的组合物投药结束后将实验组及对照组的兔子予以牺牲，采其两侧膝关节滑膜组织(synovial tissue)并以4％paraformaldehyde予以固定；再以石蜡包埋后切成3-5μm之薄切片，并以苏木紫以及伊红染色，以显微镜及实时显像系统观察，进行组织病理学病变检查并依据
等(1997)发炎严重程度分级系统(Grade 0为正常，Grade 3为最严重)来进行评估。
The rabbits prepared in Preparation Example 6 were divided into a control group (without administration of the composition of the present invention) and an experimental group. The experimental group was administered the composition of the present invention for 60 days. The clinical symptoms, that is, palpation during palpation or lameness, began to administer. The experimental group is further divided into a high-dose group and a low-dose group, wherein the high-dose group is administered orally 24 mg/kg once a day, and the low-dose group is administered orally 8 mg/kg once a day. The composition of the present invention. After the administration of the composition of the present invention is completed, the rabbits of the experimental group and the control group are sacrificed, and the knee joint synovial tissue (synovial tissue) on both sides is collected and fixed with 4% paraformaldehyde; then embedded in paraffin and cut into Thin sections of 3-5μm, stained with hematoxylin and eosin, observed with a microscope and real-time imaging system, and performed histopathological examination and basis Et al. (1997) to assess the severity of inflammation (Grade 0 is normal, Grade 3 is the most severe).
其中，于第二次关节内注射抗原后第14天即陆续有兔子出现跛行征状，共计引起70％兔子呈现疼痛跛行临床征状(其中64％兔子于本发明的组合物投予前即呈现临床征状，另外36％兔子则于本发明的组合物给予后才出现跛行症状)。Among them, on the 14th day after the second intra-articular injection of antigens, rabbits gradually developed claudication symptoms, causing a total of 70% of rabbits to exhibit clinical symptoms of pain and claudication (of which 64% of rabbits presented before the composition of the invention was administered Clinical symptoms, the other 36% of the rabbits did not develop lameness symptoms until the composition of the present invention was administered).
此外，在本发明的组合物的投予期间，高剂量组仅有25％兔子从无跛行症状演变成有跛行症状，而低剂量组及对照组则分别有50％及66％。In addition, during the administration period of the composition of the present invention, only 25% of rabbits in the high-dose group evolved from no claudication symptoms to claudication symptoms, while the low-dose group and the control group had 50% and 66%, respectively.
而发炎严重程度的评估结果如下表1所示：The evaluation results of the severity of inflammation are shown in Table 1 below:
| A | 右膝关节Right knee |
| 高剂量High dose | 0.9±1.00.9±1.0 |
| 低剂量Low dose | 1.6±0.61.6±0.6 |
| 对照组Control group | 1.8±0.41.8±0.4 |
由肉眼检视病变及组织病理学来看，对照组兔子都有肉眼可见的病变且炎症程度亦较严重，高剂量组兔子有肉眼可见病变(均伴随有跛行征状)的比例较低且炎症程度较轻微，低剂量组则介于前述两个组别之间，因此本发明的组合物对兔子有推迟或改善病程、降低跛行发生率及炎症反应程度现象的效果。故本发明的组合物具有治疗或减缓类风湿性关节炎的效果。From the visual inspection of the lesions and histopathology, the rabbits in the control group had macroscopic lesions and more severe inflammation, and the rabbits in the high-dose group had macroscopic lesions (all accompanied by lameness symptoms) and a lower proportion of inflammation The lower dose group is between the two groups mentioned above, so the composition of the present invention has the effect of delaying or improving the course of disease, reducing the incidence of claudication and the degree of inflammatory response in rabbits. Therefore, the composition of the present invention has an effect of treating or slowing rheumatoid arthritis.
