Active super antigen compound biological injection preparation pain repairing T and preparation method thereof

Abstract

The invention relates to the technical field of biological preparations, and discloses a preparation method of an active super antigen compound biological injection preparation for pain repair T. The preparation method comprises the following steps: extracting a super antigen with relatively high activity on the basis of culturing a staphylococcus aureus strain; based on amplification expression of a target cytokine gene and submaxillary gland cell treatment, collecting a cytokine IL-10 and a nerve growth factor NGF, and mixing the extracted active super antigen, the cytokine IL-10, the nerve growth factor NGF, an NSAIDs derivative and an amino acid group in sequence. According to the active super antigen compound biological injection preparation pain repairing T and the preparation method thereof, a multi-target synergistic effect mechanism is formed by adopting a compound combination of an active super antigen, IL-10, NGF, an NSAIDs derivative, glutathione and taurine, so that the super antigen activates immune regulation, IL-10 inhibits inflammation, NGF promotes nerve repairing, and NSAIDs provides anti-inflammatory and analgesic effects; meanwhile, an amino acid group is matched to enhance the oxidation resistance, so that the action mechanism of pain repair is effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological preparations, and in particular to an active superantigen compound biological injection preparation pain repair T and a preparation method thereof. Background Art

[0002] Biological preparations refer to biological products made from microorganisms (bacteria, rickettsia, viruses, etc.) and their metabolites' effective antigenic components, animal toxins, human or animal blood or tissues, etc., for the prevention, treatment, and diagnosis of corresponding infectious diseases or other related diseases. They are manufactured using traditional or modern biotechnology and can have preventive, health-care, therapeutic, and diagnostic effects on various physiological symptoms of the human body. They are also called immune biological preparations.

[0003] After searching, it was found that a cell growth regulatory factor B and its preparation method are disclosed in the invention patent with Chinese patent publication number CN1424045A. The preparation method of the cell growth regulatory factor B in the invention patent is to quantitatively add leucine to the extracted natural gene and seal it with a water agent according to GMP standards to produce natural cell growth regulatory factor B, thereby activating the body to enhance immune function, increase TC, and repair and reshape aging, necrosis, degeneration, shrinkage, and fibrosis of tissue cells, and regenerate and repopulate capillaries, thereby playing a role in nerve repair and reshaping.

[0004] However, the preparation method of cell growth regulatory factor B uses a combination of natural genes and leucine, lacks a clear synergistic mechanism, the functions of the ingredients are relatively simple, and the preparation process lacks purification details, and the product quality controllability is low. Therefore, it is necessary to further improve the preparation method to increase the activity of the preparation. Therefore, an active superantigen compound biological injection preparation pain repair T and its preparation method are proposed. Summary of the Invention

[0005] (1) Technical problems solved

[0006] In response to the shortcomings of the existing technology, the present invention provides an active superantigen compound biological injection preparation pain repair T and a preparation method, which has the advantage of multiple components synergistically acting to improve the activity of the preparation, and solves the problem that the cell growth regulatory factor components in the above-mentioned background technology have relatively single functions and need further improvement in preparation control and improving immune activity effects.

[0007] (2) Technical solution

[0008] In order to achieve the above-mentioned multi-component synergistic effect to improve the activity of the preparation, the present invention provides the following technical solution: an active superantigen compound biological injection preparation pain repair T, including a preparation component composed of an active superantigen and a regulatory repair factor as active ingredients, and an auxiliary factor and an amino acid group as a small molecule excipient.

[0009] Preferably, the active superantigen is based on highly polysaccharide staphylococcus aureus metabolites, the regulatory repair factor comprises cytokine IL-10 and nerve growth factor NGF added with low-dose cyclophosphamide in a ratio of 1:4, the auxiliary factor is a non-steroidal anti-inflammatory drug NSAIDs derivative, the amino acid group comprises glutathione and taurine, and the active superantigen is prepared according to the formula (0.5-2×10 -2 ) μg of IL-10, 0.02-0.1 μg of NGF, 1.3-3.8 μg of NSAIDs derivatives, 3.1-12.4 μg of glutathione and 6.3-22.1 μg of taurine.

[0010] The method for preparing the active superantigen compound biological injection preparation pain repair T comprises the following steps:

[0011] S1. Antigen Extraction: Staphylococcus aureus is fermented in liquid culture, the cells are centrifuged and then disrupted. After fine purification and removal of endotoxins, highly active superantigens are obtained.

