An oral polypeptide formulation for the prophylaxis and treatment of rheumatoid arthritis

Abstract

The application discloses an oral polypeptide preparation for preventing and treating rheumatoid arthritis. The application provides an oral polypeptide preparation for treating or preventing rheumatoid arthritis, and an active ingredient of the oral polypeptide preparation is a polypeptide shown in SEQ ID No. 1, and the polypeptide shown in SEQ ID No. 1 is loaded in dextran particles to form the oral polypeptide preparation. The oral polypeptide preparation can effectively inhibit secretion of inflammatory cytokines in a local rheumatoid arthritis, and delay or prevent disease progression. The oral polypeptide preparation has important application prospects in treating rheumatoid arthritis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an oral polypeptide preparation for the prevention and treatment of rheumatoid arthritis. Background Technology

[0002] Mitogen-activated protein kinase (MAPK) is a crucial signaling system mediating cellular responses, participating in cell growth, differentiation, and intercellular interactions. It is ubiquitous in various organisms, including yeast and mammalian cells. Since Sturgill and colleagues identified extracellular signal-regulated kinases (ERKs) in mammalian cells in 1991, research on the MAPK signaling pathway has progressed rapidly. Different members of the MAPK family exhibit significant sequence homology, all achieving maximum activation through phosphorylation at the "TXY" dual site. Currently, five p38MAPK isoforms have been isolated. The p38 family is divided into two categories based on its responsiveness to stimuli: p38α / β / β2 and p38γ / δ. The synthesis and activation of p38 family members are not universal, but p38MAPK in different cell lines and tissues can be activated by different mediators, producing varying biological effects, and is also regulated by different inflammatory mediators. Existing research has shown that p38 kinase, a member of the mitogen-activated protein kinase (MAPK) superfamily in eukaryotic cells, can transmit various extracellular stimuli to the nucleus, thereby regulating gene expression and serving as an important signaling center in many immune-related diseases. p38 MARK participates in signal transduction in physiological and pathological processes such as inflammation, stress, development, cell growth and apoptosis, cell cycle regulation, ischemia / reperfusion injury, and myocardial hypertrophy. Inhibiting p38 MARK activity can effectively block its mediated pathological signal transduction, thereby alleviating or even eliminating pathological phenomena.

[0003] Polysaccharides are homopolysaccharides composed of glucose as a monosaccharide, with glucose units linked by glycosidic bonds. Based on the type of glycosidic bond, they can be further divided into alpha-glucans and beta-glucans. β-glucans are the most physiologically active. The active structure of β-glucans is a polysaccharide composed of glucose units, most of which are linked by β-1,3 bonds, a common glucose chain linkage. It can activate macrophages and neutrophils, thus increasing the levels of leukocytes, cytokinins, and specific antibodies, comprehensively stimulating the body's immune system. This allows the body to better prepare to fight diseases caused by microorganisms. β-glucan can rapidly restore the ability of injured lymphocytes to produce cytokines (IL-1), effectively regulating the body's immune function. Numerous experiments have shown that β-glucan can promote the production of IgM antibodies in the body, thereby enhancing humoral immunity. This glucan-activated cell activity stimulates the host's non-specific defense mechanisms, making its application in tumors, infectious diseases, and wound treatment highly promising. Extracted through a special process and free of endotoxins, β-1,3-glucan has been recognized as a safe substance by the US FDA and can be added to general food. Many reports show that oral administration of yeast β-1,3-glucan to mice can increase the antibacterial phagocytic activity of peritoneal cells.

[0004] Yeast beta-glucan is a polysaccharide found in yeast cell walls that possesses immune-enhancing activity. Beta-glucan is widely present in various fungi and plants, such as shiitake mushrooms, Ganoderma lucidum, and oats, and is a key active ingredient in their health-promoting effects. Yeast beta-glucan exhibits even stronger immune-enhancing activity and also has effects such as improving blood lipids, resisting radiation, and improving intestinal function. Summary of the Invention

[0005] The purpose of this invention is to provide an oral polypeptide preparation for the prevention and treatment of rheumatoid arthritis.

[0006] In a first aspect, the present invention claims protection for an oral polypeptide preparation for the treatment or prevention of rheumatoid arthritis.

[0007] The active ingredient of the oral polypeptide preparation for the treatment or prevention of rheumatoid arthritis claimed in this invention is the polypeptide shown in SEQ ID No. 1.

[0008] Furthermore, the oral polypeptide formulation is formed by loading the polypeptide shown in SEQ ID No. 1 into dextran particles.

