WO2016119308A1 - Antitumour preparation and preparation method thereof - Google Patents

Abstract

Provided in the present invention are an antitumour preparation and a preparation method thereof. The antitumour preparation is a preparation in the form of a hydrogel formed from an aqueous alginate solution of dispersed active ingredients of a PD-1 blocking antibody and celecoxib, and the preparation is an injectable preparation.

Description

Antitumor preparation and preparation method thereof Technical field

The invention relates to a pharmaceutical preparation and a preparation method thereof, in particular to an antitumor preparation and a preparation method thereof.

Background technique

Tumors are one of the greatest threats to human health and life. The World Health Organization reports that 14.1 million people worldwide contracted cancer in 2012, with 8.2 million deaths, exceeding the combined death toll from AIDS, malaria and tuberculosis. China's new cancer cases rank first in the world, and cancer has become the number one cause of death among Chinese residents. Every year, 2.07 million people die of cancer. At present, in clinical practice, the main treatment methods for tumors are surgical resection, chemotherapy and radiotherapy. In the treatment of malignant tumors, it is almost impossible to completely remove the diffused tumor cells by surgery. This causes recurrent tumors. Although chemotherapy and radiotherapy can assist the treatment of malignant tumors to some extent, the strong side effects cause additional damage to the body. Huge damage and a low cure rate. Most chemotherapy drugs cause irreversible damage to normal tissues and organs of the body while killing tumor cells. Therefore, traditional treatment techniques cannot solve the major threat to human health caused by tumors, especially malignant tumors.

Tumor immunotherapy technology is a kind of tumor treatment technology developed and developed based on the basic principles of tumor immunity to achieve effective killing of tumor cells by immunological means. The immune system has strong pertinence and specificity for the killing of tumor cells, so the immunological treatment technology of such diseases has the natural high efficiency and targeting unmatched by traditional therapeutic techniques. For a certain type of tumor, a successful immunological treatment program can activate the body to produce enough tumor cell characteristics of cytotoxic T lymphocytes, which can selectively kill all tumor cells. Compared with other therapies of tumors, immunotherapy is currently The only known means that is expected to completely eliminate tumor cells has no adverse effects on normal cells of the body. In recent years, with the research on tumor immune escape, the use of monoclonal antibodies to block the costimulatory molecular pathway has been gradually taken seriously as a new tumor immunotherapy, and it has opened a new chapter for tumor immunotherapy. The first drug to be used in clinical applications for blockade of negative costimulatory signaling pathways is ipilimumab as a blocker of B7/CTLA-4 costimulatory pathways, and ipilimumab has been shown to be effective in improving advanced malignant melanoma. Patient's life The rate of deposit, which was approved by the FDA in 2011 for the treatment of metastatic malignant melanoma, but Iprimima can cause serious and fatal autoimmune-related diseases. Immediately thereafter, the programmed death molecule-1 (PD-1) pathway blocker has also made a breakthrough in anti-PD-1 and PD-L1 monoclonal antibodies, which can block the inhibitory effect of PD-1 on T cells. In order to activate the tumoricidal effect of immune effector cells in tumor patients, and reduce the inhibitory T cell effect, and enhance the function of CTL to kill cancer cells has become a hot spot in immunotherapy in recent years. Multiple tumor-bearing animal experiments have shown that blocking the PD-L1/PD-1 pathway can enhance anti-tumor effects, prolong survival, and improve prognosis. At present, a variety of monoclonal antibodies against PD-L1/PD-1 pathway blockade are used in phase III clinical trials for advanced malignancies. PD-1 antibodies are due to their broad-spectrum anticancer effects on kidney cancer. The clinical efficacy of cancers such as gastric cancer, breast cancer, bladder cancer, leukemia, head and neck cancer, intestinal cancer and brain tumors is also in Phase II or Phase III clinical trials. However, due to poor stability in vivo and pleiotropic effects, continuous infusion or frequent administration is required to obtain the desired therapeutic effect. In animal experiments, the dose of PD-1 antibody is usually 250ug/mouse per mouse, and each It is administered once every two days to maintain the efficacy. Such high doses cause side effects during systemic treatment, such as side effects such as immunogenic enteritis and severe autoimmune diseases, which limits its application.