[0012] S2. Repair factor extraction: Based on the collection of cytokine IL-10, the gene sequence of the target cytokine is obtained. The gene is amplified by PCR and expressed in mammalian cells. Hamster ovary CHO cells are used as the host cell for transfection screening and secretory expression. Based on the collection of nerve growth factor (NGF), adult male mouse submandibular gland cells are obtained and homogenized with extraction buffer. The supernatant is then centrifuged to obtain the supernatant. The cytokine IL-10 and nerve growth factor (NGF) are extracted and purified by chromatography.

[0013] S3. Component mixing: The extracted active superantigen, cytokine IL-10 and nerve growth factor NGF, as well as NSAIDs derivatives and amino acid groups are mixed in order to prepare the active superantigen compound biological injection preparation pain repair T.

[0014] Preferably, the specific steps of antigen extraction in step S1 include:

[0015] 1) selecting a liquid culture medium containing glucose, yeast extract, and inorganic phosphate, controlling the pH to 6.5-7.2, setting the fermentation temperature to 30-37° C. and controlling the stirring speed to 150-400 rpm for 24-48 hours;

[0016] 2) The fermentation broth was centrifuged at high speed, and the supernatant was removed. The bacterial precipitate was collected, and the cells were resuspended in phosphate buffer in an ice bath and sonicated at a frequency of 20 to 25 kHz for 10 to 15 minutes. After disruption, the cells were centrifuged again, and the supernatant containing the superantigen components was collected.

[0017] 3) Purification was performed using DEAE column chromatography, and the target protein was separated after elution with a buffer solution having a pH of 8.0 to obtain an active superantigen.

[0018] Preferably, the step of extracting the repair factor in step S2 includes:

[0019] 1) The gene sequence of the human IL-10 gene was obtained from a public gene library. The gene was amplified by PCR and expressed in mammalian cells using the pcDNA3.1 vector. The IL-10 gene was inserted into the pcDNA3.1 vector, and a recombinant plasmid was constructed using ligase and transformed into competent cells for plasmid amplification.

[0020] 2) Using hamster ovary CHO cells as the host cell, the CHO cells were transfected by liposomes and expression strains were selected based on the resistance marker in the vector. The selected expression strains were fermented in a serum-free medium at 32-37° C., 30-60% dissolved oxygen, and pH 6.8-7.2, with glucose, glutamine, and a special feed agent added in batches:

[0021] 3) After centrifuging the culture medium, the host cells are removed and the supernatant is collected. The supernatant is purified by ion exchange chromatography to obtain the cytokine IL-10:

[0022] 4) Submandibular gland cells of adult male mice were collected and homogenized with extraction buffer in an ice bath. The supernatant was collected after further centrifugation and purified by ion exchange chromatography to obtain nerve growth factor (NGF).

[0023] Preferably, the specific steps of mixing the components in step S3 include:

[0024] 1) Active ingredient pretreatment: a. Prepare a histidine buffer containing 1% glycine and controlled at a pH of 6.5-7.0, and dissolve the active superantigen and cytokine IL-10 in the histidine buffer; b. Prepare a sodium acetate buffer containing 0.1% human serum albumin and a pH of 5.0, and dissolve nerve growth factor (NGF) in the sodium acetate buffer; c. Dissolve the NSAID derivative in a small amount of DMSO solution and dilute the NSAID concentration with PBS to less than 0.1%; d. Dissolve glutathione and taurine directly in the histidine-glycine preparation buffer;

[0025] 2) Liposome carrier preparation: Blank liposomes were prepared with a ratio of DSPC:cholesterol:PEG-DSPE = 55:35:10. After membrane extrusion, the particle size of the liposome suspension was controlled to be 100-200 nm;

[0026] 3) Macromolecule Mixing: a. Premix the active superantigen and cytokine IL-10 in histidine buffer and nerve growth factor (NGF) in sodium acetate buffer in appropriate proportions. Then, evenly mix the macromolecule mixture with the blank liposome suspension at a volume ratio of 1:10. b. Stir at 200 rpm in a 37°C water bath for 1 hour to ensure active drug loading of the liposomes. Purify the mixture by ultrafiltration to remove free protein.

[0027] 4) Small molecule mixing: slowly add the NSAIDs derivative to the drug-loaded liposome suspension, stir evenly at 500 rpm for 10 minutes, then add glutathione and taurine in sequence, control the stirring speed to 100-150 rpm, and adjust the pH to 6.8 to obtain;

[0028] 5) adding 4% w / v mannitol and 1% w / v sucrose as freeze-drying protective agents, and controlling the pH to be 6.8-7.0.