[0009] Furthermore, in the oral polypeptide formulation, each 0.5 mg of the dextran granules contains 10-250 μg (e.g., 50 μg) of the polypeptide shown in SEQ ID No. 1.

[0010] More specifically, the oral polypeptide formulation can be prepared by a method comprising the following steps:

[0011] P1. Dextran particles were extracted from live yeast cells in the logarithmic growth phase using an acid-base treatment method to obtain dextran particle powder.

[0012] P2. Dissolve the polypeptide shown in SEQ ID No. 1 in a 20% (v / v) aqueous ethanol solution to obtain a polypeptide solution; then add the dextran granules obtained in step P1 to the polypeptide solution for swelling and freeze-drying; after freeze-drying, add ultrapure water for swelling and freeze-drying, and repeat the "swell-freeze-dry" process (referring to the steps of swelling and freeze-drying with ultrapure water) until all the polypeptides are loaded into the dextran granules. Store at -20°C for later use.

[0013] The P1 process can be completed according to the following steps: Disperse the live yeast cells in the logarithmic growth phase in a 1M NaOH solution at 80°C, maintain the temperature at 80°C and stir for 1 hour, then centrifuge (e.g., at 8000g for 5 minutes) to collect the insoluble components; wash three times with ultrapure water to remove NaOH and substances decomposed by the hot alkali solution, adjust the pH to 5 with HCl, maintain the temperature at 55°C and continue stirring for 1 hour, then centrifuge (e.g., at 8000g for 5 minutes) to collect the insoluble components; wash twice with ultrapure water, four times with isopropanol, and twice with acetone; finally, vacuum dry all solvents to obtain the dextran granules in dry powder. Store at -20°C.

[0014] In P2, the peptide concentration in the peptide solution can be 1-10 μg / μL (e.g., 1 μg / μL). During the first swelling, the ratio of the dextran granules to the peptide solution can be 0.5 mg: 10-250 μL (e.g., 0.5 mg: 50 μL). The swelling is carried out at room temperature for 1 hour.

[0015] In a specific embodiment of the present invention, the yeast is brewer's yeast.

[0016] Secondly, the present invention claims protection for the method of preparing the oral polypeptide formulation described in the first aspect above.

[0017] The method for preparing the oral polypeptide formulation claimed in this invention includes P1 and P2 as described above.

[0018] Thirdly, the present invention claims protection for the use of the polypeptide and dextran particles shown in SEQ ID No. 1 in any of the following:

[0019] Q1. Prepare oral medications for treating rheumatoid arthritis or improving rheumatoid arthritis symptoms;

[0020] Q2. Prepare oral medications for the prevention of rheumatoid arthritis.

[0021] Fourthly, the present invention claims protection for the use of the oral polypeptide formulation described in the first aspect above in any of the following:

[0022] Q1. Prepare oral medications for treating rheumatoid arthritis or improving rheumatoid arthritis symptoms;

[0023] Q2. Prepare oral medications for the prevention of rheumatoid arthritis.

[0024] In a specific embodiment of the present invention, an experiment was conducted using a mouse model of rheumatoid arthritis. The oral polypeptide preparation was administered every other day, with a single dose of 50 μg polypeptide / 0.5 mg GPs, and the treatment cycle was 5 days.

[0025] Experiments have demonstrated that the oral polypeptide preparation provided by this invention can effectively inhibit the secretion of inflammatory cytokines in the local area of ​​rheumatoid arthritis, delaying or preventing disease progression. The oral polypeptide preparation provided by this invention has significant application prospects in the treatment of rheumatoid arthritis. Attached Figure Description

[0026] Figure 1 GPs loaded with peptides can protect peptides from degradation and significantly inhibit TNF-α expression levels induced by LPS and in the CIA model. (a) GPS can load peptides and protect them from degradation by gastrointestinal digestive enzymes (the Q11 peptide sequence in the figure is QQEFQFQFKQQ; the figure uses the Q11 peptide as an example to illustrate that GPS can protect loaded peptides from degradation, cited from J. Mater. Chem. B, 2014, 2, 5882); (b) Peptides can be loaded into GPS and effectively phagocytosed by macrophages; (c) Peptides loaded with GPS can inhibit LPS-induced TNF-α secretion levels in macrophages, with effects similar to those of peptides alone; (d) Inflammation index of rheumatoid arthritis (RA) in mice of each group; (e) TNF-α content in affected joints of CIA model mice after 50 days. It can be seen that the effect of GPs loaded with peptides is significantly stronger than that of the peptides themselves. It can be seen that oral administration of peptides alone is effective in the short term, but becomes ineffective after a long period of time due to degradation. The GPS-encapsulated peptides have targeting and sustained-release effects, and can significantly reduce the serum concentration of TNF-α over a long period of time. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] Example 1: Preparation method of glucan particles (GPs)

[0030] Genetically modified (GPs) are FDA-approved food additives, and commercially available products are already on the market, such as Biothera's Welmune supplement. Commercially available experimental GPs are also readily available, such as Invivogen's WGP. This invention uses a self-purified product obtained by purchasing live yeast (Saccharomyces cerevisiae, Baker's Yeast), culturing it to the logarithmic growth phase, and then extracting the GPs. Based on our experience, GPs obtained in this way retain their original biological activity relatively well.