实施例10异位性皮肤炎动物实验Example 10 Animal experiment of atopic dermatitis
将制备例7的实验动物分为两组，实验组有12只动物(A01-A12)接受皮肤病药物治疗外，也给予本发明的组合物每天给予一次，剂量为100mg/10kg，对照组有2只动物(B01、B02)，同样接受皮肤药物治疗，但另外分别给予类固醇(B01)或Oclacitinib(B02)，其中皮肤病治疗药物根据动物情况给予包含抗霉菌药(itrconazole)、抗生素、抗组织胺、末梢血管扩张药物(pentoxifylline)、抗氧化物(Vitamin E)等，所有实验动物在开始使用本发明的组合物后，每2周回诊评估一次，评估的工具包含兽医师检查纪录、皮肤细菌及霉菌培养。The experimental animals of Preparation Example 7 were divided into two groups. In the experimental group, 12 animals (A01-A12) received dermatological drug treatment, and the composition of the present invention was also administered once a day at a dose of 100 mg/10 kg. The control group had 2 animals (B01, B02) were also treated with skin medication, but were additionally given steroids (B01) or Oclacitinib (B02), respectively. Dermatological treatment medications were given according to the animal's condition, including antifungal drugs (itrconazole), antibiotics, and anti-tissue Amines, peripheral vasodilator drugs (pentoxifylline), antioxidants (Vitamin) E, etc., all experimental animals after the use of the composition of the present invention, every two weeks for a follow-up evaluation, evaluation tools include veterinarian examination records, skin bacteria And mold culture.
(一)皮肤搔痒程度评估(1) Evaluation of skin itching
以pruritus visual analog scale(PVAS)为评估工具(Olivry,T.,Marsella,R.,Iwasaki,T.,Mueller,R.,&International Task Force On Canine Atopic Dermatitis.(2007).Validation of CADESI‐03,a severity scale for clinical trials enrolling dogs with atopic dermatitis.Veterinary dermatology,18(2),78-86.)，以0分表示不搔痒(not itchy)，而以10分表示重度搔痒(extremely itchy)。给予本发明的组合物的实验组，经兽医师检查纪录，结果如图9所示，其搔痒程度均有缓解功效；对照组中则以接受Oclacitinib药物者(B02)，其搔痒缓解程度和接受本发明的组合物的动物相近。Using pruritus visual analog (PVAS) as an evaluation tool (Olivry, T., Marsella, R., Iwasaki, T., Mueller, R., & International Task Force On On Canine Atopic Dermatitis. (2007).Validation of CADESI-03, a scale for clinical trials, enrolling dogs with a topic dermatitis. Veterinary dermatology, 18 (2), 78-86.), with 0 points for not itchy, and 10 points for extremely itchy (extremely itchy). The experimental group administered with the composition of the present invention has been examined by a veterinarian and the results are shown in Fig. 9. The degree of itching is alleviated; the control group is those who receive Oclacitinib (B02), the degree of itching relief and acceptance The composition of the present invention has similar animals.
(二)皮肤病兆严重程度(2) The severity of skin disease signs
皮肤病兆严重程度评估是经过兽医师检查、纪录，详细评估方法包括经由兽医师讨论、伍氏灯检查、皮毛采集显微镜下检查及皮肤压 片检查等，结果如图10所示，实验组多有明显改善，对照组中则以接受Oclacitinib药物的犬只(B02)获得明显改善。The evaluation of the severity of skin disease signs is carried out by the veterinarian. The detailed evaluation methods include discussion by the veterinarian, wood lamp examination, examination under the microscope of fur collection, and skin compression examination. The results are shown in Figure 10. There was a significant improvement, and in the control group, the dogs (B02) who received Oclacitinib were significantly improved.
(三)细菌及霉菌培养(3) Culture of bacteria and mold
以采样刷刷取动物身上皮毛上的皮屑，再以培养基进行细菌及霉菌的培养。Use the sampling brush to take the dander on the fur of the animal, and then culture the bacteria and mold with the medium.
