A serine protease that selectively degrades mucin and its applications

Abstract

This application relates to a serine protease that selectively degrades mucin and its applications. The serine protease is serine protease EatA, whose protein includes a passenger domain with serine protease EatA activity. The amino acid sequence of the passenger domain has at least 90% identity with the amino acid sequence shown in SEQ ID NO. 2; preferably, it is the amino acid sequence shown in SEQ ID NO. 2. The serine protease EatA can selectively degrade mucin in disease-associated mucus and has high and durable stability. Therefore, the serine protease and pharmaceutical compositions including the serine protease can be used as drugs or active pharmaceutical ingredients for the treatment of mucin-secreting cancers such as pseudomyxoma peritonei (PMP) and other diseases involving mucin.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to a serine protease that selectively degrades mucin and its application in mucin degradation. Background Technology

[0002] Mucins are a class of high-molecular-weight, highly glycosylated proteins expressed by secretory cells in the human body. These cells are typically found in the epithelial and glandular tissues of the gastrointestinal tract, respiratory tract, lungs, kidneys, ovaries, breasts, and pancreas. In normal physiological environments, mucins play a lubricating and protective role on the surface of human tissues. The expression and composition of mucins are strictly regulated. However, under certain pathological conditions, such as cancer and inflammation, the expression and composition of mucins become uncontrolled and altered. The excessive accumulation of these mucins has a significant negative impact on patients. Abnormal mucin expression can manifest as severe clinicopathological features in both cancerous and non-cancerous diseases. These diseases include pseudomyxoma peritonei, chronic obstructive pulmonary disease, mucinous colorectal cancer, and mucinous ovarian cancer.

[0003] Pseudomyxoma peritoneum is a type of cancer with an unclear etiology, but clinical diagnosis often indicates its origin in organs and tissues such as the ovary, appendix, and intestinal mucin glands. Cancer cells eventually colonize the peritoneum and the peritoneal lateral surface of abdominal organs. The tumor continues to grow and secrete large amounts of mucin, particularly mucin MUC2, a major gel-forming mucin in the small and large intestines. Gel-forming mucins form gel-like, jelly-like polymers under specific pH, salt concentration, and concentrations of their family of mucins. The core region of the MUC2 molecule is highly glycosylated, exhibiting extremely high resistance to degradation by common proteases such as proteinase K and gastric trypsin. The covalent interactions at the C-terminus and N-terminus of the protein chain contribute to the formation of MUC2 dimers and polymers, respectively. This gel-like mucin encapsulates clusters of cancer cells and other lesions, gradually developing into diffuse metastases, forming pseudomyxoma peritoneum with mucus permeating the peritoneum. Because this type of cancer cell is encapsulated by a large amount of gel-like mucin, conventional drug delivery methods make it difficult for drugs to reach the target site, resulting in poor therapeutic efficacy. Furthermore, the large amount of peritoneal mucin distributed in the patient's body causes harm. In severe cases, a large amount of mucin can compress organs, leading to intestinal obstruction complications and endangering the patient's life.

[0004] Chronic obstructive pulmonary disease (COPD) is a chronic lung disease and the third leading cause of death worldwide (2019, WHO). It is caused by abnormalities in the small airways of the lungs, restricting airflow in and out of the lungs. Causes of COPD include long-term exposure to harmful gases and particles, as well as personal factors such as childhood events affecting lung development and genetic factors. COPD causes persistent and progressive respiratory symptoms, including shortness of breath, cough, and sputum production. Several processes lead to narrowing of the airways. Parts of the lungs may be damaged; patients with this disease experience abnormal mucus secretion in the respiratory tract, which obstructs the airways, causing inflammation and swelling of the airway walls. COPD is sometimes also referred to as "emphysema" or "chronic bronchitis." Emphysema typically refers to damage to the small alveoli at the ends of the airways in the lungs. Chronic bronchitis refers to a chronic cough caused by airway inflammation, accompanied by sputum production.

[0005] Mucinous ovarian cancer is a disease with an unclear etiology. It mostly originates from mucinous epithelial cells and causes bilateral ovarian involvement, ovarian surface involvement, extracellular mucin, destructive stromal infiltration, nodular growth, ovarian hilum involvement, vascular invasion, signet ring cells, and extensive necrosis. More than 90% of ovarian mucinous tumors contain a large amount of mucin in their cells.

[0006] Furthermore, diseases such as mucinous colorectal cancer and other mucinous disorders are characterized by the extensive expression of mucus. Therefore, clearing mucus is necessary in the treatment of diseases involving mucin to provide better outcomes for patients. For example, in the treatment of peritoneal pseudomyxoma, open surgery can remove the mucus, but this is often difficult to completely remove, and the long and repeated open surgeries are a burden on patients. Therefore, mucolytic agents have been developed and used to treat mucin-related diseases. These agents can physically and chemically break down and destroy the structure of mucin, removing the gel-like structure, allowing the mucin to be easily absorbed from the body, eliminated by the body, or facilitated by drugs targeting cancer cells to reach target cells and exert their anti-cancer effects. Clinically, patients preparing for surgery often have extensive intra-abdominal tumors and hardened mucus, leading to severe organ adhesions, increasing the difficulty of surgery and the incidence of adverse events. Therefore, softening or even dissolving the mucus before surgery can reduce the degree of organ adhesion and lower surgical risks to some extent.