[0031] The extraction method employed an acid-base treatment approach. First, 500 mL of live yeast (OD600 value between 0.6 and 0.8) in the logarithmic growth phase was separated from the culture medium by centrifugation at 8000g for 20 min, and the supernatant was discarded. A fresh 1M NaOH aqueous solution was prepared and heated to 80℃. The centrifuged live yeast was then dispersed in 100 mL of the hot NaOH solution and stirred at 80℃ for 1 h. After treatment, the solution was centrifuged at 8000g for 5 min, and the insoluble fraction was collected and washed three times in 100 mL of Milli-Q water (ultrapure water) to remove NaOH and substances decomposed by the hot alkali solution. The pH of the 100 mL solution was then adjusted to 5 with HCl and stirred at 55℃ for 1 h. The insoluble matter was then collected by centrifugation at 8000g, washed twice with Milli-Q water, four times with 50 mL of isopropanol, and twice with acetone. All solvents were removed under vacuum to obtain GPs, which were stored at -20℃.

[0032] Example 2: Loading peptides into GPs

[0033] The peptide shown in SEQ ID No. 1 (MKK3b) was dissolved in a 20% (v / v) ethanol / water solution to a concentration of 1 μg / μL, with a volume of approximately 20-50 μL. Then, the GPs powder prepared in Example 1 was placed in the peptide solution according to the predetermined peptide / GPs loading ratio (1 mg GPs loading 100 μg peptide). The solution was allowed to swell at room temperature for 1 hour, followed by complete lyophilization. After lyophilization, 10 μL of Milli-Q water was added to continue swelling for 1 hour, followed by another lyophilization. This swelling-lyophilization process was repeated approximately 3-5 times until all the peptides were loaded (if the peptides were not fully loaded, ciliated peptide lyophilized fibers would be present on the outside of the dextran particles after lyophilization. The absence of ciliated peptide lyophilized fibers indicates that the peptides have been fully loaded). Finally, the GPs loaded with MKK3b peptides were lyophilized and stored at -20°C for later use.

[0034] The Q11 peptide (Q11 peptide sequence: QQEFQFQFKQQ) was labeled with Cy5, and GPs were labeled with DTAF. The Q11 peptide was then loaded into the GPs according to the method described above. The GPs loaded with the Q11 peptide were then digested with simulated gastric juice and simulated small intestinal juice, with or without the enzyme (the formulation and preparation methods of the simulated gastric juice and simulated intestinal juice are publicly available in the European Pharmacopoeia (07 / 2010:51701 Recommendations on Dissolution Testing. Section 2.12 of the Methods section of J. Mater. Chem. B, 2014, 2, 5882 describes the formulation and preparation methods in detail). Samples were taken before and after digestion for fluorescence imaging. After digestion, the ratio of the fluorescence intensity of Cy5 containing the peptide to the fluorescence intensity of the DTAF-labeled GPs was measured; the ultraviolet absorption of Cy5 in the supernatant after digestion was also measured. The results are as follows: Figure 1 As shown in (a), the first fluorescence image illustrates the MKK3b peptide loaded into GPs; the second inset shows the GPs containing the fluorescent peptide after digestion with simulated gastric and small intestinal fluids, centrifuged. The fluorescence intensity of Cy5 containing the peptide and the fluorescence intensity of the DTAF-labeled GPs were measured, and the ratio was unchanged before and after digestion, suggesting that GPs protect the loaded peptide from enzymatic digestion; the third inset shows the supernatant after digestion, measuring the UV absorption of Cy5. If the peptide is digested, the UV absorption of Cy5 in the supernatant will be high, and vice versa. This comparison further verifies that GPs can protect the loaded peptide from degradation by digestive enzymes.

[0035] Example 3: Macrophage phagocytosis of GPs containing peptides can effectively inhibit TNF-α secretion.

[0036] The test peptide was MKK3b peptide; the test GPs containing MKK3b peptide were the products prepared in Example 2; the test GPs were the products prepared in Example 1.