Therefore, how to provide an anti-drug preparation using PD-1 antibody, but having good in vivo stability, having an improved pharmacological effect and having few side effects, has become a problem to be solved.

Summary of the invention

The present invention provides an antitumor preparation which is an antitumor preparation in the form of a hydrogel formed by blocking an aqueous solution of an antibody and celecoxib with an active ingredient PD-1. The formulation has good in vivo stability, a markedly enhanced inhibition of tumor cell growth and metastasis, an expanded anti-tumor profile, and reduced toxic side effects on the body.

The invention also provides a preparation method of an anti-tumor preparation, which comprises dispersing an active ingredient PD-1 blocking antibody and celecoxib in an alginate solution, and forming an injectable anti-tumor preparation in the form of a hydrogel. . The anti-tumor preparation prepared by the method is applied to an individual, and the PD-1 blocking antibody and celecoxib exhibit good in vivo stability and prolonged sustained release effect, which is more conducive to the exertion of the active ingredient and lowering It uses the dose.

Non-steroidal anti-inflammatory drugs, such as Celecoxib, have been shown to inhibit a variety of Tumor cell proliferation, promote cell apoptosis, inhibit tumor neovascularization and prevent tumorigenesis and development, and prevent tumor growth, inhibit tumor growth and metastasis, and achieve good results in animal experiments. However, its clinical application is still limited, mainly because many problems remain to be solved, such as longer medication time and larger dosage. Mouse experiments show that it is usually necessary to administer a dose of 25 mg/kg body weight of celecoxib per day. It has anti-tumor effect, resulting in large side effects in the gastrointestinal tract and cardiovascular, and narrow anti-tumor spectrum (mainly used for prevention and early treatment of colorectal cancer). There have been no reports of non-steroidal anti-inflammatory drugs, such as celecoxib combined with PD-1 blocking antibody, which can reduce the amount of drug, expand the anti-tumor spectrum, and reduce the side effects of the body.

The present invention provides an antitumor preparation which is an antitumor preparation in the form of a hydrogel formed by dissolving an aqueous solution of an active ingredient PD-1 blocking antibody and celecoxib, which is an injection. preparation.

In the embodiment of the invention, the alginate has a molecular weight of from 75,000 to 300,000 g.

Further, further, the alginate solution mixture is crosslinked in the presence of a crosslinking agent. The crosslinking agent is a divalent metal cation. Further, the divalent metal cation is provided by a sulfate or a hydrochloride of a divalent metal, which is calcium or magnesium. Further, the divalent metal cation is provided by CaSO ₄ , MgCl ₂ or CaCl ₂ .

Further, the anti-tumor preparation is an anti-tumor preparation for melanoma, breast cancer, kidney cancer, gastric cancer, bladder cancer, leukemia, head and neck cancer, intestinal cancer or brain tumor.

The invention provides a method for preparing an anti-tumor preparation, comprising:

Dissolving alginate in physiological saline to prepare an alginate solution;

Adding the active ingredient PD-1 blocking antibody and celecoxib to the alginate solution, and dispersing it in the alginate solution to obtain a sea alginate solution mixture;

The alginate solution mixture is crosslinked to form an antitumor preparation in the form of a hydrogel, which is an injection preparation.

Further, the alginate has a molecular weight of 75,000 to 300,000 g. For the alginate used in the present embodiment, those skilled in the art can select as needed within the above molecular weight range. One of the above molecular weight ranges of one or more alginate can also be used directly by those skilled in the art.

Further, the alginate solution mixture is crosslinked in the presence of a crosslinking agent. The crosslinking agent is a divalent metal cation. Further, the divalent metal cation is provided by a sulfate or a hydrochloride of a divalent metal, which is calcium or magnesium. Further, the divalent metal cation is provided by CaSO ₄ , MgCl ₂ or CaCl ₂ .

Further, the anti-tumor preparation is an anti-tumor preparation for melanoma, breast cancer, kidney cancer, gastric cancer, bladder cancer, leukemia, head and neck cancer, intestinal cancer or brain tumor.