[0007] Therefore, mucolytic agents play an important role in the treatment of mucin-related diseases, and there is a need to provide a new protease and its formulation that can better degrade disease-related mucins. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this application provides a serine protease EatA that selectively degrades mucin, based on the discovered degradation activity of the serine protease EatA. This serine protease EatA can effectively degrade mucin in mucus-related diseases. When applied to patients, it can exert its mucus-degrading function, thus making it well-suited for the treatment of mucus-related diseases.

[0009] To this end, the first aspect of this application provides a serine protease that selectively degrades mucin, the serine protease being a serine protease EatA, the protein of which includes a passenger domain having the activity of the serine protease EatA, the amino acid sequence of which is an amino acid sequence having at least 90% identity with SEQ ID NO.2.

[0010] In some specific embodiments, the amino acid sequence of the passenger domain is an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identity with SEQ ID NO.2.

[0011] In some preferred embodiments, the amino acid sequence of the passenger domain is the amino acid sequence shown in SEQ ID NO.2. The specific sequence of SEQ ID NO.2 is shown below:

[0012] In this application, since the passenger domain of the serine protease EatA protein, when correctly folded, exhibits the activity of serine protease EatA (i.e., selective degradation of mucin activity), and this activity is formed by the histidine-aspartic-serine catalytic triplet, in some specific embodiments, the serine protease EatA is a serine protease EatA-EatAp containing only the passenger domain.

[0013] In some embodiments, the serine protease EatA further includes at least one of an N-segment signal peptide and a C-terminal β-barrel structure.

[0014] In some embodiments, the protein amino acid sequence of the serine protease EatA having the passenger domain and the C-terminal β-barrel structure is an amino acid sequence that has at least 90% identity with SEQ ID NO.3.

[0015] In some specific embodiments, the protein amino acid sequence of the serine protease EatA having the passenger domain and the C-terminal β-barrel structure is an amino acid sequence that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identity with SEQ ID NO.3.

[0016] In some preferred embodiments, the amino acid sequence of the serine protease EatA, having the passenger domain and C-terminus β-barrel structure, is the amino acid sequence shown in SEQ ID NO.3. The specific sequence of SEQ ID NO.3 is shown below:

[0017] In some embodiments, the full-length amino acid sequence of the serine protease EatA, which has the passenger domain, the N-segment signal peptide, and the C-terminal β-barrel structure, is an amino acid sequence that has at least 80% identity with SEQ ID NO.1.

[0018] In some specific embodiments, the full-length amino acid sequence of the serine protease EatA having the passenger domain, the N-segment signal peptide, and the C-terminal β-barrel structure is an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% identity with SEQ ID NO. 1.

[0019] In some preferred embodiments, the full-length amino acid sequence of the serine protease EatA, which has the passenger domain, the N-segment signal peptide, and the C-terminal β-barrel structure, is an amino acid sequence with at least 90% identity to SEQ ID NO.1.

[0020] In some preferred embodiments, the full-length amino acid sequence of the serine protease EatA, having the passenger domain, the N-segment signal peptide, and the C-terminal β-barrel structure, is the amino acid sequence shown in SEQ ID NO.1. The specific sequence of SEQ ID NO.1 is shown below:

[0021] In this application, the serine protease EatA is a self-transporting protein derived from enterotoxigenic Escherichia coli of the Enterobacteriaceae family. Its full-length protein includes an N-terminal signal peptide, a C-terminal β-barrel structure, and a passenger domain in the middle of the protein chain. The passenger domain possesses serine protease activity, which is formed by a histidine-aspartic acid-serine catalytic triplet. The serotype of the Enterobacteriaceae family is O78:H11, and the enterotoxigenic Escherichia coli strain is H10407. In this application, the serine protease EatA is defined as: a protein comprising the passenger domain fragment EatA-EatAp with serine protease activity as shown in SEQ ID NO.2, the full-length serine protease EatA as shown in SEQ ID NO.1, and a protease fragment without a signal peptide as shown in SEQ ID NO.3.

[0022] In this application, when the serine protease EatA is expressed using Escherichia coli as an expression vector, EatA is a full-length membrane protein. It will transfer the middle part (passenger domain) to the outside of the membrane through autotransport and release the passenger domain through self-cleavage. The protein released into the culture medium is the target protein of EatA with serine protease activity (the serine protease EatA-EatAp with only the passenger domain).

[0023] Mucolytic agents are protease-containing reagents that can influence and act on mucins, thereby degrading gel-like mucus by inducing proteolytic hydrolysis of glycoproteins and mucins. Serine protease EatA, a member of the Enterobacteriaceae family of serine autotransporters, has been used as an antigen to prevent severe diarrhea caused by Enterobacteriaceae, triggering an immune response and producing antibodies upon injection. The inventors of this application have discovered that the serine protease EatA has a relatively long stable period in vitro and can disrupt the gel-like physical state of mucins, forming a liquid state. Therefore, the serine protease EatA of this application can be used to prepare mucolytic agents.

[0024] It should be noted that homologs of the serine protease EatA can be found in other species and bacterial strains using conventional and known search methods in the art. The sequence similarity of homologs can be determined using conventional algorithms. For example, when comparing the similarity of two proteins, calculations can be performed using the widely used blastN and blastX programs. These programs, whose algorithms are derived from Karlin and Altschul, allow the introduction of a small number of vacancies to achieve the best fit of the percentage of sequence identity between the two nucleic acid or protein molecules. Therefore, based on the above, the serine protease EatA described in this application can also be replaced by homologs with a high degree of identity (at least 80%) with its amino acid sequence.

