A medicament containing a mucosal adhesive protein and its preparation method and use

Abstract

The application discloses a biological macromolecular drug for tumor treatment, which has tissue protein adhesion, can continuously improve the oxidative stress environment of a tumor site by prolonging the time at the tumor site, can inhibit tumor growth by tumor microenvironment regulation, can also be used as an auxiliary therapeutic agent to enhance the curative effect of other tumor treatments, and has a good clinical transformation prospect.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and more particularly to a biopharmaceutical for the treatment of tumors. Background Technology

[0002] The tumor microenvironment plays a crucial role in tumor growth, invasion, metastasis, and treatment. In the tumor microenvironment, peroxide levels are significantly higher than in normal tissues. Excessive peroxides can damage DNA, proteins, and lipids through oxidation, promoting gene mutations in tumor cells and disease progression. Hydrogen peroxide is a prevalent peroxide in the tumor microenvironment; therefore, existing technologies can decompose endogenous hydrogen peroxide to generate oxygen, increasing local oxygen concentration and improving the hypoxic microenvironment at the tumor site.

[0003] Catalase is an endogenous protein that efficiently breaks down hydrogen peroxide to produce oxygen. However, catalase has limited ability to accumulate and retain at tumor sites, usually requiring the assistance of other components, such as gels or nanocarriers. However, the introduction of such exogenous components carries other risks, and complex formulations are not conducive to drug development. Therefore, a simpler and more efficient method is needed to achieve the therapeutic effect of catalase at tumor sites. Summary of the Invention

[0004] To address the problems in the prior art, this invention provides a biological macromolecular drug for tumor treatment. The biological macromolecular drug has a simple composition, can remain at the tumor site for a relatively long time, and effectively improves the hypoxic microenvironment of the tumor, thereby achieving the therapeutic effect of tumor treatment.

[0005] Specifically, the present invention provides a drug containing a mucosal adhesion protein, wherein the mucosal adhesion protein includes catalase, and the catalase is chemically grafted with adhesion groups.

[0006] Furthermore, the adhesive group is selected from one or more of thiol, disulfide bond, and thioether.

[0007] Furthermore, the molar ratio of the adhesive group to the mucosal adhesive protein is (0-100):1, and is not 0:1.

[0008] The present invention also provides a tumor treatment preparation comprising a mucosal adhesion protein.

[0009] Furthermore, it also includes one or more of surfactants, osmotic pressure regulators, stabilizers, metal ion chelators, and thickeners.

[0010] Furthermore, the mucosal adhesive protein accounts for no less than 0.1 wt% of the mass fraction of the formulation.

[0011] Furthermore, the mucosal adhesive protein accounts for no more than 3.0 wt% of the mass fraction of the formulation.

[0012] Furthermore, the concentration of the mucosal adhesive protein is 0.01–5 mg / mL.

[0013] Further, the concentration of the mucosal adhesion protein is not greater than 0.02 mg / mL, 0.05 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.12 mg / mL, 0.15 mg / mL, 0.17 mg / mL, 0.2 mg / mL, 0.22 mg / mL, 0.25 mg / mL, 0.28 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, or 0.45 mg / mL. / mL, 0.5mg / mL, 0.55mg / mL, 0.6mg / mL, 0.65mg / mL, 0.7mg / mL, 0.75mg / mL, 0.8mg / mL, 0.85mg / mL, 0.9mg / mL, 0.95mg / mL, 1mg / mL, 1.5mg / mL, 2.mg / mL, 2.5mg / mL, 3mg / mL, 3.5mg / mL, 4mg / mL, 4.5mg / mL, 5mg / mL.

[0014] The present invention also provides a method for preparing a mucosal adhesion protein, characterized in that the surface of catalase is chemically modified.

[0015] The present invention also provides the application of a mucosal adhesion protein or a preparation containing a mucosal adhesion protein in the preparation of a tumor therapeutic agent.

[0016] The present invention also provides the use of a mucosal adhesion protein or a preparation containing a mucosal adhesion protein in the preparation of a drug for enhancing tumor radiotherapy.

[0017] Furthermore, the tumor is a solid tumor.

[0018] According to the technical solution of the present invention, the following beneficial effects are achieved:

[0019] 1) The mucosal adhesion proteins described in this application are naturally occurring components in living organisms. Even after synthesis through bioengineering systems and purification, they still have good biocompatibility. Furthermore, no other chemical components such as polymers are used as carriers. The protein's ability to be locally retained is increased only through surface modification. The composition is simple and the safety is high.