[0037] The mouse macrophage cell line RAW264.7 was stabilized and passaged three times after resuscitation. Then, it was seeded at a density of 20,000 cells per well in 6-well plates overnight. The next day, 10 times the cell count of macrophages loaded with Cy5-labeled MKK3b peptides (Cy5 labeling of peptides is a well-known and routine procedure in this field) were added. The cells were incubated at 37°C for 8 hours. Unphased macrophages were washed away with PBS, and imaging under a microscope showed that the Cy5-labeled MKK3b peptides were significantly taken up by the RAW264.7 cells. Results are shown below. Figure 1 (b)

[0038] Meanwhile, RAW264.7 cells were seeded in 96-well plates at a density of 5000 cells per well overnight. The next day, the following treatments were administered: control group (solvent only, i.e., phosphate-buffered saline PBS); MKK3b peptide group (10 μg); GPs group (0.1 mg); LPS stimulation group (0.1 μg / mL); LPS+GPs group (0.1 μg / mL LPS + 0.1 mg GPs); LPS+MKK3b peptide group (0.1 μg / mL LPS + 10 μg MKK3b); peptide-loaded GPs (10 μg peptide / 0.1 mg GPs); and LPS+peptide-loaded GPs group (0.1 μg / mL LPS + 10 μg peptide / 0.1 mg GPs). After 8 hours, the cell culture supernatant was collected to measure the TNF-α concentration. Results are shown below. Figure 1 (c)

[0039] Example 4: Establishment of a CIA mouse model

[0040] Six- to eight-week-old male DBA / 1 mice were used (see: https: / / www.jax.org / strain / 000670). DBA / 1 mice are relatively sensitive to chicken, bovine, and porcine type II collagen. Bovine type II collagen (Chondrex) was completely dissolved at a concentration of 2 mg / mL in 0.1 M glacial acetic acid solution and incubated overnight at 4°C. Subsequently, it was mixed with an equal volume of complete Freund's adjuvant (Sigma) and thoroughly emulsified by sonication to obtain a collagen emulsion of 1 mg / mL. The collagen emulsion (100 μL / mouse) was injected intradermally (intradermal, not subcutaneous) 2-3 cm from the base of the tail in the DBA / 1 mice for the first injection. Ulceration at the injection site is normal and usually occurs within 7-10 days. A second injection of the same volume of emulsion was given on day 21. Rheumatoid arthritis (RA) inflammatory indices were recorded every other day starting one day before injection and continued until day 50. Significant progression of RA is generally observed around 28 days. Day 0 is defined as the first injection of collagen emulsion. RA inflammation index score: 0 points: no redness or swelling; 1 point: mild redness and swelling of the ankle or toe; 2 points: severe redness and swelling of the joint or toe; 3 points: joint stiffness or ankylosis. The limbs of each mouse were observed individually, and the cumulative score was recorded.

[0041] Example 5: Grouping of CIA mice and determination of joint TNF-alpha

[0042] The test peptide was MKK3b peptide; the test GPs containing MKK3b peptide were the products prepared in Example 2; the test GPs were the products prepared in Example 1.

[0043] Mice with the CIA model established in Example 4 were divided into groups of 5. On days 24, 26, and 28 (day 0 being the first injection of collagen emulsion during CIA modeling), mice were administered oral orally via gavage (100 μL of physiological saline) (i.e., the CIA model group), GPs (0.5 mg GPs), MKK3b peptide (50 μg), GPs containing MKK3b peptide (50 μg peptide / 0.5 mg GPs), and dexamethasone (0.5 mg / kg body weight) via tail vein injection, respectively. Before oral or gavage, mice were deprived of water and food for 4 hours. After treatment via gavage or injection, water and food were restored. Normal mice were used as controls.

[0044] On the one hand, the inflammatory index of rheumatoid arthritis (RA) in each group of mice was measured according to the method in Example 4, such as Figure 1 As shown in (d).

[0045] On the other hand, mice in each group were euthanized by cervical dislocation at day 50 (day 0 for the first injection of collagen emulsion when modeling the CIA model). Affected joints were harvested, weighed, and then ground into a powder in 0.5 mL of HBSS on ice. The supernatant was collected by centrifugation, and the concentration of TNF-alpha was determined using an ELISA kit (eBioscience). Results are shown below. Figure 1 Middle (e).