[0025] In some embodiments, the optimal reaction conditions for the serine protease EatA to degrade mucin are: a reaction temperature of 34–37°C, a pH of 7–8, and a reaction coefficient of 1–1,000,000. For example, in some specific embodiments, the reaction temperature for the serine protease EatA to degrade mucin is 37°C, the pH is 7.2–7.4 (a pH of 7.2 to 7.4 is more consistent with the human body environment, especially the pH of tissues and the peritoneum), and the reaction coefficient is 100–10,000.

[0026] In this application, when the reaction conditions are controlled within the above-mentioned range, the serine protease EatA exhibits the best degradation effect on mucin in in vitro experiments.

[0027] Since the mucin degraded in this application is a gel-like semi-solid, initially insoluble in water but exhibiting slow hydration, and the relative average density of the mucin and water used in the embodiments of this application is close to 1.0, we define the following when measuring the concentration: Reaction coefficient: In a fixed 1000 μL (1, y) reaction system containing 1000 μL (1, x) mucin, the mucin density is 1 (1 g / mL, ρ), and the system contains a total mass concentration of 1 μg / mL (1 μg, z) of serine protease EatA. After incubation at 37°C for 10 hours, a mucin solubility value w = 100% indicates complete degradation (w represents the percentage of dissolved mucus mass). At this point, the reaction coefficient is 1. Therefore, the formula for calculating the reaction coefficient is: Formula 1: ------------------Reaction coefficient y>x, y~δ: that is, when the mass ratio of enzyme to substrate mucin is known, the volume of the reaction system cannot exceed its corresponding reaction coefficient, and the numerical value of the reaction system volume should always be greater than the numerical value of the mucin volume.

[0028] Therefore, it can be foreseen that the range of reaction coefficients used will be 1 to 1,000,000, such as 1 to 1,000, 1,000 to 1,000, 1,000 to 10,000, 10,000 to 100,000, 100,000 to 1,000,000, etc.

[0029] The above reaction coefficients are constructed based on logic. Since mucin spontaneously hydrates and slowly dissolves in large volumes of aqueous solution, to reduce the impact of hydration on enzymatic degradation, the reaction coefficients refer to the nonlinear effect of the reaction system on the substrate mucin when all mucus is degraded into a soluble liquid 10 hours after the addition of the serine protease EatA. They do not directly represent enzyme activity. Their values ​​are related to the reaction volume, the amount of substrate mucin, the enzyme's concentration and activity, and the reaction time. Because mucin has different densities under different pathological conditions, its volume was used as the standard for uniform calculation.

[0030] It should be noted that the reaction coefficients given in the examples are preferred embodiments and are not intended to specify or limit the dosage and concentration of EatA when it degrades mucus / mucin.

[0031] In some embodiments, the serine protease EatA has specific serine protease activity for selectively degrading mucins.

[0032] In this application, the serine protease EatA specifically degrades mucins but does not degrade non-target proteins (such as extraneous proteins in mucus). Previous studies have shown that EatA does not degrade common human glycoproteins such as gelatin, lactoferrin, and CD43, nor non-human proteins such as BSA and BSM. The inventors' experimental results further demonstrate that EatA does not degrade extraneous proteins in clinically derived mucins, but selectively degrades gel-like mucins, transforming them from a semi-solid gel into a solution that is easily absorbed and transferred or may be absorbed by the body. Therefore, the serine protease EatA in this application can be effectively used to degrade mucins in mucus-related diseases without damaging other proteins in the body.

[0033] In some embodiments, the degraded mucin is at least one of the following: peritoneal pseudomyxoma mucus, chronic obstructive pulmonary disease mucus, mucinous ovarian cancer mucus, mucinous colorectal cancer mucus, and appendiceal cancer mucus; preferably, it is present in peritoneal pseudomyxoma mucus.

[0034] In this application, the mucin degraded by the serine protease EatA is not a denatured and purified mucin (such as MUC2), but rather a mucin present in disease-associated mucus. Unlike denatured and purified mucin (such as MUC2), the mucin present in disease-associated mucus participates in the formation of mucus with tissues, particularly forming gel-like mucus in PMP. MUC2 forms large aggregates with itself and other mucin family molecules, and the mucin family exhibits different modification patterns in diseases. Therefore, compared to denatured and purified mucin, the mucin present in disease-associated mucus has a more complex structure and is more difficult to degrade. For example, elastase and trypsin can degrade mucin molecules purified to the SDS-PAGE level, but their degradation efficiency for mucin after mucus formation is low. The serine protease EatA described in this application can efficiently degrade mucin in mucus-associated diseases, and thus can be used in mucus-associated diseases expressing the above-mentioned mucins, especially those expressing MUC2-type mucin, such as pseudomyxoma peritonei, to degrade the mucin, eliminate gel properties, and achieve specific therapeutic effects.

[0035] In this application, the specific therapeutic effects include, but are not limited to, making mucin easier to clear, exposing cancer cells or lesions, promoting the effects of other chemotherapy drugs, and reducing the form of patient involvement.

[0036] When using the serine protease EatA from this application to treat mucinous diseases such as peritoneal pseudomyxoma, the serine protease EatA described in this application has long-lasting and stable activity. Therefore, after being injected into the human peritoneal cavity, it exhibits long-lasting mucin-degrading activity, reducing the mass and volume of gel-like solids formed by a large amount of mucin under minimally invasive or even non-invasive conditions. Compared to open surgery, it is easier to perform, less invasive, and avoids the infection and damage to the patient that may result from open surgery. The mucin-degrading effect of serine protease EatA can be achieved by injecting it into specific pathological areas within the patient's body. The injection method can be tailored to different disease types and target areas. For example, in the case of covered pseudomyxoma, it can be achieved through injection, directly injecting a specific concentration of EatA protease into the peritoneal cavity, or into the tumor or cancerous cyst, thereby degrading mucin within the peritoneal cavity or target area, ultimately achieving a certain therapeutic effect. This therapeutic effect can be a decrease in the amount of mucin in the environment in which the mucin exists, and a simultaneous improvement in the patient's condition.

