WO2021042777A1

WO2021042777A1 - Multi-component gel sustained-release pharmaceutical composition for treatment of tumors

Info

Publication number: WO2021042777A1
Application number: PCT/CN2020/093377
Authority: WO
Inventors: 刘庄; 金秋桐; 赵琪
Original assignee: 苏州百迈生物医药有限公司
Priority date: 2019-09-03
Filing date: 2020-05-29
Publication date: 2021-03-11
Also published as: CN112516073A

Abstract

Disclosed in the present invention is a multi-component gel sustained-release pharmaceutical composition for the treatment of tumors, characterized by comprising active components and auxiliary materials, wherein the active components comprise a chemotherapy drug, an immune adjuvant or an immune checkpoint inhibitor causing the immunogenic death of tumor cells; the auxiliary materials comprise a gel-forming matrix and a buffer liquid; the gel-forming matrix comprises one or more of serum albumin, fibrinogen, sodium alginate, calcium alginate, collagen, chitosan, butyl glycan, gelatin, starch, hyaluronic acid, sodium carboxymethyl cellulose, sodium glycerophosphate, cyanoacrylate, acrylamide, polyoxyethylene, polyethylene glycol, polyvinyl alcohol, poloxamer, polycaprolactone, and steroid compounds. A method for preparing the sustained-release gel is simple, and the gel prepared can slowly release a drug, which effectively enhances the synergy of the drug, thereby having good application prospects in the field of resisting tumors.

Description

Multi-component gel sustained-release pharmaceutical composition for treating tumor

Technical field

This patent relates to the field of tumor drugs, in particular to a multi-component pharmaceutical composition for treating tumors.

Background technique

As one of the major malignant diseases in the world, cancer is a serious threat to human health. According to its different nature and different sites of invasion, tumors respond differently to various treatments. Therefore, most patients need comprehensive treatment. The most common treatment is to remove the tumor with surgery or to fight cancer cells with chemotherapy. However, surgery cannot guarantee the complete removal of tumor lesions, especially in the vicinity of key organs that cannot be resected. The recurrence, invasion and secondary metastasis of residual tumor tissues will pose a great threat to the life and health of patients. As one of the main treatment methods, chemotherapy has poor selectivity. While achieving the therapeutic effect, different degrees of toxic and side effects often occur. Even in some cases, chemotherapy drugs can destroy the body's normal immune system and cause immune suppression.

However, there are also some chemotherapy drugs that can cause the immunogenic death of tumor cells, thereby activating the body's immune response to tumors, inhibiting tumor growth and recurrence, such as anthracyclines (such as adriamycin, epirubicin) , Mitoxantrone, etc.), oxaliplatin, cyclophosphamide, bortezomib, gemcitabine, pentafluorouracil and toxins (such as maytansine).

The struggle between the immune system and tumor cells is a dynamic process of long-term game. Immune cells can detect and kill tumor cells, but they may also be "deceived" by tumor cells, making T cells unable to recognize tumor cells. In order to survive and grow, tumor cells can adopt different strategies to suppress the body's immune system and not kill cells normally, so that they can survive the various stages of the anti-tumor immune response. The above-mentioned characteristics of tumor cells are also called immune escape. Although the tumor itself has an immune escape mechanism to protect itself, immune checkpoint inhibitors such as anti-CTLA4, anti-PDL1, and anti-PD1 can remove the camouflage of tumor cells, prompting T cells to recognize tumor cells and kill tumor cells. In addition, immune adjuvants such as imiquimod, CpG oligonucleotides, monophosphoryl lipid A and requimod and other Toll-like receptor agonists can help antigen-presenting cells present antigens and enhance the body’s immune response. Stimulate the immune system more effectively.

On the basis of the immunogenic death of tumor cells caused by chemotherapeutics, the combined use of immune adjuvants enhances the immune response, and immune checkpoint inhibitors restore the body's normal anti-tumor response, which can achieve better therapeutic effects-chemotherapy can kill first Some cancer cells expose related antigens to improve the sensitivity of tumors to immunotherapy; immune adjuvants can better present tumor-related antigens produced by chemotherapy to T cells, thereby amplifying the immune response; checkpoint inhibitor antibodies can inhibit tumors The immune escape allows immune cells to better kill tumor cells.