[0029] (3) Beneficial effects

[0030] Compared with the prior art, the present invention provides an active superantigen compound biological injection preparation pain repair T and a preparation method thereof, which has the following beneficial effects:

[0031] 1. The active superantigen compound biological injection preparation pain repair T and its preparation method use a compound combination of active superantigens, IL-10, NGF, NSAIDs derivatives and glutathione and taurine to form a multi-target synergistic mechanism, which promotes superantigens to activate immune regulation, IL-10 to inhibit inflammation, NGF to promote nerve repair, and NSAIDs to provide anti-inflammatory and analgesic effects. At the same time, it is combined with an amino acid group to enhance antioxidant capacity, thereby effectively improving the mechanism of pain repair.

[0032] 2. This active superantigen compound biological injection preparation pain repair T and its preparation method, by adopting gene expression, liposome drug delivery and ion exchange chromatography purification technology, strictly controls the preparation steps and parameters, which can effectively ensure the high activity and low toxicity of the prepared product, thereby effectively improving the treatment effect and reducing side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The present invention is a flow chart of the preparation method of the active superantigen compound biological injection preparation. DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0035] Example 1

[0036] In this embodiment, the activity of the components in the biological injection preparation is detected, including:

[0037] 1) Superantigen activity detection is achieved through T cell activation assay and cytokine release assay. The specific method of T cell activation assay is as follows:

[0038] 1) Human peripheral blood mononuclear cells were co-cultured with different concentrations of the preparation at 37°C and 5% CO2 for 24 hours;

[0039] 2) Based on the expression of T cell surface activation markers CD69 / CD25 detected by flow cytometry, the activation rate was calculated to ensure that the T cell activation rate was ≥15%;

[0040] 3) positive control with the superantigen Staphylococcal enterotoxin B of known activity;

[0041] 2) The specific method for detecting cytokine release is as follows:

[0042] The concentrations of IL-2, IFN-γ, and TNF-α in the culture supernatant were determined by ELISA to verify the SDCC effect of superantigen-dependent cytokine circulation and ensure that the amount of IFN-γ released should reach 80% to 120% of the positive control;

[0043] 3) The specific detection method for cytokine IL-10 activity is:

[0044] 1) LPS-stimulated THP-1 macrophages were co-cultured with the formulation, and the reduction ratios of TNF-α and IL-6 in the culture medium were measured by ELISA. The TNF-α inhibition rate was determined to be ≥70% at an IL-10 concentration of 10 ng / mL to verify the anti-inflammatory effect.

[0045] 2) Determine the binding affinity of IL-10 to the IL-10 receptor based on surface plasmon resonance, ensure the KD value is ≤1 nM, and perform receptor binding experiments;

[0046] 4) The method for detecting the activity of nerve growth factor NGF is as follows:

[0047] 1) Rat pheochromocytoma PC12 cells were co-cultured with a formulation containing NGF for 5 days. The proportion of cells with neurites >50 μm in length was counted under a microscope to ensure that the differentiation rate induced by 50 ng / mL NGF was ≥60%. PC12 cell differentiation experiments were performed.

[0048] 2) Detection of TrkA receptor tyrosine phosphorylation levels based on Western Blot analysis to verify activation of the NGF signaling pathway and perform TrkA receptor phosphorylation detection;

[0049] 5) Activity detection methods for NSAIDs derivatives include:

[0050] 1) Using a COX-1 / COX-2 enzyme activity assay kit, determine the inhibition rate of the prodrug on the enzyme after conversion to its active form and perform a COX enzyme inhibition experiment.

[0051] 2) Inflammation was simulated using umbilical vein endothelial cells, and the PGE2 secretion inhibition rate was detected. The PGE2 secretion inhibition rate at a concentration of 10 μM was determined to be ≥50%, and an in vitro anti-inflammatory experiment was performed;

[0052] 6) Methods for detecting the antioxidant activity of glutathione (GSH) and taurine include:

[0053] 1) Determine the scavenging rate of DPPH free radicals by GSH and taurine in the preparation, and determine that the DPPH free radical scavenging rate in 5 mM GSH is ≥ 80%.

[0054] 2) Neuronal cells were treated with H2O2 to verify the inhibitory effect of the preparation on reactive oxygen species (ROS). Cell oxidative stress experiments were performed to determine whether the DCF fluorescence intensity was reduced by ≥50% as determined by flow cytometry.