[0037] It is worth noting that when the serine protease EatA described in this application degrades the mucus in mucus-related diseases, the degraded mucus becomes a water-like liquid, eliminating the gel-like properties of the mucus. This makes the mucus in the patient's lesion area easier to remove, allowing it to be aspirated from the body using a needle or specific aspiration instruments. Furthermore, it can facilitate the direct delivery of other therapeutic drugs, such as anticancer drugs, to the lesions and cancer cells, thereby improving treatment efficacy, reducing patient suffering and burden, and improving quality of life. Compared with existing mucin-degrading formulations in the art (e.g., formulations including bromelain), the serine protease EatA described in this application has superior properties, exhibiting better selectivity, not degrading non-target proteins, higher degradation efficiency, and degradation products that better meet expectations, such as being easier to aspirate with pipettes.

[0038] A second aspect of this application provides a pharmaceutical composition comprising the serine protease as described in the first aspect of this application.

[0039] In some embodiments, the pharmaceutical composition further includes other reagents selected from at least one of chemotherapy and radiotherapy reagents, enzymes, and chemical salt reagents.

[0040] In this application, the serine protease EatA can be used in combination with one or more other therapeutic agents, such as common cytotoxic chemotherapy drugs like cisplatin and gemcitabine, allowing the drugs to target cancer cells more directly and effectively, thus improving the efficacy of chemotherapy. Furthermore, the mucin-degrading function of the serine protease EatA is due to its protease activity inhibited by PMSF; therefore, in practice, it can be used in conjunction with enzymes possessing other mucin glycan degradation activities and certain chemical salt reagents.

[0041] In some specific embodiments, the chemotherapy and radiotherapy reagents are selected from at least one of gemcitabine, cisplatin, adriamycin, fluorouracil, paclitaxel, paclitaxel, and oxaliplatin; the enzymes are selected from at least one of N-acetylgalactosidase, galactosidase, glucosidase, sialidase, and specific endoplasminase; and the chemical salt reagents are selected from at least one of sodium bicarbonate, carboxycysteine, N-acetylcysteine, and ambroxol.

[0042] The third aspect of this application provides the use of the serine protease as described in the first aspect of this application or the pharmaceutical composition as described in the second aspect in the preparation of a medicament for treating mucus-related diseases, and / or a medicament for improving the therapeutic effect of mucus-related diseases and the quality of life of patients.

[0043] Because of its protease activity, the serine protease EatA can be used as a drug or drug component for treating mucus-related diseases. Therefore, this application provides a novel use of the serine protease EatA in mucus-related diseases such as peritoneal pseudomyxoma and chronic obstructive pulmonary disease. By applying an effective dose of the serine protease EatA to the lesion area of ​​the aforementioned mucus-related diseases, such as the abdominal cavity and respiratory tract, the degradation of mucin in the lesion area, or the reduction or elimination of gel-like protein, allows for the easy removal of large amounts of mucin, thereby achieving the goal of treating mucus-related diseases. Furthermore, by applying an effective dose of the serine protease EatA to degrade mucin, the encapsulation and isolation of cancer cells and lesions by mucus is eliminated, enhancing the efficacy of cytotoxic drugs and promoting the accessibility of other drugs, such as chemotherapy drugs cisplatin and gemcitabine, to the lesions and cancer cells, thereby improving the treatment effect of mucus-related diseases and the quality of life of patients. Therefore, the serine protease described in the first aspect or the pharmaceutical composition described in the second aspect of this application can be used in the preparation of drugs for treating mucus-related diseases, and / or drugs for improving the treatment effect and quality of life of patients with mucus-related diseases. Drugs including the serine protease described in the first aspect or the pharmaceutical composition described in the second aspect can degrade mucin and destroy the gel-like protein formed by mucin, making it easier to remove, and can promote other therapeutic drugs such as anticancer drugs to reach the lesion and cancer cells directly, thereby improving the therapeutic effect, reducing the suffering and burden on patients, and improving their quality of life.

[0044] In this application, the aforementioned mucin-related diseases, in addition to including mucinous carcinoma as defined by the World Health Organization: a malignant tumor composed of gastrointestinal cells containing intracytoplasmic mucin, can also be considered as abnormal regulation related to mucin expression in cells, tissues, and organs under normal healthy conditions due to changes in function or environment, leading to excessive mucus production, the formation of pathological tissue, or the secretion of mucin that coats the outside of cells, tissues, and organs, blocking the realization of normal physiological functions, such as immune recognition, or the infiltration and access of therapeutic drugs.

[0045] In some embodiments, the mucus-related disease is selected from at least one of chronic obstructive pulmonary disease, mucinous colorectal cancer, lung cancer, liver cancer, gastric cancer, appendix cancer, peritoneal cancer, prostate cancer, colorectal cancer, small bowel cancer, lymphoma, ovarian cancer, adenocarcinoma, and asthma; the ovarian cancer includes mucinous ovarian cancer, and the peritoneal cancer includes pseudomyxoma peritoneum. The appendix cancer may be, for example, mucinous appendiceal adenocarcinoma.