However, chemotherapeutics, immune adjuvants, and immune checkpoint inhibitors all have certain side effects. The current conventional medication methods, such as intravenous injection, cannot fully exert the efficacy of tumor treatment drugs and reduce side effects. The continuous development of new original drugs for the treatment of tumors is a very valuable research direction, but how to make the existing drugs for the treatment of tumors be fully effective, kill tumors more effectively, inhibit tumor metastasis and recurrence, and effectively reduce side effects at the same time. Very valuable research direction. How to make the existing chemotherapeutics, immune adjuvants and immune checkpoint inhibitors that can cause the immunogenic death of tumor cells to more effectively kill tumors and inhibit tumor metastasis and recurrence is a very challenging research direction.

Summary of the invention

The purpose of the present invention is to provide a multi-component gel sustained-release pharmaceutical composition for the treatment of tumors and a preparation method. The sustained-release gel of the pharmaceutical composition contains chemotherapeutic drugs and immune agents that cause the immunogenic death of tumor cells. Adjuvants and immune checkpoint inhibitors can kill tumors more effectively, and inhibit tumor metastasis and recurrence. The slow-release gel has a simple preparation method, and the gel can slowly release the drug, which effectively enhances the principle of drug synergy , Has a good application prospect in the field of anti-tumor.

In order to achieve the above objectives, the present invention provides the following technical solutions:

A multi-component gel sustained-release pharmaceutical composition for treating tumors, which is characterized in that it comprises active components and adjuvants, the active components including chemotherapeutics, or immune adjuvants, or immune adjuvants that cause immunogenic death of tumor cells, or Immune checkpoint inhibitors;

The auxiliary material includes a gel-forming matrix and a buffer;

The gel-forming matrix includes serum albumin, fibrinogen, sodium alginate, calcium alginate, collagen, chitosan, butyltosan, gelatin, starch, hyaluronic acid, sodium carboxymethylcellulose, sodium glycerophosphate , Cyanoacrylate, acrylamide, polyoxyethylene, polyethylene glycol, polyvinyl alcohol, poloxamer, polyvinyl lactone, one or more of steroids.

As a preferred solution of the multi-component gel sustained-release pharmaceutical composition for treating tumors of the present invention: the gel-forming matrix includes: the first solution has a concentration of 1-400 mg per milliliter of fibrinogen solution, and the second The solution is a thrombin solution with a concentration of 5-200IU per milliliter. Active ingredients are added to the first solution or the second solution, and the first solution and the second solution are mixed to form a gel sustained-release pharmaceutical composition;

Or the gel-forming matrix includes, the first solution is a dexamethasone sodium phosphate solution with a concentration of 5-200 mg/ml, and the second solution is a calcium ion buffer solution with a calcium ion concentration of 0.4-16 mg/ml. The active ingredient is added to the first solution or the second solution, and the first solution and the second solution are mixed to form a gel sustained-release pharmaceutical composition.

As a preferred solution of the multi-component gel sustained-release pharmaceutical composition for treating tumors of the present invention: the fibrinogen solution of the first solution is 10-200 mg/ml, and the concentration of the thrombin solution of the second solution is It is 20-100IU per milliliter.

As a preferred solution of the multi-component gel sustained-release pharmaceutical composition for treating tumors of the present invention: the buffer is calcium ion buffer or sodium ion buffer, and the ion concentration is 0.4-16 mg/ml; or The buffer is acetate buffer or hydrochloric acid buffer, with a concentration of 0.4-40 mg per milliliter.

As a preferred solution of the multi-component gel sustained-release pharmaceutical composition for the treatment of tumors of the present invention: the multi-component gel sustained-release pharmaceutical composition is a multi-component gel sustained-release drug for the treatment of breast cancer tumors The composition, the gel-forming matrix comprises a first solution and a second solution, the first solution is a fibrinogen solution dissolved in deionized water with a final concentration of 10-200 mg/ml;

The second solution is a calcium ion buffer solution with a final concentration of 20-100IU/mL thrombin solution;

Add doxorubicin, imiquimod, and a-PDL1 to the first solution or the second solution, mix well, and mix the mixed first solution and the second solution to form a gel sustained-release pharmaceutical composition.

As a preferred solution of the multi-component gel sustained-release pharmaceutical composition for treating tumors of the present invention: the gel-forming matrix is a mixture of fibrinogen and thrombin to form a gel, and the first solution has a concentration of 10-200 mg For each milliliter of fibrinogen solution, the second solution is thrombin with a final concentration of 20-100 IU/mL, and the immune adjuvant emulsion is added to the first solution or the second solution and mixed. The immune adjuvant emulsion is imiquimol For special emulsion, add imiquimod R837 and deionized water to a ball milling tank for ball milling for 2-3 hours to obtain a uniformly dispersed imiquimod emulsion. The imiquimod particles have a particle size of 20-300 microns. The first solution containing the immune adjuvant emulsion is mixed with the second solution to form a gel.