[0046] The results of each embodiment are as follows Figure 1 As shown. Figure 1 This study demonstrates that peptide-loaded GPs protect peptides from degradation and significantly inhibit TNF-α expression levels induced by LPS and in CIA models. (a) GPS can load peptides and protect them from degradation by gastrointestinal digestive enzymes; (b) Peptides can load GPS and be effectively phagocytosed by macrophages; (c) Peptides loaded with GPS can inhibit LPS-induced TNF-α secretion by macrophages, with effects similar to peptides alone; (d) MKK3b peptide alone can effectively delay rheumatoid arthritis in the short term, but progression recurs after drug withdrawal; while oral peptide formulations long-term prevented the progression of rheumatoid arthritis in mouse CIA models, with effects similar to dexamethasone; (e) TNF-α levels in affected joints of CIA model mice after 50 days show that GPs loaded with peptides are significantly more effective than peptides alone. It can be seen that oral peptides alone are effective in the short term, but become ineffective after long-term degradation. In contrast, the peptides encapsulated by GPS have targeting and sustained-release effects, significantly reducing serum TNF-α concentrations over a long period.

[0047] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. <110> Beijing University <120> An oral polypeptide preparation for the prevention and treatment of rheumatoid arthritis <130> GNCLN212376 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> twenty four <212> PRT <213> Artificial sequence <400> 1 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Lys Gly Lys Ser 1 5 10 15 Lys Arg Lys Lys Asp Leu Arg Ile 20

Claims

1. An oral polypeptide preparation for the treatment or prevention of rheumatoid arthritis, characterized in that: The active ingredient of the oral polypeptide preparation is the polypeptide shown in SEQ ID No. 1; The oral polypeptide formulation is formed by loading the polypeptide shown in SEQ ID No. 1 into dextran particles; In the oral polypeptide formulation, each 0.5 mg of the dextran granules contains 10-250 μg of the polypeptide shown in SEQ ID No. 1; The oral polypeptide formulation is prepared by a method comprising the following steps: P1. Dextran particles were extracted from yeast cells in the logarithmic growth phase using an acid-base treatment method to obtain dextran particle powder. P2. Dissolve the polypeptide shown in SEQ ID No. 1 in an aqueous ethanol solution with a volume percentage of 20% to obtain a polypeptide solution; then add the dextran granules obtained in step P1 to the polypeptide solution for swelling and freeze-drying; after freeze-drying, add ultrapure water for swelling and freeze-drying, and repeat the "swelling-freeze-drying" process until all the polypeptides are loaded into the dextran granules. The P1 process is performed according to the following steps: dispersing the logarithmic growth phase yeast cells in a 1 M NaOH solution at 80°C, stirring at 80°C for 1 hour, then centrifuging to collect the insoluble components; washing with ultrapure water, adjusting the pH to 5, stirring at 55°C for another hour, then centrifuging to collect the insoluble components; washing sequentially with ultrapure water, isopropanol, and acetone; finally, vacuum drying to obtain the dextran granules powder. In P2, the peptide concentration in the peptide solution is 1-10 μg / μL; and in P2, during the first swelling, the ratio of the dextran granule powder to the peptide solution is 0.5 mg: 10-250 μL.

2. The method for preparing the oral polypeptide formulation according to claim 1, characterized in that, It is prepared according to a method including the following steps: P1. Dextran particles were extracted from yeast cells in the logarithmic growth phase using an acid-base treatment method to obtain dextran particle powder. P2. Dissolve the polypeptide shown in SEQ ID No. 1 in an aqueous ethanol solution with a volume percentage of 20% to obtain a polypeptide solution; then add the dextran granules obtained in step P1 to the polypeptide solution for swelling and freeze-drying; after freeze-drying, add ultrapure water for swelling and freeze-drying, and repeat the "swelling-freeze-drying" process until all the polypeptides are loaded into the dextran granules. The P1 process is performed according to the following steps: dispersing the logarithmic growth phase yeast cells in a 1 M NaOH solution at 80°C, stirring at 80°C for 1 hour, then centrifuging to collect the insoluble components; washing with ultrapure water, adjusting the pH to 5, stirring at 55°C for another hour, then centrifuging to collect the insoluble components; washing sequentially with ultrapure water, isopropanol, and acetone; finally, vacuum drying to obtain the dextran granules powder. In P2, the peptide concentration in the peptide solution is 1-10 μg / μL; and in P2, during the first swelling, the ratio of the dextran granule powder to the peptide solution is 0.5 mg: 10-250 μL.

3. The use of the oral polypeptide formulation of claim 1 in any of the following: Q1. Prepare oral medications for treating rheumatoid arthritis or improving rheumatoid arthritis symptoms; Q2. Prepare oral medications for the prevention of rheumatoid arthritis.

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