Further, in the solution of the present application, since celecoxib is insoluble in water in a powder form, it can be subjected to ultrasonication in the process of dispersing it in an alginate solution.

Further, the process of preparing the antitumor preparation of the present invention should be carried out under aseptic conditions.

The antitumor preparation provided by the present invention can be administered by subcutaneous injection to inhibit tumorigenesis or kill tumor cells.

The antitumor preparation of the present invention may be a unit preparation. The unit preparation is a preparation which satisfies the active ingredient required for one administration, such as a unit (needle) injection or the like. The amount of drug required for a single administration of a patient can be conveniently obtained by calculating the product of the patient's body weight and the unit weight dose required for the patient to take the drug once. For example, in the preparation of a drug, it is generally considered that the adult body weight is 50-70 kg, and the dose can be determined initially by an equivalent dose conversion relationship between the experimental animal and the human body weight dose. For example, it can be based on the guidance provided by the FDA, SFDA and other drug regulatory agencies, or refer to (Huang Jihan, et al., "Equivalent dose conversion between animals and animals and humans in pharmacological tests", "Chinese Clinical Pharmacology and Therapeutics" , 2004Sep; 9 (9): 1069-1072) to determine. In an embodiment of the present invention, the human and mouse doses can be converted according to the body surface area conversion coefficient of 0.0026 for humans and mice.

In one embodiment of the invention, a mouse weighing 25 g is injected subcutaneously with 0.2 ml of an antitumor preparation of the invention, wherein the PD-1 blocking antibody is 100 ug and the celecoxib is 625 ug, ie: 4 mg. /kg mouse body weight of PD-1 blocking antibody, 25mg/kg mouse body weight of celecoxib, can be maintained once every two weeks.

The pharmaceutical manufacturer can obtain the content of the active ingredient in the unit preparation for one administration of the adult according to the above conversion method, and apply it to the pharmaceutical process. In the technical solution of the present invention, according to the equivalent dose conversion relationship and the normal body weight of the human, and the comprehensive drug safety, cost and efficacy, preferably, the unit preparation contains 20-30 mg of the PD-1 blocking antibody and 130-200mg Celecoxib.

In the preparation method of the antitumor preparation of the present application, the solution concentration of the alginate solution may be in the range of 10-40 mg/mL, and the PD-1 blocking antibody may be dispersed in the above alginate in an amount of 500 to 1000 μg/mL. In the solution, the celecoxib may be dispersed in the above alginate solution in an amount of 3.75 to 7.5 mg/mL. Applicants may appropriately select the solution concentration of the alginate solution according to the specifications of the antitumor preparation to be prepared, for example, the amount of the PD-1 blocking antibody and celecoxib to be contained in the unit preparation, and PD-1 blocks the antibody, The amount of celecoxib dispersed in the alginate is achievable by those skilled in the art.

The present invention also provides a method for treating or preventing a tumor comprising the step of administering an antitumor preparation to an individual having a tumor or an individual having a tendency to develop a tumor, the antitumor preparation being blocked by the active ingredient PD-1 An antitumor preparation in the form of a hydrogel formed by breaking an antibody and an aqueous solution of alginate of celecoxib, and the antitumor preparation is an injection preparation.

Further, the anti-tumor preparation further comprises a crosslinking agent for crosslinking the aqueous alginate solution.

Further, the alginate has a molecular weight of 75,000 - 300,000 g, and the crosslinking agent is a divalent metal cation.

Further, the anti-tumor preparation is a unit preparation.

Further, the unit preparation contains a therapeutically effective amount of 20-30 mg of PD-1 blocking antibody and 130-200 mg of celecoxib.

An individual having a tendency to develop a tumor in the present invention refers to an individual who is genetically at risk for a tumor or an individual who exhibits a risk of developing a tumor on other physical indicators.

The solution provided by the invention has the following advantages:

(1) The antitumor preparation prepared by the method of the invention can make PD-1 blocking antibody and celecoxib have good stability in vivo, and has an extended sustained release effect, thereby being more beneficial to the effect of the drug. , reduce the dose.