[0055] Example 2

[0056] In this embodiment, the safety of biological agents is evaluated and tested, including:

[0057] 1) Endotoxin detection

[0058] Endotoxin content was tested based on the Limulus Amebocyte Lysate (LAL) method to ensure that the endotoxin content was less than 0.1 EU / mg. 2) Host protein residue detection

[0059] Detection of residual HCP using anti-host cell protein HCP antibodies based on ELISA method, and ensure that CHO cell HCP residual is less than 100ppm;

[0060] 3) Cytokine Storm Risk Detection

[0061] 1) In conjunction with acute toxicity studies in mice, a single high-dose intravenous injection of approximately 10 times the therapeutic dose was administered, and body temperature, body weight, and peak serum cytokines, including IL-6 and TNF-α, were monitored to detect sudden temperature rise or death.

[0062] 2) Combined with the in vitro PBMC overactivation experiment, the PBMCs were co-cultured with a formulation at a dose greater than 10 times the therapeutic dose to detect whether the release of IL-2 and IFN-γ exceeded the safety threshold;

[0063] 4) Local and systemic toxicity testing

[0064] 1) Combined with the rabbit skin local irritation test, the redness, swelling, and induration were observed after a single subcutaneous injection;

[0065] 2) Combined with repeated dose toxicity studies in rats, blood biochemistry including ALT, AST, creatinine, and spleen and lymph node pathology were measured after 4 consecutive weeks of administration;

[0066] 5) Immunogenicity testing

[0067] 1) Based on the anti-drug antibody (ADA) test, detect whether neutralizing antibodies against SAG, IL-10, or NGF are produced in the animal serum after drug administration, and ensure that the positive rate is less than 5%;

[0068] VI) Nanocarrier Safety Testing

[0069] 1) Incubate the preparation with a 2% rabbit red blood cell suspension at 37°C for 1 hour, and measure the hemolysis rate to ensure that the hemolysis rate is <5%;

[0070] 2) Detect elevated levels of complement C3a and C5a in human serum and perform complement activation experiments.

[0071] The beneficial effects of the present invention are as follows: the active superantigen compound biological injection preparation pain repair T and its preparation method form a multi-target synergistic mechanism by adopting a compound combination of active superantigens, IL-10, NGF, NSAIDs derivatives and glutathione and taurine, so as to promote superantigens to activate immune regulation, IL-10 to inhibit inflammation, NGF to promote nerve repair, and NSAIDs to provide anti-inflammatory and analgesic effects, while simultaneously cooperating with an amino acid group to enhance antioxidant capacity, thereby effectively improving the mechanism of action of pain repair. By adopting gene expression, liposome drug delivery and ion exchange chromatography purification technology, and strictly controlling the preparation steps and parameters, the high activity and low toxicity of the prepared product can be effectively ensured, thereby effectively improving the therapeutic effect and reducing side effects.

[0072] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Active superantigen compound biological injection preparation pain repair T, characterized by: The preparation components include active super antigens and regulatory repair factors of active ingredients, as well as auxiliary factors and amino acid groups of small molecule excipients.

2. The active superantigen compound biological injection preparation pain repair T according to claim 1, characterized in that: The active superantigen is based on highly aggregated staphylococcus aureus metabolites, the regulatory repair factor includes cytokine IL-10 and nerve growth factor NGF added with low-dose cyclophosphamide in a ratio of 1:4, the auxiliary factor is a non-steroidal anti-inflammatory drug (NSAID) derivative, and the amino acid group includes glutathione and taurine. The active superantigen is prepared according to the formula (0.5-2×10 -2 ) μg of IL-10, 0.02-0.1 μg of NGF, 1.3-3.8 μg of NSAIDs derivatives, 3.1-12.4 μg of glutathione and 6.3-22.1 μg of taurine.

3. The preparation method of active superantigen compound biological injection preparation pain repair T is characterized by: The following steps are included: S1. Antigen Extraction: Staphylococcus aureus is fermented in liquid culture, the cells are centrifuged and then disrupted. After fine purification and removal of endotoxins, highly active superantigens are obtained. S2. Repair factor extraction: Based on the collection of cytokine IL-10, the gene sequence of the target cytokine is obtained. The gene is amplified by PCR and expressed in mammalian cells. Hamster ovary CHO cells are used as the host cell for transfection screening and secretory expression. Based on the collection of nerve growth factor (NGF), adult male mouse submandibular gland cells are obtained and homogenized with extraction buffer. The supernatant is then centrifuged to obtain the supernatant. The cytokine IL-10 and nerve growth factor (NGF) are extracted and purified by chromatography. S3. Component mixing: The extracted active superantigen, cytokine IL-10 and nerve growth factor NGF, as well as NSAIDs derivatives and amino acid groups are mixed in order to prepare the active superantigen compound biological injection preparation pain repair T.