As a preferred solution of the multi-component gel sustained-release pharmaceutical composition for treating tumors of the present invention: the gel-forming matrix is mixed with a solution of dexamethasone sodium phosphate and calcium ion to form a gel, and the first solution contains adriamycin. DOX, imiquimod R837, dexamethasone sodium phosphate solution of antibody a-PDL1, the concentration of dexamethasone sodium phosphate solution is 5-200 mg/ml, the second solution is calcium ion buffer, the calcium ion solution The concentration is 0.4-16 mg per milliliter; the first solution and the second solution are mixed to form a gel.

As a preferred solution of the multi-component gel sustained-release pharmaceutical composition for treating tumors of the present invention, it is characterized in that the dosage form of the pharmaceutical composition is ointment, suppository, spray, and solution.

The present invention provides a multi-component gel sustained-release pharmaceutical composition for treating tumors, including active components and adjuvants. The active components include the following raw materials: chemotherapeutics that cause immunogenic death of tumor cells, immune adjuvants , Immune checkpoint inhibitors.

Preferably, the auxiliary materials include the following raw materials, a gel-forming matrix, and a buffer.

Preferably, the chemotherapeutics that cause the immunogenic death of tumor cells are anthracyclines (such as doxorubicin, epirubicin, mitoxantrone, etc.), oxaliplatin, cyclophosphamide, bortezomib One or more of, gemcitabine, pentafluorouracil and toxins (such as maytansine) can be used clinically and kill tumor cells to turn dead tumor cells into related antigens, activate anti-tumor immune responses, and can target different The chemotherapeutics used for the type of tumor are selected in a targeted manner; the immune adjuvant is imiquimod, CpG oligonucleotides, monophosphoryl lipid A and requimod and other Toll-like receptor agonists One or several types that can help antigen-presenting cells to present antigens, so the immune adjuvant can better present tumor-associated antigens produced by chemotherapy to T cells, thereby amplifying the immune response; the checkpoint inhibitor antibody is anti-CTLA4 One or more of anti-PDL1 and anti-PD1 can inhibit the immune escape of tumors, so that immune cells can better kill tumor cells.

Preferably, the matrix includes serum albumin, fibrinogen, sodium alginate, calcium alginate, collagen, chitosan, butyltosan, gelatin, starch, hyaluronic acid, sodium carboxymethylcellulose, glycerophosphate One or more of sodium, cyanoacrylate, acrylamide, polyoxyethylene, polyethylene glycol, polyvinyl alcohol, poloxamer, polyvinyl lactone, steroids, the concentration is 0.1-40wt% .

The matrix is the excipient and carrier of the drug, and the matrix can be mixed to form a gel or mixed with an ionic solution to form a gel.

Preferably, the buffer is a calcium ion buffer or a sodium ion buffer, and the ion concentration is 0.4-16 mg/mL.

Preferably, the buffer is acetate buffer or hydrochloric acid buffer with a concentration of 0.4-40 mg/mL.

Preferably, the dosage form of the pharmaceutical composition is ointment, gel, suppository, spray or solution.

The present invention provides the effect of the pharmaceutical composition for treating tumors as described in the above scheme in the preparation of drugs for treating tumors.

Taking the gelation of fibrinogen and thrombin as an example, the present invention provides a drug sustained-release gel for the treatment of tumors. Each 1 mL includes the following raw materials: 5 mg of chemotherapeutics, 1.87 mg of immune adjuvants, and 0.5 of immune checkpoint inhibitors. mg, fibrinogen 10-200mg, thrombin 20-100IU, purified water 0.5-0.9mL and the remaining buffer.

Taking the gelation of fibrinogen and thrombin as an example, the present invention provides a method for preparing the sustained-release gel for treating tumors as described in the above scheme, which includes the following steps.

1) Mix fibrinogen and purified water to swell to obtain solution A;

2) Mix and dissolve thrombin and buffer to obtain solution B;

3) Stir and mix the chemotherapeutics, immune adjuvants, and immune checkpoint inhibitors that cause immunogenic death with the solution A obtained in step 1) or with the solution B obtained in step 2);

4) Load the new solution A and solution B into the double-drug mixer respectively, mix them and inject them into the affected area at room temperature to form a sustained-release gel in situ.

Preferably, such as the gel forming method by adding salt, a polymer solution and a salt buffer can be mixed to form a gel.