[0020] 2) The mucosal adhesion protein described in this application can remain inside the tumor for a long time, continuously catalyze the decomposition of hydrogen peroxide to generate oxygen, or remove substances that promote tumor progression in the tumor microenvironment by consuming glutathione and hydrogen peroxide, while improving tumor hypoxia and oxidative stress, significantly inhibiting tumor growth and prolonging survival.

[0021] 3) The method for preparing the mucosal adhesion protein described in this application is simple, easy to purify, and has little impact on enzyme activity. It can be mass-produced and has great commercial value. Attached Figure Description

[0022] Figure 1 These are SDS-PAGE electrophoresis band images of each group of samples in Example B1;

[0023] Figure 2 This is a statistical chart of enzyme activity detection in different samples in Example B2;

[0024] Figure 3 These are mass spectra of different samples in Example B3, with the horizontal axis representing molecular weight (in Da) and the vertical axis representing intensity counts (in counts).

[0025] Figure 4 This is a statistical result of the percentage of catalase retained in the tumor at different times after injection in Example C1;

[0026] Figure 5 These are the tumor growth curves of each group of mice in Example C2;

[0027] Figure 6 These are the survival curves of the mice in each group in Example C2;

[0028] Figure 7 These are the tumor growth curves of mice in each group in Example C3. Detailed Implementation

[0029] To further illustrate the technical means and effects of the present invention in order to achieve the intended purpose, the following detailed description, in conjunction with the accompanying drawings and preferred embodiments, describes the specific implementation methods, steps, structures, features and effects of the biopharmaceutical drugs proposed according to the present invention.

[0030] The foregoing and other technical contents, features, and effects of the present invention will be clearly presented in the following detailed description of preferred embodiments with reference to the accompanying drawings. Through the description of the specific embodiments, a more in-depth and specific understanding can be gained of the technical means and effects adopted by the present invention to achieve its intended purpose. However, the accompanying drawings are for reference and illustration only and are not intended to limit the present invention.

[0031] To illustrate the technical solutions of the invention in more detail, specific embodiments are provided below to demonstrate the technical effects. It should be emphasized that these embodiments are for illustrative purposes only and not for limiting the scope of the invention.

[0032] The experimental materials used in the examples are as follows:

[0033] name supplier Item number Catalase activity assay kit Azure Sky S0051 PAGE gel preparation kit Shanghai Yamei Biotechnology Co., Ltd. PG113

[0034] In this invention, the term "mucosal adhesive protein" refers to a protein with adhesive groups, such as thiol-grafted proteins, maleimide-grafted proteins, and disulfide-grafted proteins.

[0035] Example A: Preparation of mucosal adhesive proteins

[0036] Example A1:

[0037] A mucosal adhesion protein C1, wherein the protein is catalase and the adhesive group is a thiol group, is prepared as follows:

[0038] Add an appropriate amount of catalase to 5 mmol / L EDTA, then add 2 mg / mL of Traut's reagent working solution. The molar ratio of catalase to Traut's reagent is 1:100. React at 25℃ for 4 hours to obtain mucosal adhesion protein C1.

[0039] Example A2:

[0040] A mucosal adhesion protein C2, wherein the protein is catalase and the adhesive group is a maleimide group, is prepared as follows:

[0041] Add an appropriate amount of catalase to 5 mmol / L EDTA, then add Sulfo-SMCC (sodium salt of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester). The mass ratio of catalase to Sulfo-SMCC is 20:1. React at 37℃ for 30 minutes to obtain mucosal adhesion protein C2.

[0042] Example A3:

[0043] A mucosal adhesion protein C3, wherein the protein is catalase and the adhesive group is a disulfide bond, is prepared as follows:

[0044] Add an appropriate amount of catalase to Sulfo-LC-SPDP (sulfosuccinimide-6-[3-(2-pyridyldithio)propionamide]hexanoic acid), with a catalase to Sulfo-LC-SPDP mass ratio of 5:1, and react at 25℃ for 1 hour to obtain mucosal adhesion protein C3.