(2) Applicant's study found that the anti-tumor preparation of the present invention is administered to an individual by injection, that is, the PD-1 blocking antibody and celecoxib are simultaneously encapsulated into the alginate hydrogel to provide the individual, as compared to Administration of alginate hydrogel containing only PD-1 blocking antibody and/or alginate hydrogel containing only celecoxib increases the percentage of survival of tumor-bearing mice and promotes the production of active CD8 T cells The raw side has an unexpected synergy.

(3) The anti-tumor preparation provided by the invention enhances the acquired immune response against tumor cells by the body, blocks the immunosuppressive pathway, activates the effector cells to specifically kill the tumor cells, and has almost no harm to the normal tissues and cells of the body. Compared with the damage caused by surgery, chemotherapy and radiotherapy, the advantages are obvious.

(4) The material used in the solution of the present invention has a wide range of sources, and can be extracted from seaweed in large amounts, and a significantly reduced PD-1 blocking antibody and celecoxib dose can be used to obtain a good tumor suppressing effect, remarkable Reduce the cost of medication for patients.

(5) The anti-tumor preparation provided by the invention can be used for treating solid tumors and inhibiting tumor growth; and can be used for treating tumors with malignant metastasis; and for adjuvant treatment of post-operative radiotherapy and chemotherapy for malignant tumors; The treatment of tumors that cannot be surgically removed has a wide range of applications.

DRAWINGS

Figure 1 is a flow chart showing the preparation of the antitumor preparation of the present invention.

Figure 2A shows the concentration of celecoxib in the serum of each group of mice, and Figure 2B shows the content of celecoxib in the tumor tissues of each group of mice. Figure 2C shows the ratio of cytotoxic T lymphocytes (CD8 and IFN-γ double positive) and regulatory T lymphocytes (CD4 and FoxP3 double positive) in each group of mice. Figure 2D shows the percent survival of each group of mice.

Figures 3A-3B show the results of detection of CD8 T cells that are effective in killing tumor cells in the tumor microenvironment.

Figure 4A shows the expression level of relative messenger RNA of the COX-2 gene. Figure 4B shows the expression level of the relative messenger RNA of the IL-1β gene. Figure 4C shows the expression level of the relative messenger RNA of the IL-6 gene. Where * indicates a p value of <0.05, and ** indicates a p value of <0.01.

Figure 5A shows a picture of the tumor appearance of mice of each group on days 0, 10, 13, 17, 20, and 22. Figure 5B shows tumor size changes for mice of each group on days 0, 10, 13, 17, 20, and 22. Where * indicates a p value <0.05.

Figure 6A shows a picture of a pulmonary metastases from each group of mice. Figure 6B shows the number of lung metastases in each group of mice. Where $ indicates that the group is compared with the group a; # indicates that the group is compared with the group b. *, $, # indicates p value <0.05; $$, ## indicates p value <0.01.

detailed description

Example 1. Preparation of an antitumor preparation of the present invention.

The antitumor preparation of the present invention can be prepared according to the procedure shown in Fig. 1.

Step (1), using a physiological saline solution to prepare a high molecular weight of 10-40 mg / mL (SLG100, Mw = 200,000 - 300,000 g / mol) and a low molecular weight (SLG20, Mw = 75,000 - 220,000 g / mol, FMC Biopolymer, USA The alginate solution of Philadelphia) was stirred overnight at 4 ° C with a magnetic stirrer and filtered through a filter having a pore size of 0.22 μm. Take high and low molecular weight alginate solution in a ratio of 1:3 and mix well;

Step (2)-a, 3.75 to 7.5 mg of celecoxib powder (LC laboratory, LC Laboratories, Massachusetts, USA) is weighed per ml of alginate solution and mixed;

Step (2)-b, using a probe type ultrasonic instrument (QSONICA, USA) to ultrasonically 60 seconds for 60 seconds, so that the celecoxib powder is uniformly dispersed in the alginate solution to obtain a mixed system;