[0046] The serine protease EatA described in this application has been shown in studies to possess highly efficient and specific degradative activity against mucus originating from peritoneal pseudomyxoma. Furthermore, mucin associated with peritoneal pseudomyxoma is expressed in large or small amounts in other mucinous diseases, depending on the disease type. Treatable peritoneal pseudomyxomas are often caused by cancer cells from the colorectal, appendix, and ovary metastasizing to the peritoneal cavity, resulting in the formation of pseudomyxomas due to the excessive secretion of mucus by these tumor cells. These cancer cells have diverse origins, including the colorectal, rectal, pancreas, breast, lung, gallbladder, and ovary, but numerous clinical reports indicate their origin in the appendix. These tumor cells secrete large amounts of mucus into the peritoneal cavity, which is difficult to degrade, ultimately leading to massive accumulation, damaging other organs, and increasing the severity and mortality of the disease. Conventional open surgery to remove mucin often requires multiple procedures due to incomplete treatment and the progressive nature of the disease, leading to increased morbidity and mortality.

[0047] In the above definitions of mucus in mucus-related diseases, the mucus in pseudomyxoma peritoneum refers to a colorless, transparent to amber-colored jelly-like semi-solid gel-like substance formed after a tumor or cancer forms on the peritoneal surface. It is composed mainly of MUC2, MUC5AC, and MUC5B and exhibits three phenotypes: soft, medium-firm, and firm. Its characteristic feature is the proliferation of intra-abdominal neoplastic mucin-secreting cells on the peritoneal surface, producing mucinous ascites. The mucus in mucinous ovarian cancer refers to a white to amber-colored, sometimes chocolate-brown, semi-solid substance formed after secretion from the pathological area of ​​a tumor or cancer occurring in the ovary. Clear to transparent mucus with extremely high viscosity, its viscosity ranging from thousands to tens of thousands of mPa·s is a syrupy semi-solid or fluid, characterized by a large amount of encapsulated mucin-based viscous mixture adhering to the surface of ovarian mucinous cancer cells or glandular cysts; the mucus of chronic obstructive pneumonia, also known as sputum, is a mucous substance secreted by the epithelial / glandular cells of the lungs or respiratory tract, characterized by mucin-based mucus adhering to the surface of the respiratory tract and trachea, forming mucus. The mucus of chronic obstructive pneumonia differs from that of sputum under normal physiological conditions in that it has a higher content and viscosity, often leading to lung obstruction and tracheal obstruction.

[0048] Generally, the route of administration of the drug should be determined by taking into account the recipient's physical characteristics, including the type of disease and health condition, which is readily apparent to clinicians and relevant technical personnel. In some embodiments, the route of administration of the drug is selected from injection, oral administration, and spray administration.

[0049] In the administration routes described above in this application, injection administration may be, for example, intraperitoneal injection; oral administration may be, for example, fast-dissolving tablets; and spray administration may be, for example, nasal or oral inhalation as an aerosol. Specifically, as an injection administration route, the drug carrier may be a pharmaceutically acceptable carrier or excipient such as physiological saline, phosphate buffer, Tris-HCl buffer, Ringer's reagent, glucose injection buffer, and water-propylene glycol solution. Methods for preparing compositions and drugs for parenteral and intraperitoneal administration are readily adaptable to those skilled in the art.

[0050] In this application, the effective dosage of the aforementioned drug should be determined by a variety of factors, such as the type of mucin-related disease to which it is applied, the amount of mucus, and the expected duration of treatment. For example, an appropriate dosage may depend on a variety of factors, including but not limited to an individual's physical characteristics, such as age, weight, and sex; whether the drug is used as a single agent or as an adjuvant therapy; the progression of the disease or condition being treated, i.e., the pathological state; and other factors that are obvious to a person skilled in the art.

[0051] The drug of this application is applicable to any patient and individual. In some embodiments, the patient is a mammal, most typically a human patient. However, it is clear that other mammalian individuals will also benefit from this invention where applicability permits. Therefore, these patients and individuals can be mice, rats, cats, dogs, bears, cattle, horses, and other mammals with therapeutic value.

[0052] The beneficial technical effects of this application are as follows: The serine protease described in this application is the serine protease EatA. The serine protease EatA can selectively degrade disease-related mucins and has long-term stability. Therefore, the serine protease and the pharmaceutical composition including the serine protease can be used as a drug or active pharmaceutical ingredient to treat cancers that secrete mucins, such as pseudomyxoma peritonei (PMP), and other diseases involving mucins. Attached Figure Description

[0053] Figure 1 This is a graph showing the degradation results of mucin in the control group of Example 1 without the addition of EatA to the gel-like mucin.

[0054] Figure 2 This is a diagram showing the degradation results of mucin after adding EatA to gel-like mucin in the experimental group of Example 1.

[0055] Figure 3 This is a diagram showing the results of the degradation of mucin by bromelain (BroM) and serine protease EatA in Example 2.

[0056] Figure 4 This is a temperature curve of the degradation of mucin by the serine protease EatA in Example 3.

[0057] Figure 5 This is a pH curve of the degradation of mucin by the serine protease EatA in Example 3.

[0058] Figure 6 This is a time curve of the degradation of mucin by the serine protease EatA in Example 3.

[0059] Figure 7 This is a diagram showing the degradation results of bromelain (BroM) and serine protease EatA on the mixed proteins inside the mucin in Example 4.

[0060] Figure 8 The graph shows the final amount of mucin remaining in the experimental group (mucus + EatA), the control group without enzyme (mucus + PBS), and the experimental group (mucus + bromelain) in Example 5.

