Use of a pharmaceutical combination preparation in the preparation of a medicament for treating severe 2019-nCoV infection

Abstract

The application discloses application of a pharmaceutical combination preparation in preparation of a medicine for treating severe patients infected with 2019-nCoV and belongs to the technical field of medicines. It is found for the first time that ACE-his MSC and CAR-NK anti-His can be combined to prepare the medicine for treating severe patients infected with 2019-nCoV, which not only opens up a new application field of ACE-his MSC and CAR-NK anti-His, but also opens up a new medicine for treating severe patients infected with 2019-nCoV, and has positive pharmaceutical value and wide social significance.

Description

Technical Field

[0001] The present invention relates to the use of a drug combination preparation in preparing a drug for treating patients with severe 2019-nCoV infection, and belongs to the field of pharmaceutical technology. Background Art

[0002] The 2019 novel coronavirus (2019-nCoV) is a new strain of coronavirus discovered in humans in 2019. Clinical manifestations of infection are primarily fever, fatigue, and a dry cough, with a small number of patients experiencing nasal congestion, runny nose, and diarrhea. Severe cases often develop breathing difficulties after a week, while critically ill patients may develop acute respiratory distress syndrome, septic shock, difficult-to-correct metabolic acidosis, and coagulopathy. Some patients have mild onset, even without fever, and generally recover within a week. Most patients have a good prognosis, but a small number develop critical illness or even death, most commonly in the elderly and those with underlying chronic diseases. The main challenge in treating the disease is how to reduce and eliminate the novel coronavirus in the body.

[0003] 2019-nCoV belongs to the genus β-coronavirus. It has an envelope, and its particles are round or oval, often pleomorphic, with a diameter of 60-140 nm. The S protein is one of the main proteins of the virus, and its encoding gene is used for virus typing. It infects human respiratory epithelial cells through a molecular mechanism where the S protein interacts with human ACE2.

[0004] In the treatment of novel coronavirus infection, antiviral therapy is the top priority. Currently, there is still no effective antiviral drug for 2019-nCoV in humans, and screening and research efforts are ongoing. The main options are as follows:

[0005] (1) Small molecule drugs

[0006] For example, chloroquine phosphate, arbidol, remdesivir, darunavir, favipiravir and lopinavir.

[0007] (2) Serum from patients who have recovered from novel coronavirus infection

[0008] The plasma of recovered patients contains a large amount of protective antibodies, which can be used to treat critically ill patients. For example, using the plasma of recovered patients to perform "plasma therapy" on critically ill patients has shown significant improvement in clinical symptoms, with all test indicators improving across the board, and no obvious adverse reactions. However, "plasma therapy" also carries risks, such as inconsistent serum antibody levels among recovered patients, the possibility that the serum may contain other potentially dangerous pathogens that may cause infection, and even adverse reactions to the body. At the same time, due to the limited number of recovered patients, and the need to extract sufficient antibody serum from multiple recovered patients to cure a critically ill patient, this method objectively determines that it cannot be applied on a large scale, let alone "help the people of the country resist the virus."

[0009] (3) Vaccines

[0010] The novel coronavirus is a new pathogen, making vaccine development challenging and time-consuming. Currently, multiple technical approaches are being pursued simultaneously, including inactivated vaccines, recombinant protein vaccines, and nucleic acid vaccines. Some of these vaccines have entered the animal testing stage. The primary challenge in vaccine development is achieving both efficacy and safety. Developing a new vaccine first requires extracting the viral strain, a step previously accomplished by numerous research centers at astonishing speed. However, this is only the beginning of vaccine development; subsequent steps include cultivating the strain, modifying the virus, attenuating it, conducting animal testing, and conducting human clinical trials, a process that typically takes several years. To stimulate the production of corresponding antibodies, the vaccine must share many characteristics with the virus. However, beyond producing antibodies, will such a "very virus-like" vaccine cause side effects after entering the human body? Clinical trials are particularly time-consuming, as they test the safety and efficacy of vaccines.

[0011] (4) Stem cells

[0012] Stem cell therapy can inhibit overactivation of the immune system, promote endogenous repair by improving the microenvironment, inhibit the progression of acute lung inflammation, and alleviate symptoms of respiratory distress. After rigorous preclinical safety and efficacy evaluations and rigorous quality control, stem cell products have been used in several critically ill patients, adhering to current standards for stem cell clinical application and drug clinical trials, and have shown preliminary safety and effectiveness. Studies have shown that stem cell technology, used in the treatment of H7N9 avian influenza, can improve the condition of patients with critical infections caused by the novel coronavirus, suggesting that stem cell therapy could be a viable option for critically ill patients infected with the novel coronavirus.