Step (3), 500-1000 μg of PD-1 blocking antibody per ml of the alginate solution (BioXCell, USA) is added to the mixed system obtained in the step (2)-b, and the shaker is shaken and thoroughly mixed;

Step (4)-a, weigh calcium sulfate solid powder (CaSO ₄ , Sinopharm Chemical Reagent Co., Ltd.), mix it with purified water at a mass to volume ratio of 0.21 g/ml, and autoclave to obtain a calcium sulfate suspension ( If aggregation occurs during use, coarse particles are generated, which can be ultrasonicated by an ultrasonic machine to form a uniform suspension);

Step (4)-b, sucking the mixture obtained in the step (3) with a syringe with a screw;

Step (4)-c, adding another suspension of CaSO _{4 to} the syringe with screw, adding 40 μL of CaSO ₄ suspension per mL of alginate solution;

Step (5), through the joint and the screw, the two syringes are tightly connected to eliminate excess air as much as possible;

Step (6), quickly push the syringe 20 times each other;

Step (7), standing for 1 minute, the antitumor preparation of the present invention in the form of a hydrogel is completed. The syringe can be injected by unscrewing the syringe containing the hydrogel and attaching the injection needle. Or can The prepared antitumor preparation was stored at 4 °C.

Example 2: In vivo sustained release effect and drug effect prolongation effect of antitumor preparation provided by the invention

1. The antitumor preparation was prepared in the same manner as in Example 1 so that the anti-tumor preparation contained 500 ug of PD-1 blocking antibody and 3125 ug of celecoxib per ml of the antitumor preparation.

C57BL/6 mice were purchased from Beijing Huakangkang Biotechnology Co., Ltd., each weighing about 25 grams. B16-F10 melanoma cells were purchased from the American Type Culture Collection.

2. Construction of a mouse B16 melanoma model.

B16-F10 melanoma cells are recognized as the most invasive tumor cells. After each subcutaneous injection of 2.5×10 ⁴ B16-F10 melanoma cells in C57BL/6 mice, the protoplasts can be produced after 1 week. A melanoma that then grows rapidly and causes the mouse to die. The C57BL/6 mouse melanoma model is a mature animal model widely used to evaluate the anti-tumor effect of drugs. The inventors will subcutaneously inject B16-F10 melanoma cells purchased from the ATCC (American Type Culture Collection) with C57BL/6 male mice weighing about 25 grams at 6-8 weeks of age (purchased from Beijing Huakangkang Biotechnology). Technology Co., Ltd.), about 7-10 days later, melanoma formation can be seen at the site of injection of cells.

3. The anti-tumor preparation provided by the invention has an effect of sustained release effect and prolonged effect of the drug in vivo.

3.1 After one week of inoculation of melanoma cells in C57BL/6 mice, the following three groups of mice were set up:

In addition to the oral celecoxib group, mouse serum and tumor tissue were taken 24 hours after administration, the other two Mouse sera and tumor tissues were taken on the first day, the third day, the 7th day and the 14th day after administration, and the mouse serum was detected by liquid chromatography-mass spectrometry (HPLC-MS). The concentration of celecoxib in the homogenate of the tumor tissue was 3 in each group at each time point.

Figure 2A shows the concentration of celecoxib in the serum of each group of mice. Figure 2B shows the celecoxib content in tumor tissues of each group of mice. It can be seen that the serum concentration of celecoxib in the experimental group injected with the antitumor preparation of the present invention is significantly higher than that of the control group at least within one week; the celecoxib content in the tumor tissue is significantly higher at least within two weeks. In the control group.

3.2 After one week of inoculation of melanoma cells in C57BL/6 mice, the following 4 groups of mice were set up:

After treatment of each group of mice as in the above table, inguinal lymph nodes were sampled on the first day, the third day, the 7th day and the 14th day after treatment, and the cytotoxic T lymphocytes were detected by flow cytometry. The number of cells (double positive for CD8 and IFN-γ) and regulatory T lymphocytes (double positive for CD4 and FoxP3) were calculated and the number of experimental animals per group at each time point was 3. The activation of cytotoxic T cells (CTL) and the dynamic changes of inhibition of regulatory T cells (Treg) were detected by flow cytometry to reflect the sustained release effect of PD-1 blocking antibody.