Preferably, the polymer solution can be sodium alginate, calcium alginate, collagen, chitosan, butyl glycan, gelatin, starch, hyaluronic acid, sodium carboxymethyl cellulose, sodium glycerophosphate, cyanoacrylate, Acrylamide, polyoxyethylene, polyethylene glycol, polyvinyl alcohol, poloxamer, polyvinyl lactone, steroids, etc., with a concentration of 0.1-40 wt%.

Preferably, the salt buffer solution may be a calcium ion buffer solution or a sodium ion buffer solution, and the ion concentration is 0.4-16 mg/mL. Preferably, the salt buffer may be acetate buffer or hydrochloric acid buffer, with a concentration of 0.4-40 mg/mL. Preferably, the polymer solution may be dexamethasone sodium phosphate solution.

Preferably, such as a gel forming method that occurs a chemical reaction-fibrinogen and thrombin can be selected for gel forming. Thrombin cleaves fibrinogen to release fibrin A peptide and B peptide to form fibrin monomers. Fibrin monomers can be polymerized into unstable soluble fibrin fibers by hydrogen bonding and electrostatic attraction. With the participation of calcium ions, a gel mass is further formed.

In this system, such a sustained-release gel is used for the second treatment after surgery or directly for the treatment of tumors.

In addition, it can also be used as ointment, suppository, spray (mixed to form a gel after spraying), solution (mixed in situ to form a gel) and other dosage forms to achieve similar effects to gels.

This multi-component sustained-release gel mainly contains four components, which are the auxiliary materials to form the gel, the chemotherapeutics that cause immunogenic death, the immune adjuvant, and the immune checkpoint inhibitor.

In this patent, the gel, as a drug carrier, can be used for subcutaneous local injection or spraying, etc., to maximize the concentration of the drug in the tumor tissue area. In this system, the gel is loaded with chemotherapeutics, immune adjuvants, and immune checkpoint inhibitors at the same time, which can enhance the immune response in situ, improve the therapeutic effect, and make immune cells better kill tumor cells. At the same time, the active ingredients in the gel will be slowly released from the gel over time, reducing toxic and side effects, and greatly reducing the number of administrations and medication cycles.

There are several ways to form a gel: 1. Change the temperature. Many substances dissolve in hot water, and their solubility decreases when they are cooled, and they can form gels when they are connected to each other by collisions. 2. Add non-solvent. Some substances can form a gel by adding a suitable precipitant, for example, if ethanol is added to the gel solution, it can form a gel. 3. Add salt. Adding an appropriate electrolyte solution to a sol with greater hydrophilicity and asymmetric ionic shape can form a gel. For example, adding KCl electrolyte to a ferric hydroxide sol can solidify the system into a gel. 4. A chemical reaction occurs. When a chemical reaction occurs to generate insoluble matter, if a large number of small crystal grains are generated at the same time and the shape of the crystal grains is asymmetric, it is beneficial to form a gel, such as a silica gel. Some macromolecular solutions can also form gels during the reaction, such as protein deformation, from spherical molecules to fibrous molecules.

Although there are many gel forming mechanisms, all kinds of gel forming mechanisms have more or less shortcomings or side effects when applied to drug treatment. The research and development can effectively improve the efficacy of drugs, enhance synergistic effects, and have side effects on the body. A small, easy-to-use, and easy-to-produce gel composition for treating tumor drugs is a technical problem that is worth solving.

Based on the above several gel forming principles, we can design a variety of multi-component gels, which can effectively improve the efficacy of drugs, enhance synergistic effects, have less side effects on the body, and are easy for doctors to use and easy to produce. When the different components are mixed together, a gel can be quickly formed in situ, and the above-mentioned chemotherapeutics that cause immunogenic death, immune adjuvants, and immune checkpoint inhibitors as the active ingredients can be loaded into the gel. It kills tumors more effectively, and inhibits tumor metastasis and recurrence.

The method of the present invention can form a gel in situ on the tumor affected area. First, the chemotherapeutic agent kills the tumor cells and causes their immunogenic death, and activates the tumor-specific immune response; secondly, the immune adjuvant enhances the antigen-presenting cell The ability to further enhance the corresponding immune response; finally, the use of immune checkpoint inhibitors makes immunotherapy more effective in killing tumors, thereby inhibiting tumor metastasis and recurrence.

The creativity of this scheme lies in the use of gelled raw materials to wrap chemotherapeutics, immune adjuvants and immune checkpoint inhibitors, gelatinize in situ and slowly release these components, simultaneously induce tumor-specific immune responses and enhance the immune effect, which is beneficial to patients. The damage is low, and it can also assist surgery for large-area preventive spraying of potential target areas after surgery, further inhibiting tumor metastasis and recurrence, and greatly contributing to the synergistic effect of multi-component drugs.