4. The active superantigen compound biological injection preparation pain repair T and its preparation method according to claim 3, characterized in that: The specific steps of antigen extraction in step S1 include: 1) selecting a liquid culture medium containing glucose, yeast extract, and inorganic phosphate, controlling the pH to 6.5-7.2, setting the fermentation temperature to 30-37° C. and controlling the stirring speed to 150-400 rpm for 24-48 hours; 2) The fermentation broth was centrifuged at high speed, and the supernatant was removed. The bacterial precipitate was collected, and the cells were resuspended in phosphate buffer in an ice bath and sonicated at a frequency of 20 to 25 kHz for 10 to 15 minutes. After disruption, the cells were centrifuged again, and the supernatant containing the superantigen components was collected. 3) Purification was performed using DEAE column chromatography, and the target protein was separated after elution with a buffer solution having a pH of 8.0 to obtain an active superantigen.

5. The active superantigen compound biological injection preparation pain repair T and its preparation method according to claim 3, characterized in that: The steps of extracting the repair factor in step S2 include: 1) The gene sequence of the human IL-10 gene was obtained from a public gene library. The gene was amplified by PCR and expressed in mammalian cells using the pcDNA3.1 vector. The IL-10 gene was inserted into the pcDNA3.1 vector, and a recombinant plasmid was constructed using ligase and transformed into competent cells for plasmid amplification. 2) Using hamster ovary CHO cells as the host cell, the CHO cells were transfected by liposomes and expression strains were selected based on the resistance marker in the vector. The selected expression strains were fermented in a serum-free medium at 32-37° C., 30-60% dissolved oxygen, and pH 6.8-7.2, with glucose, glutamine, and a special feed agent added in batches: 3) After centrifuging the culture medium, the host cells are removed and the supernatant is collected. The supernatant is purified by ion exchange chromatography to obtain the cytokine IL-10: 4) Submandibular gland cells from adult male mice were collected and homogenized with extraction buffer in an ice bath. The supernatant was collected after further centrifugation and purified by ion exchange chromatography to obtain nerve growth factor (NGF).

6. The active superantigen compound biological injection preparation pain repair T and its preparation method according to claim 3, characterized in that: The specific steps of mixing the components in step S3 include: 1) Active ingredient pretreatment: a. Prepare a histidine buffer containing 1% glycine and controlled at a pH of 6.5-7.0, and dissolve the active superantigen and cytokine IL-10 in the histidine buffer; b. Prepare a sodium acetate buffer containing 0.1% human serum albumin and a pH of 5.0, and dissolve nerve growth factor (NGF) in the sodium acetate buffer; c. Dissolve the NSAID derivative in a small amount of DMSO solution and dilute the NSAID concentration with PBS to less than 0.1%; d. Dissolve glutathione and taurine directly in the histidine-glycine preparation buffer; 2) Liposome carrier preparation: Blank liposomes were prepared with a ratio of DSPC:cholesterol:PEG-DSPE = 55:35:

10. After membrane extrusion, the particle size of the liposome suspension was controlled to be 100-200 nm; 3) Macromolecule Mixing: a. Premix the active superantigen and cytokine IL-10 in histidine buffer and nerve growth factor (NGF) in sodium acetate buffer in appropriate proportions. Then, evenly mix the macromolecule mixture with the blank liposome suspension at a volume ratio of 1:

10. b. Stir at 200 rpm in a 37°C water bath for 1 hour to ensure active drug loading of the liposomes. Purify the mixture by ultrafiltration to remove free protein. 4) Small molecule mixing: slowly add the NSAIDs derivative to the drug-loaded liposome suspension, stir evenly at 500 rpm for 10 minutes, then add glutathione and taurine in sequence, control the stirring speed to 100-150 rpm, and adjust the pH to 6.8 to obtain; 5) adding 4% w / v mannitol and 1% w / v sucrose as freeze-drying protective agents, and controlling the pH to be 6.8-7.0.

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