[0013] Vaccines are antibodies produced by the body in response to a virus. Once the virus enters the body, the antibodies bind to the virus, ultimately mediating immune responses and destroying it. However, finding a vaccine that mimics the viral immunogen is a complex and time-consuming process. Furthermore, as a novel construct, the safety of the vaccine requires clinical research to demonstrate. Therefore, there is a need for a new treatment for severe COVID-19 infection to address the shortcomings of existing technologies. Summary of the Invention

[0014] The purpose of the present invention is to overcome the deficiencies of the prior art and provide the use of a pharmaceutical combination formulation in the preparation of a drug for treating patients with severe 2019-nCoV infection. The present invention is the first to discover that the combination of ACE-his MSC and CAR-NK anti-His can be used to prepare a drug for treating patients with severe 2019-nCoV infection. This not only opens up new application areas for ACE-his MSC and CAR-NK anti-His, but also opens up new drugs for treating patients with severe 2019-nCoV infection, which has positive pharmaceutical value and broad social significance.

[0015] The technical solution of the present invention to solve the above technical problems is as follows: use of a drug combination preparation in the preparation of a drug for treating patients with severe 2019-nCoV infection, wherein the drug combination preparation is composed of ACE-his MSC and CAR-NK antiHis.

[0016] The principle of using the drug combination preparation of the present invention in the preparation of drugs for treating patients with severe 2019-nCoV infection is:

[0017] Mesenchymal stem cells (MSCs) are a type of multipotent cell with the ability to self-replicate and differentiate. Under certain conditions, they can differentiate into a variety of functional cells. On the one hand, MSCs secrete keratinocyte growth factor, which enhances the ability of sodium channel proteins in lung epithelial cells to transport to the apical membrane, thereby improving the lung's ability to clear alveolar exudate and reducing pulmonary edema. On the other hand, they secrete angiopoietin-1, which maintains cell adhesion factors and intercellular connections, inhibiting the formation of stress fibrin, thereby improving the survival rate of alveolar capillary endothelial cells, enhancing vascular stability, and preventing the penetration of blood proteins into the alveolar spaces, thereby enhancing the ability of the alveolar epithelium to eliminate pulmonary edema. In addition, mesenchymal stem cells, in addition to their self-replication and multidirectional differentiation properties to repair damaged tissues, possess unique hematopoietic support and immune regulation functions. For example, they secrete anti-inflammatory factors to reduce excessive inflammatory responses and cytokine storms, and secrete chemokines to mobilize the body's regulatory response. They secrete cytokines to nourish and repair damaged cells, helping them maintain and restore normal function. As the most important stromal cells in the microenvironment, they provide a resting or activated microenvironment for hematopoietic stem and progenitor cells, promoting hematopoietic reconstruction and homeostasis, and safeguarding the overall balance of the blood system. However, there are currently no reports of mesenchymal stem cells being used to prepare drugs to treat patients with severe 2019-nCoV infection.

[0018] Natural killer cells (NK cells) are granular lymphocytes and are an important type of innate immune system lymphocyte in the body. Their phenotype is generally CD3-CD16+CD56+, and they are mainly derived from bone marrow CD34+ lymphocytes. NK cells do not express specific antigen recognition receptors and are a third type of lymphocyte that is different from T and B lymphocytes. Compared with other lymphocytes, the killing activity of NK cells is not restricted by MHC and does not require pre-sensitization with antigens, so it is called natural killer activity. When the chimeric antigen receptor (CAR) is fused with tumor-associated antigen antibodies and expressed on the cell membrane surface of NK cells, it can give NK cells the ability to specifically recognize corresponding tumor cells. At this time, the NK cells are called CAR-NK cells.

[0019] This invention uses mesenchymal stem cells (MSCs) as a tool, simultaneously expressing ACE2 and HIS-tagged proteins on the MSCs. These modified MSCs are then infused into patients with severe 2019-nCoV infection, competitively attracting 2019-nCoV to the MSCs. Anti-HIS CAR-NK cells then specifically recognize the HIS-tagged protein and produce grandenzymes, which kill all HIS-tagged MSCs. These MSCs are then eliminated through immune clearance, effectively destroying and reducing 2019-nCoV. During this process, the patient's body develops viral immunity. Furthermore, human cells do not express the HIS-tagged protein, minimizing damage to normal human cells and ensuring safety.