[0045] Example B: Test case of mucosal adhesion proteins

[0046] Example B1: Molecular weight characterization of mucosal adhesion proteins

[0047] 1) BCA (disulfide bond thiol protein colorimetric method) is used to detect protein concentration and is used for quantitative sample loading.

[0048] 2) Mix the mucosal adhesion protein with the unmodified protein control and loading buffer, heat at 100°C for 5 minutes to prepare the SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) sample, and load 10 μg of protein for electrophoresis.

[0049] 3) After electrophoresis, the gel was stained with Coomassie Brilliant Blue for 30 minutes, destained with ultrapure water, and photographed for record.

[0050] The experimental groups are as follows:

[0051] Control Example B1.1: Unmodified catalase;

[0052] Example B1.2: Mucosal adhesion protein C1 prepared in Example A1;

[0053] Example B1.3: Mucosal adhesion protein C2 prepared in Example A2;

[0054] Example B1.4: Mucosal adhesion protein C3 prepared in Example A3.

[0055] Figure 1 These are SDS-PAGE electrophoresis band images of each group of samples. The results show that, compared with Control Example B1.1, the protein samples described in Examples B1.2-B1.4 did not show significant changes in band position and no other impurities. This indicates that the mucosal adhesive proteins described in this application did not undergo aggregation, dissociation, or other effects on the protein structure due to surface adhesive modifications, and the molecular weight of the proteins did not change significantly.

[0056] Example B2: Enzyme activity detection of mucosal adhesion proteins

[0057] Mucosal adhesion protein samples C1-C3 and unmodified protein (catalase) control were weighed quantitatively and prepared into 2 μg / mL sample solutions. Enzyme activity was detected according to the instructions of the detection kit. The samples were grouped according to the scheme of Example B1 and numbered as Control Example B2.1, Example B2.2, Example B2.3, and Example B2.4, respectively.

[0058] Figure 2This is a statistical chart of enzyme activity detection in different samples in Example B2. The results show that the enzyme activity data of the samples described in Examples B2.2-B2.4 are similar to those of Control Example B2.1, indicating that the mucosal adhesion modification process has little effect on protease activity. The mucosal adhesion protein described in this application has catalase activity and is expected to be able to perform the original catalase function and be further used for reactive oxygen species scavenging.

[0059] Example B3: Mass spectrometry characterization of mucosal adhesion proteins

[0060] Mucosal adhesion protein samples C1-C3 and unmodified protein (catalase) control were weighed quantitatively and prepared into 1 mg / mL sample solutions. The samples were detected by liquid chromatography-mass spectrometry (LC-MS) and grouped according to the scheme of Example B1, and numbered as Control Example B3.1, Example B3.2, Example B3.3, and Example B3.4, respectively. Figure 3 These are mass spectra of different samples in Example B3.

[0061] The results showed that in Control Example B3.1, the unmodified catalase peak was located at 59754 Da, and this characteristic peak signal was strong with few extraneous peaks. In contrast, the characteristic peaks of the three groups of catalases in Examples B3.2-B3.4 showed significant shifts. The shifted peaks represent single subunits of catalase modified with different numbers of adhesive groups. The relative content of mucosal adhesive proteins at this grafting rate can be calculated using the peak area. Figure 3 The data show that the corresponding adhesive groups were successfully grafted onto the surface of the three mucosal adhesion proteins C1-C3. Through mass spectrometry characterization, the ratio of the surface-modified groups to catalase can be estimated. The products are detectable, which is beneficial for large-scale production and quality control.

[0062] Example B4: Characterization of the degree of modification of adhesive groups on the surface of mucosal adhesive proteins

[0063] Samples with different feed ratios were prepared according to the preparation method of Example A1, and the mass spectra of the samples were detected by the method of Example B3. The degree of modification, i.e. the molar ratio of thiol groups to catalase, was calculated. The results are shown in Table 1.

[0064] Table 1: Statistical table of Calculation of Thiol Modification Degree on Catalase Surface Obtained by Changing Feed Ratio in Example A1

[0065]

[0066] The results showed that the number of thiol groups modified on the surface of catalase increased with the increase of the feed ratio, indicating that within a certain range, products with different degrees of modification can be obtained by adjusting the feed ratio. Generally, the more adhesive groups modified, the easier and more stable the binding of mucosal adhesive proteins to mucosal surface proteins. However, considering the cost of raw materials and the final therapeutic effect, it is necessary to balance the relationship between the degree of modification and the dosage, and select mucosal adhesive proteins with appropriate degrees of modification for the treatment of specific diseases as needed.