Figure 2C shows cytotoxic T lymphocytes (CD8 and IFN-γ double positive) in each group of mice. The ratio of regulatory T lymphocytes (CD4 and FoxP3 double positive). The results showed that the experimental group in which the antitumor preparation group of the present invention was injected was at least two weeks, and the effect of prolonging the effect was significantly higher than that of the respective control groups.

The anti-tumor preparation provided by the present invention is described by the data of the present embodiment, and the effective drug concentration and effect for a long time are maintained while reducing the dose.

3.3 After one week of inoculation of melanoma cells in C57BL/6 mice, the following 4 groups of mice were set up:

Figure 2D shows the percent survival of each group of mice. It is indicated that the administration of the antitumor agent group of the present invention (formed by simultaneously encapsulating the PD-1 antibody and celecoxib into the alginate hydrogel) gives an active ingredient group which is coated into the alginate hydrogel in a stepwise manner ( That is, administration of an alginate hydrogel containing only the PD-1 antibody, and simultaneous administration of an alginate hydrogel containing only celecoxib, resulted in a significantly increased percentage of mouse survival. This indicates that the antitumor preparation of the present invention does not simply use the three components of celecoxib, PD-1 antibody and alginate hydrogel, and celecoxib and PD-1 antibodies must be simultaneously embedded in alginic acid. In a saline gel (i.e., the antitumor preparation of the present invention), a superior antitumor effect can be produced.

Example 3 The antitumor preparation of the present invention can promote the production of active CD8 T cells

1. The antitumor preparation was prepared in the same manner as in Example 1 so that the anti-tumor preparation contained 500 ug of PD-1 blocking antibody and 3125 ug of celecoxib per ml of the antitumor preparation.

2. The same mouse melanoma model as in Example 2 was constructed.

After one week of inoculation of melanoma cells in C57BL/6 mice, the following 4 groups of mice were set up:

After treatment of each group of mice as in the above table, the percentage of active CD8 cells in the tumor microenvironment was detected by flow cytometry one week later.

Figures 3A and 3B show the results of detection of CD8 T cells that are effective in killing tumor cells in the tumor microenvironment. It was shown that administration of the antitumor agent group of the present invention (formed by simultaneously encapsulating PD-1 antibody and celecoxib into alginate hydrogel) can greatly promote the production of active CD8 T cells. This indicates that the antitumor preparation of the present invention is not only used as a drug sustained release carrier in the treatment of tumors, but also the volume and space occupied by the body is a place for promoting the production of antitumor immune cells. Both celecoxib and PD-1 antibody are present in the "site" and they are activated The synergistic effect of the plaque cells can be produced.

Example 4 The anti-tumor preparation provided by the present invention has reduced toxic side effects.

1. The antitumor preparation was prepared in the same manner as in Example 1 so that the anti-tumor preparation contained 500 ug of PD-1 blocking antibody and 3125 ug of celecoxib per ml of the antitumor preparation.

2. The same mouse melanoma model as in Example 2 was constructed.

3. To test the toxic side effects of the antitumor preparation provided by the present invention on the body.

One week after inoculation of melanoma cells in C57BL/6 mice, the following groups of mice were set up:

After treating each group of mice as in the above table, the mice were further cultured for one week, the mice were sacrificed, the subcutaneous tumors were removed, total RNA was extracted according to the RNA extraction reagent, and COX-2 was analyzed by real-time PCR. The relative expression level of the messenger RNA of the four genes IL-1β and IL-6. The results are shown in Figures 4A-4C, which is the expression level of the relative messenger RNA of the COX-2 gene. Figure 4B is the expression level of relative messenger RNA of the IL-1β gene. Figure 4C shows the level of expression of the relative messenger RNA of the IL-6 gene. It can be seen that the experimental group injecting the antitumor preparation group of the present invention has a significantly reduced expression of inflammatory factors as compared with the alginate hydrogel group injected with the PD-1 antibody. It is shown that the antitumor preparation provided by the present invention has a significantly reduced toxic side effect on the body.