[0061] Figure 9The images show the peritoneal examination results of mice in the experimental group (mucus + EatA), the control group without enzyme (mucus + PBS), and the experimental group (mucus + bromelain) in Example 5.

[0062] Figure 10 This is a box plot showing the degradation of mucus from ovarian mucinous carcinoma and mucinous appendiceal carcinoma by the serine protease EatA in Example 6.

[0063] Figure 11 This is a schematic diagram showing the results of the serine protease EatA in Example 6 degrading mucus from mucinous appendiceal adenocarcinoma (MAA). Detailed Implementation

[0064] To make this application easier to understand, the following detailed description will be provided with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of this application. Unless otherwise specified, the raw materials or components used in this application can be obtained commercially or by conventional methods.

[0065] Because mucins from different sources, and even mucins associated with mucinous diseases, exhibit different expression patterns and post-translational modifications, this application utilizes various mucins and mixtures thereof from clinical pathological sources. Preferably, mucins derived from clinical pseudomyxoma peritoneum are used. This application first collects gel-like mucins from the peritoneal cavity of patients with clinical pseudomyxoma peritoneum. These are precipitated with 30–100% ammonium sulfate. After precipitation, cells, tissue debris, adipose tissue, etc., that may have been contaminated during collection are removed. The mucin is then dialyzed using a 100 kDa dialysis bag. Dialysis is terminated after confirming the sulfate concentration in the dialysate and inside the dialysis bag is below 200 mg / L using sulfate test strips, revealing the gel-like mucin. This sample is collected as a substrate for in vitro experiments to examine the degradation capacity of the serine protease EatA on the mucin. In some examples, unpurified mucins taken directly from the peritoneal cavity of clinical patients are used for experiments.

[0066] Example 1 In a 500 μL reaction system, 0.30 g of gel-like mucin was accurately weighed. Approximately 80 μg of serine protease EatA (reaction coefficient 133) was added to the experimental group, while no serine protease EatA was added to the control group. Both groups were incubated at 37°C for 3 hours. After incubation, the degradation of the mucin was observed. The degradation results of the mucin in the control and experimental groups are shown below. Figure 1 and Figure 2 As shown.

[0067] from Figure 1 and Figure 2It can be seen that the control group without the addition of serine protease EatA still had lumpy gel-like protein molecules after 3 hours, which could not be aspirated by pipette tip; compared with the control group, the experimental group with the addition of serine protease EatA showed obvious degradation of gel-like mucin. The gel-like mucin in the experimental group was degraded into a water-like solution that could be aspirated by pipette tip, and the lumpy gel disappeared under a stereomicroscope.

[0068] Example 2 Bromelain, as a broad-spectrum protease, possesses extensive degradation activity and can degrade MUC2 mucin, showing some application in anti-inflammatory and even mucus degradation. Based on this, this example compares the degradation activities of bromelain (BroM) and serine protease EatA on gel-like mucin with the same substrate / enzyme ratio.

[0069] Accurately weigh 0.10 g of mucin and place it in a 500 μL phosphate-buffered saline (PBS) reaction system. Add two enzymes (bromelain (BroM) and serine protease EatA) and react at 37°C for 6 hours. Invert all the product onto a 40 μm cell filter, and use absorbent paper to remove the liquid from the other side of the filter. Carefully invert the gel-like product three to four times, removing as much flowing liquid as possible. Weigh the remaining gel-like mucin. Repeat the above experiment with three samples at each sampling point. The results are as follows: Figure 3 As shown. From Figure 3 It was observed that within 6 hours, the mass loss of gel-like mucin increased with increasing EatA enzyme concentration, and the remaining amount decreased rapidly, indicating degradation of the gel-like mucin. When 40 μg of EatA was added, the average mass loss of the gel-like mucin was 90.33% (calculated from an initial 0.1 g), indicating almost complete degradation. Furthermore, due to the water absorption of the gel-like mucin, its mass increased initially. In contrast, under the same substrate / enzyme ratio, bromelain resulted in a negative mass loss of gel-like mucin after water absorption, with the mass loss becoming negative as BroM enzyme concentration increased, indicating low degradation efficiency and minimal degradation of the gel-like mucin.

[0070] Example 3 This embodiment characterizes the working environment of the serine protease EatA in degrading mucin by obtaining degradation curves under pH-dependent, temperature-dependent, and different reaction times.

[0071] During the experiment, 0.10 g of gel-like mucin was accurately weighed for each individual reaction, and 20 μg of the serine protease EatA was added. All reactions were carried out in a 500 μL solution system. The remaining gel-like mucin was weighed using the same method as in Example 2. The control group was conducted under the same conditions except that no serine protease EatA was added. Three reactions were set up for each group for statistical replication. Due to the hydration properties of the gel-like mucin, it absorbs water and swells in solution, resulting in a certain weight increase even in the control group at the final weighing.

[0072] The experiment investigating the temperature gradient was conducted in physiological saline at pH 7.1, and the results are as follows: Figure 4 In the pH experiment, the reaction system used was a solution system with different pH values ​​prepared based on 0.9% physiological saline, and the reaction temperature was 37℃. The results are as follows: Figure 5 The reaction time for the two experiments mentioned above was 6 hours.

[0073] In experiments concerning degradation time, based on temperature and pH curves, the degradation was carried out at 37°C in PBS buffer with pH 7.2–7.4. The results are shown below. Figure 6 .