[0067] Example C: Effect Example

[0068] Example C1: Detection of tumor retention of mucosal adhesion proteins

[0069] Radiolabeling of protein samples: Dissolve 200 μg of catalase control or mucosal adhesion protein sample C3 in sodium acetate buffer at pH 4, then add 1 mCi of... 177 Lu, after mixing, place in a 37°C heated and shaken for 1 hour.

[0070] A subcutaneous tumor model was established in Balb / C Nude mice by subcutaneously inoculating tumor cells. Labeled mucosal adhesion protein sample C3 or catalase control was injected into the tumor. At different time points after sample injection, the radiation signal intensity at the mouse tumor site was detected by in vivo gamma imaging. Using the signal intensity at the first time point as 100%, the relative percentage of residual radiation intensity at each time point was calculated and plotted as a line graph. The experimental groups were as follows:

[0071] Control Example C1.1: Tumor-bearing mice were injected with catalase control at a dose of 200 μg / mouse;

[0072] Example C1.2: Tumor-bearing mice were injected with mucosal adhesion protein sample C3 at a dose of 200 μg / mouse;

[0073] The results showed that, at various time points within 96 hours after intratumoral injection, the retention amount of mucosal adhesion protein C3 in mouse tumors in Example C1.2 was significantly higher than that of unmodified catalase in Control Example C1.1. This indicates that the mucosal adhesion protein described in this application can bind to mucosal proteins in the tumor stroma, thereby significantly prolonging its action time at the tumor site, continuously consuming endogenous hydrogen peroxide, and effectively improving the hypoxic environment and alleviating oxidative stress.

[0074] Example C2: Tumor Treatment Based on Mucosal Adhesion Proteins

[0075] A CT26 tumor model was established according to the method described in Example C1, and the antitumor effect of the mucosal adhesion protein described in this application was verified. The experimental groups are as follows:

[0076] Control C2.1: Tumor-bearing mice, without treatment;

[0077] Example C2.2: Tumor-bearing mice were injected with catalase control at a dose of 50 μg / mouse;

[0078] Example C2.3: Tumor-bearing mice were injected with mucosal adhesion protein sample C3 at a dose of 50 μg / mouse;

[0079] The first intratumoral injection sample was recorded as day 0. Tumor volume in mice was recorded every 2 days, and the results were statistically analyzed as follows: Figure 5 As shown in the figure. The results indicate that the tumor growth rate in Examples C2.2 and C2.3 was slower than that in Control Example C2.1, suggesting that intratumoral injection of catalase can improve the tumor microenvironment and kill tumor cells, thus exhibiting an anti-tumor effect. Furthermore, the slower tumor growth rate in Example C2.3 compared to Example C2.2 indicates that the mucosal adhesion protein exhibits better anti-tumor activity. The modification of the adhesion groups enhances the tumor retention of the mucosal adhesion protein, thereby achieving long-term regulation of the tumor microenvironment and achieving better anti-tumor efficacy compared to unmodified catalase.

[0080] The survival curves of the mice after treatment were further recorded and the mean survival time of the mice was calculated. The results are as follows: Figure 6 As shown in the table below. The results indicate that the average survival time of mice in Example C2.3 was significantly longer than that of the other two groups, suggesting that the mucosal adhesion protein can exert a sustained anti-tumor effect after injection and significantly prolong the survival of mice.

[0081] Table 2: Survival rate statistics of mice in different embodiments

[0082] Group number Example C2.1 Example C2.2 Example C2.3 time Survival rate (%) Survival rate (%) Survival rate (%) Day 13 85.71 100 100 Day 15 71.43 100 100 Day 17 71.43 85.71 100 Day 19 57.14 71.43 100 Day 23 28.57 28.57 85.71 Day 25 28.57 28.57 71.42 Day 27 0 14.29 57.14 Day 29 0 14.29 28.57 Day 36 0 0 28.57

[0083] Example C3: Mucosal adhesion protein combined with radiotherapy for tumor treatment

[0084] A CT26 tumor model was established according to the method described in Example C1, and the anti-tumor effect of the mucosal adhesion protein described in this application combined with radiotherapy was verified. The experimental groups are as follows:

[0085] Control C3.1: Tumor-bearing mice, without treatment;

[0086] Example C3.2: Tumor-bearing mice, X-ray therapy;

[0087] Example C3.3: Tumor-bearing mice, intratumoral injection of catalase (25 μg / mouse) combined with X-ray treatment;

[0088] Example C3.4: Tumor-bearing mice were treated with intratumoral injection of mucosal adhesion protein sample C1 (25 μg / mouse) combined with X-ray therapy.