Example 5: The antitumor preparation provided by the invention is used for inhibiting tumor growth of B16 melanoma

1. The antitumor preparation was prepared in the same manner as in Example 1 so that the anti-tumor preparation contained 500 ug of PD-1 blocking antibody and 3125 ug of celecoxib per ml of the antitumor preparation.

2. The same mouse melanoma model as in Example 2 was constructed.

3. The antitumor preparation provided by the present invention is used for the anti-B16 melanoma tumor growth effect.

One week after inoculation of melanoma cells in C57BL/6 mice, the following groups of mice were set up:

On the 0th, 10th, 13th, 17th, 20th, and 22nd day after treatment of each group of mice in the above table, the long diameter (length) and short diameter (width) of melanoma were measured with a vernier caliper, and the appearance of the tumor was photographed.

Figure 5A shows a picture of the tumor appearance of mice of each group on days 0, 10, 13, 17, 20, and 22. Figure 5B shows the size changes of tumors of mice of each group on days 0, 10, 13, 17, 20, and 22.

As can be seen from Figs. 5A to 5B, the experimental group in which the antitumor preparation of the present invention was subcutaneously injected was able to inhibit the growth of malignant melanoma in situ tumors as compared with the control group. The anti-tumor effect of the anti-tumor preparation of the invention is better than that of the alginate hydrogel alone occluding celecoxib or PD-1 blocking antibody, and is also superior to the saline mixed celecoxib and PD-1 blocking. The antibody group, that is, all the components in the antitumor preparation of the present invention, is necessary for obtaining the antitumor effect of the present application, and an effect that the PD-1 blocking antibody alone or celecoxib is not administered alone is obtained.

Example 6: The anti-tumor preparation provided by the invention is used for controlling the metastasis of 4T1 malignant breast cancer

1. The antitumor preparation was prepared in the same manner as in Example 1 so that the anti-tumor preparation contained 500 ug of PD-1 blocking antibody and 3125 ug of celecoxib per ml of the antitumor preparation.

Balb/c mice were purchased from the Medical Laboratory Animal Center of Wuhan University, each weighing about 25 grams. Mouse breast cancer cells were purchased from the American Type Culture Collection.

2. Construct a 4T1 malignant breast cancer metastasis model.

The 4T1 breast cancer metastasis model is the most commonly used experimental model for studying malignant tumor metastasis. The inventors injected 4T1 mouse breast cancer cells (purchased from the American Type Culture Collection) in vitro into the second breast site on the right side of Balb/c mice with a female body weight of about 25 grams at 6-8 weeks old. The amount of cells injected by only one mouse was 1 × 10 ⁶ . A week later, it was seen that the breast site was injected with obvious breast tumors, and after 4-5 weeks, obvious lung metastases were observed. By observing the appearance of the lungs in mice after invasion by cancer cells, and counting the number of lung metastases, qualitative and quantitative analysis of cancer cell metastasis can be performed.

3. The antitumor preparation provided by the present invention inhibits the effect of tumor metastasis.

One week after the above Balb/c mice were inoculated with 4T1 breast cancer cells, the following groups of mice were set up:

Twenty-five days after the treatment of each group of mice in the above table (day 32 after inoculation of cancer cells), the mice in each group were sacrificed by cervical dislocation, and the whole lung was taken, and the number of tumors on the surface of the lungs was counted from the lungs. The tissue was surgically stripped of the metastatic tumor, and the whole lung and the exfoliated metastatic tumor of the mouse were photographed.

Figure 6A shows a picture of a pulmonary metastases from each group of mice. Figure 6B shows the number of lung metastases in each group of mice.

As can be seen from Figs. 6A to 6B, the injection of the antitumor preparation group of the present invention showed an excellent effect of inhibiting tumor metastasis as compared with the control group, and the number of lung metastases was suppressed by 70%. Compared with the injection group of the present invention, the number of metastases was significantly increased significantly in the control group lacking any one component, and the effect of inhibiting tumor metastasis was far less than the present invention, indicating all the components in the antitumor preparation of the present invention. It is necessary to obtain the anti-tumor effect desired by the present application.