[0020] The beneficial effects of the use of the drug combination preparation of the present invention in the preparation of drugs for treating patients with severe 2019-nCoV infection are:

[0021] 1. The present invention discovered for the first time that the combination of ACE-his MSC and CAR-NK antiHis can be used to prepare drugs for treating patients with severe 2019-nCoV infection. This not only opens up new application areas for ACE-his MSC and CAR-NK antiHis, but also opens up new drugs for treating patients with severe 2019-nCoV infection, which has positive pharmaceutical value and broad social significance.

[0022] 2. The combination of ACE-his MSC and CAR-NK antiHis is used to prepare drugs for the treatment of severe patients infected with 2019-nCoV. The drugs have significant effects, strong safety, and low toxicity and side effects.

[0023] On the basis of the above technical solution, the present invention can also be improved as follows.

[0024] Furthermore, in the drug, ACE-his MSC is used first, and CAR-NK antiHis is used 24 hours later. The dose of the ACE-his MSC is 1 million cells / kg body weight, and the dose of the CAR-NK antiHis is 10 million cells / kg body weight.

[0025] A further beneficial effect of the above is that the present invention has found through experiments that, among the drugs for treating severe patients infected with 2019-nCoV, ACE-his MSC and CAR-NK antiHis can achieve the best efficacy by using the above-mentioned usage and dosage.

[0026] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0027] A further beneficial effect of the above is that ACE-his MSC and CAR-NK antiHis can be prepared together with a pharmaceutically acceptable carrier to treat patients with severe 2019-nCoV infection.

[0028] Furthermore, the carrier is any one or a mixture of two or more of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant and a lubricant.

[0029] Further beneficial effects of the above-mentioned systems include: carriers can alter how drugs enter and distribute within the body, control their release rate, and deliver them to targeted organs. Various drug carrier release and targeting systems can reduce drug degradation and loss, mitigate side effects, improve bioavailability, control drug release rates, and minimize or avoid peaks and valleys in blood drug concentrations, ensuring a more stable and sustained drug efficacy.

[0030] Furthermore, the dosage form of the drug is any one of an external preparation, an oral preparation and an injection preparation.

[0031] The above further beneficial effects are: the drug for treating severe patients infected with 2019-nCoV can be prepared into a variety of dosage forms and is suitable for a variety of administration routes, such as topical preparations, oral preparations or injection preparations, and the injection administration can be intradermal, subcutaneous, intramuscular, local or intravenous.

[0032] Furthermore, the external preparation is a spray or an aerosol.

[0033] Further advantages of the above-mentioned method include: the topical preparation is a spray or aerosol, which is convenient to use and quick in effect; it can maintain the cleanliness and sterility of the drug and improve its stability; because the drug is contained in a sealed container, it is protected from contact with air, moisture, and light, thereby reducing the possibility of contamination and deterioration; it can also reduce pain (such as in patients with burns and sensitive skin diseases) and infection during topical application; the sprayed mist particles are tiny, can directly reach the site of action or absorption, and are evenly distributed, resulting in a small dosage and minimal side effects.

[0034] Furthermore, the oral preparation is any one of granules, capsules, tablets and vesicles.

[0035] The further beneficial effect of adopting the above is that oral preparation is the most commonly used route, and its greatest advantages are safety, convenience and economy. The dosage forms of oral medicine include granules, capsules, tablets and vesicles.

[0036] Furthermore, the injection preparation consists of ACE-his MSC, CAR-NK antiHis, a co-solvent and a main solvent.

[0037] Furthermore, the cosolvent is any one of Tween-80, propylene glycol, glycerol, ethanol and PEG-400, or a mixture of two or more thereof.

[0038] Furthermore, the main solvent is 0.9g / 100mL sodium chloride solution or water for injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the structure of anti-his CAR-NK.

[0040] Figure 2 The Fc segment of the anti-his antibody is an extracellular structure that recognizes antigens and specifically recognizes the corresponding antigens.

[0041] Figure 3 Schematic diagram of CARNK cells killing target cells.

[0042] Figure 4 Schematic diagram of continuous recording of NK cells killing PC3 cells.