[0089] X-ray therapy was performed as follows: 24 hours after intratumoral injection of the sample, the mouse tumor site was locally irradiated with X-rays at a dose of 2 Gy. Repeat irradiation with a 2 Gy dose was performed 3 and 5 days after sample injection. The average tumor volume of each group of mice was recorded 21 days after treatment. The results are as follows: Figure 7 As shown in Table 3, the tumor inhibition rate was calculated on day 18 after treatment.

[0090] Table 3: Statistical table of average tumor inhibition rate in mice from different embodiments

[0091] Grouping Compare with Example C3.1 Example C3.2 Example C3.3 Example C3.4 Tumor inhibition rate 0％ 41.7％ 47.5％ 82.1％

[0092] The results showed that the tumor inhibition rate of Example C3.4 was significantly higher than that of Control Example C3.1 and the radiotherapy-only group of Example 3.2, indicating that catalase can improve tumor hypoxia, thereby enhancing radiotherapy and exhibiting a significant tumor inhibition effect. The tumor inhibition rate of Example C3.4 was significantly higher than that of Example C3.2, indicating that compared with the catalase control, the mucosal adhesive protein remained at the tumor site for a longer time, resulting in a more lasting improvement in tumor hypoxia and demonstrating better combined efficacy.

[0093] Obviously, the above embodiments are merely examples for clear illustration and are not intended to limit the implementation.

[0094] While the foregoing disclosure has discussed some inventive embodiments that are currently considered useful through various examples, it should be understood that such details are for illustrative purposes only, and the appended claims are not limited to the disclosed embodiments. Rather, the claims are intended to cover all modifications and equivalent combinations that conform to the substance and scope of the embodiments of this application.

[0095] Similarly, it should be noted that, in order to simplify the description of the present application and thus aid in the understanding of one or more embodiments of the invention, the foregoing description of the embodiments of the present application sometimes combines multiple features into a single embodiment, drawing, or description thereof. However, this disclosure method does not imply that the subject matter of the application requires more features than those mentioned in the claims. In fact, the embodiments contain fewer features than all the features of the single embodiments disclosed above.

[0096] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are sometimes modified by the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed according to the characteristics required by individual embodiments.

[0097] Finally, it should be understood that the embodiments described in this application are merely illustrative of the principles of the embodiments of this application. Other modifications may also fall within the scope of this application. Therefore, alternative configurations of the embodiments of this application are considered as examples and not limitations, and are regarded as consistent with the teachings of this application. Accordingly, the embodiments of this application are not limited to the embodiments explicitly described and illustrated in this application.

Claims

1. A drug containing a mucosal adhesion protein, characterized in that, The mucosal adhesive protein is a catalase chemically grafted adhesive group.

2. The drug according to claim 1, characterized in that, The adhesive group is selected from one or more of thiol, disulfide bond, and thioether.

3. The drug according to claim 1, characterized in that the molar ratio of the adhesive group to the mucosal adhesive protein is (0-100):1, and is not 0:

1.

4. A tumor treatment preparation comprising the mucosal adhesion protein as described in claims 1 to 3.

5. The formulation according to claim 4, characterized in that, It also includes one or more of surfactants, osmotic pressure regulators, stabilizers, metal ion chelators, and thickeners.

6. The formulation according to claim 5, characterized in that, The mucosal adhesion protein accounts for no less than 0.01 wt% of the mass fraction of the preparation.

7. The formulation according to claim 5, characterized in that, The concentration of the mucosal adhesion protein is 0.01–5 mg / mL.

8. A method for preparing the mucosal adhesion protein according to any one of claims 1-3, characterized in that, Chemical modification of the surface of catalase.

9. The use of the drug according to claims 1 to 3 or the formulation according to claims 5 to 8 in the preparation of tumor therapeutic formulations.

10. The use of the medicament according to claims 1 to 3 or the formulation according to claims 5 to 8 in the preparation of a drug for enhancing tumor radiotherapy.

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