Example 7 The antitumor preparation of the present invention is equally effective for melanoma and other tumor types other than breast cancer

1. Experimental materials and reagents

Different tumor-bearing mice include: kidney cancer mice, gastric cancer mice, bladder cancer mice, leukemia mice, head and neck cancer mice, intestinal cancer mice, brain tumor mice, respectively, purchased from Wuhan University Medical Laboratory Animal Center, Weighing about 25 grams.

2, the experimental steps

2.1. The antitumor preparation was prepared in the same manner as in Example 1 so that the anti-tumor preparation contained 500 ug of PD-1 blocking antibody and 3125 ug of celecoxib per ml of the antitumor preparation.

Construction of tumor-bearing mice: The method of constructing a tumor-bearing mouse model in this example is a conventional method in the art, and these tumor-bearing mouse models are mature animal models widely used to evaluate the anti-tumor effect of drugs.

2.2. Experiment with various tumor-bearing mice as follows

The experimental results show that the kidney cancer mice, the gastric cancer mice, the bladder cancer mice, the leukemia mice, the head and neck cancer mice, the intestinal cancer mice, and the brain tumor mice to which the antitumor preparation of the present invention is administered are compared with the respective control groups. Mice, which exhibit excellent tumor growth inhibition and reduced toxic side effects.

Claims (15)

An antitumor preparation characterized by being an antitumor preparation in the form of a hydrogel formed by blocking an aqueous solution of an antibody and celecoxib with an active ingredient PD-1, the antitumor preparation being an injection preparation. The antitumor preparation according to claim 1, wherein the antitumor preparation further comprises a crosslinking agent for crosslinking the aqueous alginate solution. The antitumor preparation according to claim 2, wherein the alginate has a molecular weight of 75,000 to 300,000 g, and the crosslinking agent is a divalent metal cation. The antitumor preparation according to claim 1, wherein the antitumor preparation is a unit preparation. The antitumor preparation according to claim 4, wherein the unit preparation contains a therapeutically effective amount of 20-30 mg of PD-1 blocking antibody and 130-200 mg of celecoxib. The antitumor preparation according to any one of claims 1 to 5, wherein the antitumor preparation is for melanoma, breast cancer, kidney cancer, stomach cancer, bladder cancer, leukemia, head and neck cancer, and intestinal cancer. Or an antitumor agent for brain tumors. A method for preparing an antitumor preparation, comprising: Dissolving alginate in physiological saline to prepare an alginate solution; Adding the active ingredient PD-1 blocking antibody and celecoxib to the alginate solution, and dispersing it in the alginate solution to obtain a sea alginate solution mixture; The alginate solution mixture is crosslinked to form an antitumor preparation in the form of a hydrogel, which is an injection preparation. The method according to claim 7, wherein the alginate solution mixture is crosslinked in the presence of a crosslinking agent. The method of claim 8 wherein said alginate has a molecular weight of from 75,000 to 300,000 g and said crosslinking agent is a divalent metal cation. The method according to any one of claims 7 to 9, wherein the antitumor agent is for melanoma, breast cancer, kidney cancer, stomach cancer, bladder cancer, leukemia, head and neck cancer, intestinal cancer or brain Antitumor preparation of tumor. A method for treating or preventing a tumor, characterized by comprising a tumor The process of administering an antitumor preparation to an individual having a tendency to develop a tumor in the form of a hydrogel formed by an aqueous solution of an alginate dispersed with the active ingredient PD-1 blocking antibody and celecoxib A tumor preparation, and the anti-tumor preparation is an injection preparation. The method according to claim 11, wherein said antitumor preparation further comprises a crosslinking agent for crosslinking said aqueous alginate solution. The method according to claim 12, wherein the alginate has a molecular weight of from 75,000 to 300,000 g, and the crosslinking agent is a divalent metal cation. The method according to claim 11, wherein the antitumor agent is a unit preparation. The method according to claim 14, wherein the unit preparation contains a therapeutically effective amount of 20-30 mg of PD-1 blocking antibody and 130-200 mg of celecoxib.

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