结果如表2、表3显示，实验组于试验后，其细菌和霉菌培养多为阴性，显示当皮肤屏障修复完整后，较不易让健康的皮肤受到细菌或霉菌感染，而试验前后所培养出的细菌主要以克雷伯氏肺炎菌(Klebsiella pneumoniae)、金黄色葡萄球菌(Staphylococcus aureus)及革兰氏阳性杆菌，与Kristensen等人在1978年调查正常犬只皮肤菌丛的研究中金黄色葡萄球菌流行率高的结果相似(Kristensen,S.,
N.,&Mourier,H.(1978).A study of skin diseases in dogs and cats.IV.Patterns of flea infestation in dogs and cats in Denmark.Nordisk veterinaermedicin,30(10),401)。
The results are shown in Table 2 and Table 3. After the test, the bacteria and mold culture of the experimental group were mostly negative, indicating that when the skin barrier repair is complete, it is less likely to cause healthy skin to be infected by bacteria or mold. The bacteria are mainly Klebsiella pneumoniae, Staphylococcus aureus, and Gram-positive bacillus, and Kristensen et al. investigated the normal dog skin flora in 1978. The results with high cocci prevalence are similar (Kristensen, S., N., & Mourier, H. (1978). A study of skin diseases in dogs and cats. IV. Patterns of flea infestation in dogs and cats in Denmark. Nordisk veterinaermedicin, 30(10), 401).
表2皮屑的细菌及霉菌培养结果Table 2 Results of bacterial and mold culture of dander
P：阳性(positive)、N：阴性(negative)、NA：无法获得(not available)P: positive (positive), N: negative (negative), NA: not available (not available)
表3皮毛采样的细菌培养结果Table 3 Bacterial culture results of fur sampling
因此，有使用本发明的组合物的动物都会让皮肤快速修复且维持皮肤健康状态，本发明的组合物具有治疗或减缓异位性皮肤炎的效果。Therefore, the animals using the composition of the present invention will allow the skin to repair quickly and maintain the skin health state. The composition of the present invention has the effect of treating or alleviating atopic dermatitis.
实施例11本发明的组合物的清除自由基能力Example 11 free radical scavenging ability of the composition of the present invention
自由基不仅会造成皮肤老化、对于皮肤较敏感或是皮肤疾病的患者亦可能造成发炎、或破坏皮肤的阻隔作用并伴随搔痒症状的发生。DPPH(1,1-diphenyl-2-picrylhydrazyl)是一稳定的自由基，常用于测定物质的供氢、捕捉自由基的能力，本实验利用DPPH测定本发明的组合物的清除自由基能力，步骤如下：Free radicals can not only cause skin aging, patients who are sensitive to the skin or skin diseases, but also cause inflammation, or destroy the barrier effect of the skin and accompany itching symptoms. DPPH (1,1-diphenyl-2-picrylhydrazyl) is a stable free radical, which is often used to measure the ability of a substance to supply hydrogen and capture free radicals. In this experiment, DPPH was used to determine the free radical scavenging ability of the composition of the present invention. Steps as follows:
(1)将本发明的组合物先溶于0.1M柠檬酸配制成浓度250mg/mL的溶液(pH3.5)，再稀释成0.25、0.5、1、1.5、2、4mg/mL的浓度。(1) The composition of the present invention is first dissolved in 0.1 M citric acid to prepare a solution (pH 3.5) with a concentration of 250 mg/mL, and then diluted to a concentration of 0.25, 0.5, 1, 1.5, 2, 4 mg/mL.
(2)取90μL的50mM Tris-HCl、pH 7.4溶液到96孔盘。(2) Take 90 μL of 50 mM Tris-HCl, pH 7.4 solution to a 96-well dish.
(3)加入10μL的步骤(1)不同浓度的本发明的组合物，并以0mg/mL为对照组。(3) Add 10 μL of step (1) the composition of the present invention at different concentrations, and use 0 mg/mL as a control group.
(4)加入200μL的0.1mM DPPH(Sigma，D9132)溶液后均匀混合。(4) Add 200 μL of 0.1 mM DPPH (Sigma, D9132) solution and mix evenly.
(5)避光环境下于室温静置30分钟后，利用酵素免疫分析测读仪读取517nm的吸光值。(5) After standing at room temperature for 30 minutes in the dark, read the absorbance at 517nm using an enzyme immunoassay reader.