[0074] from Figure 4 It can be seen that in the control group without added enzyme, the mass of gel-like mucin did not change significantly after incubation with changes in temperature. However, in the experimental group with added enzyme, all gel-like mucin was degraded, and the enzyme activity was at its maximum between 34 and 37°C. Since the control group also showed a certain trend of degradation (mass loss) above 41°C, it may indicate the effect of temperature on the protein, and it cannot be concluded that the increased degradation efficiency at this stage is solely related to the enzyme.

[0075] from Figure 5 The results showed that the degradation of gel-like mucin by the serine protease EatA under different pH conditions had the highest degradation efficiency around pH 8. In contrast, the mass loss of the control group remained almost unchanged under different pH conditions.

[0076] from Figure 6 It can be seen that after the addition of serine protease EatA, the mass loss of gel-like mucin first decreased to a negative value, indicating that it absorbed water and increased its mass. As time increased, the mass loss increased, and reached 80% at 10.5 hours. This indicates that the serine protease EatA has a continuous effect on the degradation of the substrate gel-like mucin as the incubation time increases.

[0077] In summary, the optimal working environment for the serine protease EatA to degrade gel-like mucin is: a reaction temperature of 34–37°C and a pH of 7–8.

[0078] Example 4: Specificity of EatA degradation by serine protease Untreated mucin from clinical patients was simply cleaned to remove cells and tissue. 0.20 g of the mucin was accurately weighed and placed in 1 mL of PBS solution. 3-4 magnetic beads (1 mm in diameter) were added, and the mixture was homogenized by shaking at -30°C to disrupt the gel-like morphology of the mucin and release patient-derived contaminating proteins mixed within the gel-like mucin. 100 μL of the homogenized mucin solution was taken, and 2-10 μg of protease (bromelain or serine protease EatA) was added to a 200 μL reaction system for reaction. After 6 hours, samples were taken for SDS-PAGE analysis to determine the total protein content. This total protein represents patient-derived contaminating proteins in the peritoneal / peritoneal mucin. The results are as follows: Figure 7 As shown.

[0079] from Figure 7 It can be seen that in the group with BroM (bromelain) added, at a dosage of a few micrograms of BroM enzyme, the peritoneal proteins of the patients showed a degradation trend that changed with the increase of enzyme dosage, that is, in the BroM added group, the miscellaneous proteins were degraded. The control group (the three middle wells marked with "0") showed the same protein bands as the group with added serine protease EatA, indicating that the addition of serine protease EatA does not degrade these peritoneal proteins during the degradation of mucin, demonstrating its specificity in the degradation of mucin.

[0080] Example 5: The ability of serine protease EatA to degrade mucin in vivo Based on the good results of the in vitro experiments, this embodiment conducted in vivo animal experiments to demonstrate the in vivo degradation ability of the serine protease EatA on mucin.

[0081] The experiment included 20 male C57 mice aged 6-8 weeks. Five groups were established: experimental group (mucus + EatA, 5 mice), control group (mucus + PBS, 5 mice), bromelain experimental group (mucus + bromelain, 5 mice), negative sham-operated group (open surgery only, no mucin or enzyme added, 3 mice), and control group (open surgery, EatA protease added, 2 mice). Based on previous studies in rats and the reference average mass of intraperitoneal mucin in patients with pseudomyxoma peritoneum, as well as the response coefficient in in vitro experiments, the maximum allowable intraperitoneal injection volume in mice was 2 mL. 0.30 g of human patient-derived gel-like mucin, simply washed with PBS buffer, was implanted in the mice to observe and confirm their viability. 24 hours later, equal masses of bromelain and serine protease EatA dissolved in PBS (80 μg each) were injected. After 48 hours, the mice were sacrificed and laparotomized (post-laparotomy peritoneal examination results are shown below). Figure 9 (As shown) All gel-like mucin was removed from the mice and weighed using the method described in Example 2. The amount of gel-like mucin remaining in the three groups (experimental group (mucus mucus + EatA), control group without enzyme (mucus + PBS), and experimental group (mucus mucus + bromelain)) with gel-like mucin as a substrate was counted and compared. The results are shown in […]. Figure 8 .

[0082] from Figure 8 It was found that the mice group supplemented with the serine protease EatA had the smallest remaining mass of gel-like mucin, indicating that it was degraded most effectively, with a degradation efficiency of 78% (calculated based on an initial inoculation dose of 0.30 g; in the peritoneal cavity, the gel-like mucin will still absorb water, causing its mass to increase). In contrast, the remaining mass of gel-like mucin in the BroM group and the control group was still greater than 0.30 g, so its degradation efficiency could not be calculated. This demonstrates that the serine protease EatA is far superior to BroM in degrading gel-like mucin in mice.

[0083] Figure 9 The red box in the image shows that the gel-like mucin migrated to different locations within the peritoneum of mice after transplantation, and also shows its final volume after degradation, compared to... Figure 8 Correspondingly.

[0084] During the experiment, the weight changes of all groups before and after the experiment were recorded, and there were no differences in the results. Abdominal examination after mouse sacrifice also showed no abnormalities. Mice injected with the serine protease EatA have survived for more than 100 days since the start of the experiment without adverse reactions. Compared with the uninjected group, the mice showed no abnormalities in behavior or weight.