[0043] Figure 5 Schematic diagram showing that when anti-his CAR-NK binds to target cells his-MSC, a large amount of Grandenzyme is produced to kill his-MSC. DETAILED DESCRIPTION

[0044] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0045] Example 1: Construction of ACE2-His MSC

[0046] This example constructs ACE2-His MSCs, comprising the following steps:

[0047] Step 1: Construct lentivirus

[0048] Step 1.1: Construction of recombinant vector ACE2-his;

[0049] Step 1.2: Construction of lentiviral vector Plenti-ACE2-his;

[0050] Step 1.3: Lentivirus packaging;

[0051] Step 2: MSC cell culture expansion

[0052] Take a fresh umbilical cord from a healthy newborn, rinse it with PBS, remove the blood vessels, peel off the Warburg jelly tissue, and cut the remaining tissue into 1mm pieces. 3The cells were cultured in α-MEM medium at 37°C and 5% CO2 in a culture incubator. After the cells were fully grown, they were digested and passaged with 0.25% trypsin. PBS was added to adjust the cell concentration to 1×10 6 / mL, and MSC cells were obtained.

[0053] Step 3: The MSC cells obtained in step 2 are infected with the lentivirus obtained in step 1 and then cultured;

[0054] Step 4: Antibiotic screening to obtain stable cell lines;

[0055] Step 5: Immunofluorescence detection.

[0056] Example 2: Construction of CAR-NK antiHis

[0057] The construction of CAR-NK antiHis in this embodiment includes the following steps:

[0058] Step 1: Construct lentivirus

[0059] Step 1.1: Construct the recombinant vector CAR-NK-anti-his;

[0060] Step 1.2: Construction of lentiviral vector Plenti-CAR-NK-anti-his;

[0061] Step 1.3: Lentivirus packaging;

[0062] Step 2: NK cell culture and expansion

[0063] 200 mL of peripheral blood was drawn, NK cells were sorted, residual red blood cells were removed, and NK cells were expanded and cultured to obtain NK cells.

[0064] Step 3: The NK cells obtained in step 2 are infected with the lentivirus obtained in step 1 and then cultured;

[0065] Step 4: Anti-his CAR-NK cell collection and storage;

[0066] Step 5: Incubation and detection of fluorescently labeled his antigen;

[0067] Step 6: Detect anti-his CAR expression by RT-PCR.

[0068] Experimental results

[0069] like Figure 1 As shown, NK cells specifically express CD244 as a transmembrane structure and NKG2D within the cell, enabling the signal to be transferred to the interior of the NK cell.

[0070] like Figure 2As shown, the Fc segment of the anti-his antibody is an extracellular structure that recognizes antigens and specifically recognizes the corresponding antigens.

[0071] like Figure 3 As shown, CFSE-labeled NK cells appear green (triangles in the image), and granzymes are fluorescently labeled red (stars in the image). Granzymes are exogenous serine proteases that are released from cytoplasmic granules released by NK cells. These granules contain granzyme proteases and other proteases. When NK cells bind to target cells, the contents of the granules are released, and the granzymes enter the target cells. Perforin enters the target cells and forms pores in the target cell membrane by polymerizing in the cell membrane, perforating the membrane. Finally, perforin perforates the granzyme membrane, triggering the release of granzymes. Within the cytoplasm, granzyme B can trigger cell death through three distinct pathways: first, triggering a chain reaction of caspases, which degrades the target cell's DNA, and then lyses it.

[0072] like Figure 4 As shown, NK cells first kill cells and then phagocytize cells by releasing granzymes, perforins and other factors. The whole process is completed in 8 hours.

[0073] like Figure 5 As shown, the CAR-NK antiHis obtained in Example 2 was combined with the ACE-his MSC obtained in Example 1 to stimulate NK to produce grandenyme, which then entered the his-MSC target cells and finally killed the target cells.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. The use of a drug combination preparation in the preparation of a drug for treating severe patients infected with 2019-nCoV, the drug combination preparation consisting of ACE2-his MSC, CAR-NK antiHis and a pharmaceutically acceptable carrier, the dose of the ACE2-his MSC is 1 million cells / kg body weight, the dose of the CAR-NK antiHis is 10 million cells / kg body weight, and the dosage form of the drug is an injectable preparation.

2. The use according to claim 1, characterized in that The carrier is any one or a mixture of two or more of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption accelerator, an adsorption carrier, a surfactant and a lubricant.

3. The use according to claim 1, characterized in that The injection preparation consists of ACE2-his MSC, CAR-NKantiHis, a co-solvent and a main solvent.

4. The use according to claim 3, characterized in that The cosolvent is any one of Tween-80, propylene glycol, glycerol, ethanol and PEG-400, or a mixture of two or more thereof.

5. The use according to claim 3, characterized in that The main solvent is 0.9g / 100mL sodium chloride solution or water for injection.

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