结果如图11所示，添加有本发明的组合物组的DPPH自由基清除能力均比对照组高，且随本发明的组合物的浓度增加，清除自由基的能力也增加，因此可降低自由基造成皮肤的发炎及相关症状的可能性。The results are shown in FIG. 11, the DPPH radical scavenging ability of the group to which the composition of the present invention is added is higher than that of the control group, and as the concentration of the composition of the present invention increases, the radical scavenging ability also increases, so the free The possibility of causing skin inflammation and related symptoms.
根据上述实施例，本发明的组合物不仅可降低巨噬细胞产生的一氧化氮自由基、降低嗜碱性球的去颗粒作用、抑制基质金属蛋白酶的分泌、抑制单核球细胞的迁移、抑制与发炎有关的NF-κB及IKK-β途径及增加自由基清除率等，并在动物实验中显示，能降低类风湿性关节炎跛行发生率、炎症程度并能减缓异位性皮肤炎造成的搔痒、病兆严重程度及加速皮肤的修复等，因此本发明的组合物可有效治疗或减缓自体免疫相关疾病，特别是类风湿性关节炎及异位性皮肤炎。According to the above examples, the composition of the present invention can not only reduce nitric oxide free radicals produced by macrophages, reduce the degranulation of basophiles, inhibit the secretion of matrix metalloproteinases, inhibit the migration and inhibition of mononuclear cells Inflammation-related NF-κB and IKK-β pathways and increase free radical scavenging rate, etc., and animal experiments have shown that it can reduce the incidence of rheumatoid arthritis lameness, the degree of inflammation and can slow down the atopic dermatitis caused Itching, the severity of disease symptoms, and accelerated skin repair, etc. Therefore, the composition of the present invention can effectively treat or slow down autoimmune-related diseases, especially rheumatoid arthritis and atopic dermatitis.
Claims (12)
- 一种组合物用于制备治疗或减缓自体免疫相关疾病的医药品的用途，其中所述组合物中含有由亚铁胺基酸螯合物烧结而成的亚铁胺基酸螯合物粒子，且所述亚铁胺基酸螯合物粒子的平均粒径为500奈米至2600奈米、平均分子量为1,500道尔顿(Dalton)至600,000道尔顿，所述医药品含有有效剂量的所述组合物及药学上可接受的载剂。Use of a composition for the preparation of a medicinal product for treatment or alleviation of autoimmune-related diseases, wherein the composition contains ferrous amino acid chelate particles sintered from ferrous amino acid chelate, And the average particle diameter of the ferrous amino acid chelate particles is 500 nm to 2600 nm, and the average molecular weight is 1,500 Dalton to 600,000 Dalton. The pharmaceutical product contains an effective dose of The composition and a pharmaceutically acceptable carrier.
- 根据权利要求1所述的用途，所述亚铁胺基酸螯合物粒子的平均分子量为1,500道尔顿至550,000道尔顿。The use according to claim 1, wherein the average molecular weight of the ferrous amino acid chelate particles is 1,500 Daltons to 550,000 Daltons.
- 根据权利要求1所述的用途，其中所述亚铁胺基酸螯合物的亚铁与胺基酸的重量比是介于1：1至1：4之间。The use according to claim 1, wherein the weight ratio of ferrous iron to amino acid of the ferrous amino acid chelate is between 1:1 and 1:4.
- 根据权利要求1所述的用途，其中所述亚铁胺基酸螯合物的亚铁与胺基酸的重量比是介于1：1.5至1：2.5之间。The use according to claim 1, wherein the weight ratio of ferrous iron to amino acid of the ferrous amino acid chelate is between 1:1.5 and 1:2.5.
- 根据权利要求4所述的用途，其中所述亚铁胺基酸螯合物是由铁化合物与胺基酸混合并历经60℃至90℃加热8小时至48小时所制得的亚铁胺基酸螯合物，其中无机铁化合物与胺基酸的重量比例是介于1：1.2至1：1.5之间。The use according to claim 4, wherein the ferrous amino acid chelate is a ferrous amino group prepared by mixing an iron compound with an amino acid and heating at 60°C to 90°C for 8 hours to 48 hours Acid chelate, wherein the weight ratio of inorganic iron compound to amino acid is between 1:1.2 and 1:1.5.