[0085] Example 6: Degradation of mucus from mucinous ovarian cancer (MOC) and mucinous appendiceal cancer (MAA) by the serine protease EatA This embodiment uses the serine protease EatA to degrade mucus from mucinous ovarian cancer and mucinous appendiceal adenocarcinoma. 0.50 mL of clinically sourced mucus from each of the two types of novel mucinous carcinoma was weighed directly into a test tube, and 50 μg of EatA serine protease dissolved in 0.25 mL of PBS buffer was added. The mixture was then incubated in a total reaction system of 750 μL at 37°C and 600 rpm for 20 h. A control group with only an equal volume of PBS was included for each type of mucinous carcinoma sample, for a total of four experimental groups, with three replicates for each group. The viscosity of the incubated samples was measured using an Anton Paar rheometer (PP25 probe) with a sample spacing of 0.45 mm. After a 20 s pre-shear at a shear frequency of 5 rad / s, each sample underwent a shear frequency of 10 rad / s, and 12 data points were collected over 60 s at 5 s intervals, resulting in a total of 36 data points per group. The experimental results are presented as box plots showing the statistical results of all data for each sample group, such as... Figure 10 As shown.

[0086] from Figure 10 It was found that the average viscosity of mucus from clinically derived mucinous ovarian cancer was approximately 2000 mPa·s, which decreased to approximately 670 mPa·s after incubation with the serine protease EatA. In contrast, the viscosity of mucus from clinically derived mucinous appendiceal adenocarcinoma (MAA) was approximately 1200 mPa·s, which decreased to approximately 7 mPa·s after the addition of mucin. Therefore, it is evident that the viscosity of mucus from these two different cancer sources decreased significantly after the reaction, demonstrating the extremely significant viscosity-reducing effect of the serine protease EatA.

[0087] Figure 11 The results of mucus degradation from mucinous appendiceal adenocarcinoma (MAA) are further illustrated. Since the appendiceal cancer mucus in this sample is transparent, its viscosity reduction can be detected by examining the homogeneity of the solution. The results after incubation show that the mucus sample solution became homogeneous after incubation with serine protease, and impurities not degraded by EatA settled to the bottom after settling. In contrast, the control group maintained a highly gel-like mucus, and internal impurities could not settle to the bottom.

[0088] Furthermore, the serine protease EatA in this application has been demonstrated in the applicant's relevant research results to have a certain degradation effect on mucus (sputum) in patients with chronic obstructive pulmonary disease and mucus in mucinous colon cancer. Based on the above examples, the serine protease in this application has good prospects for medical application and has significant advantages compared with other drugs and drug-to-produce formulations currently on the market.

[0089] It should be noted that the embodiments described above are only for explaining this application and do not constitute any limitation on this application. This application has been described with reference to typical embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to this application within the scope of the claims, and revisions can be made to the invention without departing from the scope and spirit of this application. Although the application described herein relates to specific methods, materials, and embodiments, it does not mean that this application is limited to the specific examples disclosed herein; on the contrary, this application can be extended to all other methods and applications with the same function.

Claims

1. The use of a serine protease in the preparation of a medicament for degrading mucins in mucus-related diseases, wherein the serine protease has the amino acid sequence shown in SEQ ID NO.1, and the mucus-related diseases are selected from at least one of peritoneal pseudomyxoma, mucinous appendiceal adenocarcinoma, mucinous ovarian cancer, mucinous gastric cancer, mucinous hepatic cancer, mucinous lung cancer, asthma, and mucinous colorectal cancer.

2. The application according to claim 1, characterized in that, The serine protease has specific serine protease activity and is used to selectively degrade mucins.

3. The application according to claim 1, characterized in that, The drug is used in combination with other reagents, wherein the other reagents are selected from at least one of chemotherapy and radiotherapy reagents, enzymes having other mucin-degrading activities, and chemical salt reagents selected from at least one of carboxycysteine, N-acetylcysteine, and ambroxol.

4. The application according to claim 3, characterized in that, The drug may be administered together with other reagents, separately, or in combination.

5. A pharmaceutical composition comprising a serine protease; and other reagents, said other reagents being selected from at least one of chemotherapy and radiotherapy reagents, enzymes having other mucin-degrading activities, and chemical salts selected from at least one of carboxycysteine, N-acetylcysteine, and ambroxol, wherein, The serine protease has the amino acid sequence shown in SEQ ID NO.

1.

6. The pharmaceutical composition according to claim 5, characterized in that, The chemotherapy and radiotherapy reagents are selected from at least one of gemcitabine, cisplatin, adriamycin, fluorouracil, paclitaxel, paclitaxel, and oxaliplatin; the enzymes are selected from at least one of N-acetylgalactosidase, galactosidase, glucosidase, sialidase, and specific endoplasminase.

7. The pharmaceutical composition according to any one of claims 5-6, characterized in that, The pharmaceutical composition is used to treat mucus-related diseases, wherein the mucus-related diseases are selected from at least one of peritoneal pseudomyxoma, mucus-type appendiceal carcinoma, mucus-type ovarian carcinoma, mucus-type gastric carcinoma, mucus-type liver carcinoma, mucus-type lung carcinoma, asthma, and mucus-type colorectal carcinoma.

8. Use of any pharmaceutical composition of claims 5-7 in the preparation of a medicament for treating mucus-related diseases, wherein the mucus-related diseases are selected from at least one of peritoneal pseudomyxoma, mucus-type appendiceal carcinoma, mucus-type ovarian carcinoma, mucus-type gastric carcinoma, mucus-type liver carcinoma, mucus-type lung cancer, asthma, and mucus-type colorectal cancer.

9. The application according to claim 8, characterized in that, The route of administration of the drug is selected from any one of injection, oral administration, and spray administration.

10. The application according to claim 9, characterized in that, The injection administration is an intraperitoneal injection.

Images