- 根据权利要求5所述的用途，其中所述铁化合物是硫酸亚铁、氯化亚铁、焦磷酸亚铁或其组合。The use according to claim 5, wherein the iron compound is ferrous sulfate, ferrous chloride, ferrous pyrophosphate, or a combination thereof.
- 根据权利要求1所述的用途，其中所述亚铁胺基酸螯合物为亚铁甘胺酸螯合物。The use according to claim 1, wherein the ferrous amino acid chelate is ferrous glycinate chelate.
- 根据权利要求1所述的用途，其中所述自体免疫相关疾病为类风湿性关节炎。The use according to claim 1, wherein the autoimmune-related disease is rheumatoid arthritis.
- 根据权利要求1所述的用途，其中所述自体免疫相关疾病为异 位性皮肤炎。The use according to claim 1, wherein the autoimmune-related disease is atopic dermatitis.
- 根据权利要求1或8所述的用途，其中所述有效剂量为2.5mg/kg至10mg/kg。The use according to claim 1 or 8, wherein the effective dose is 2.5 mg/kg to 10 mg/kg.
- 根据权利要求1所述的用途，所述治疗或减缓自体免疫相关疾病抑制免疫细胞的去颗粒作用。The use according to claim 1, wherein the treatment or slowdown of autoimmune-related diseases suppresses the degranulation of immune cells.
- 根据权利要求11所述的用途，所述抑制免疫细胞的去颗粒作用降低组织胺的释出量。According to the use according to claim 11, the degranulation effect of suppressing immune cells reduces the release of histamine.
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| PCT/CN2019/123869 WO2020125462A1 (en) | 2018-12-20 | 2019-12-09 | Use of composition comprising ferrous amino acid chelate particles in the preparation of medicament for treating or relieving diseases associated with nerve damage |
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| CN101102762A (en) * | 2004-12-22 | 2008-01-09 | 药物技术公司 | Compositions including iron |
| US20090035385A1 (en) * | 2004-12-22 | 2009-02-05 | Drugtech Corporation | Compositions including iron |
| CN103476419A (en) * | 2011-01-07 | 2013-12-25 | 梅里翁第三研究有限公司 | Pharmaceutical compositions of iron for oral administration |
| CN104769426A (en) * | 2012-07-27 | 2015-07-08 | 卢特波尔德药品公司 | Method of treating iron deficiency anemia |
| CN104955452B (en) * | 2013-09-05 | 2017-06-09 | 普惠德生技股份有限公司 | Purposes of the composition containing Ferrous amino acid chelates in the medicine for preparing anticancer |
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| JP5098051B2 (en) * | 2007-04-05 | 2012-12-12 | Ｓｂｉファーマ株式会社 | Mitochondrial disorder brain disease therapeutic agent and diagnostic agent |
| TWI587856B (en) * | 2016-05-26 | 2017-06-21 | Profeat Biotechnology Co Ltd | Compositions containing ferrous amino acid chelates are used in the manufacture of reduced cancer production The use of lactic acid medicine |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101102762A (en) * | 2004-12-22 | 2008-01-09 | 药物技术公司 | Compositions including iron |
| US20090035385A1 (en) * | 2004-12-22 | 2009-02-05 | Drugtech Corporation | Compositions including iron |
| CN103476419A (en) * | 2011-01-07 | 2013-12-25 | 梅里翁第三研究有限公司 | Pharmaceutical compositions of iron for oral administration |
| CN104769426A (en) * | 2012-07-27 | 2015-07-08 | 卢特波尔德药品公司 | Method of treating iron deficiency anemia |
| CN104955452B (en) * | 2013-09-05 | 2017-06-09 | 普惠德生技股份有限公司 | Purposes of the composition containing Ferrous amino acid chelates in the medicine for preparing anticancer |
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| WO2020125462A1 (en) | 2020-06-25 |
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| TW202034908A (en) | 2020-10-01 |
| TW202034907A (en) | 2020-10-01 |
| CN113260358A (en) | 2021-08-13 |
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