The multi-component gel described in this patent has a fast gelling speed, especially the combination of fibrinogen and thrombin. The concentration can be adjusted, and the gel can be gelled in tens of seconds or a minute, and it has a certain degree of adhesion and is easy to It adheres to the body organs, can stay at the lesion site for a long time, and has good biological safety. In addition, the multi-component gel described in this patent can be combined with a variety of tumor treatment drugs to treat a variety of diseases. The chemotherapeutic drugs used can be selected for different tumor types, and then the chemotherapeutic drugs can be combined with the multi-component Use a combination of gels.

Description of the drawings

Figure 1 is a record table of the gel formation of the gel in Example 4.

Figure 2 is the release curve of the drug in the gel complex in Example 4.

Figure 3 is a scanning electron micrograph of the gel composite in Example 6.

Figure 4 shows the rheological mechanical properties of the gel composite in Example 7. The rheological mechanical properties of the composite at different concentrations, where G'is the storage modulus and G" is the loss modulus.

Figure 5 is the therapeutic effect (survival rate and tumor growth curve) of the gel complex in Example 8, Figure 5-1 is the tumor signal fluorescence graph in mice, and Figure 5-2 is the survival curve graph of mice. The first group was intratumoral injection of normal saline, the second group was intratumoral injection of doxorubicin and imiquimod gel complex, and the third group was intratumoral injection of doxorubicin and imiquimod PDL1 antibody gel complex.

Figure 6 is a scanning electron micrograph of the gel composite in Example 9.

Fig. 7 is a characterization of the rheological mechanical properties of the gel composite in Example 10. The rheological mechanical properties of the composite, where G'is the storage modulus and G" is the loss modulus.

Figure 8 is the release curve of the drug in the gel complex in Example 11.

detailed description

The following is a detailed description of the gel sustained-release agent and preparation method of a sustained-release chemotherapy and immune adjuvant combined with immune checkpoint inhibitor provided by the present invention with reference to the examples and Figures 1 to 8, but they cannot be understood as a reference to the present invention. Limitation of the scope of protection of the invention.

Example 1

Preparation of immune adjuvant hydrochloride (taking imiquimod as an example):

Weigh 50-100mg imiquimod in a 50mL serum bottle, add 1 mole per liter of dilute hydrochloric acid to fully dissolve imiquimod until it is colorless and transparent, dilute with deionized water to make the final concentration of imiquimod 0.1-10 Milligrams per milliliter, lyophilized to obtain imiquimod hydrochloride lyophilized powder. The detailed process is as follows.

Preparation of immune adjuvant hydrochloride (taking imiquimod as an example). The immune adjuvant imiquimod is a hydrophobic small molecule drug, which is completely insoluble in water and soluble in organic solvent dimethyl sulfoxide. However, the injections for clinical treatment of tumors are aqueous solutions or physiological solutions, so water-insoluble imiquimod cannot be used directly as injections. Imiquimod needs to be hydrochlorized and used as a lyophilized powder before further use.

First, in this example, a large number of experiments were performed on the solubility, dispersibility and stability of imiquimod lyophilized powder acidified with different acidic buffers, and it was found that hydrochloric acid and acetic acid were suitable acidic buffers. Table 1 shows the difference in water solubility, water dispersibility and stability of imiquimod freeze-dried powder after acidification with different acid buffers. It can be seen from the table that after acidification with hydrochloric acid or lactic acid, imiquimod freeze-dried powder has good water solubility and good stability. The lyophilized powder of imiquimod after acidification with acetic acid, oxalic acid or carbonic acid is unstable in water.

酸性缓冲液Acid buffer	水溶性Water soluble	水分散性Water dispersibility	稳定性stability	可注射性Injectability
盐酸hydrochloric acid	溶Melt	均匀Evenly	稳定stable	√√
乳酸Lactic acid	溶Melt	均匀Evenly	稳定stable	√√
醋酸acetic acid	不溶Insoluble	不均匀Uneven	不稳定Unstable	×X
草酸oxalic acid	不溶Insoluble	不均匀Uneven	不稳定Unstable	×X
碳酸Carbonic acid	不溶Insoluble	不均匀Uneven	不稳定Unstable	×X

This table reflects the differences in water solubility, water dispersibility and stability of imiquimod freeze-dried powder after acidification with different acid buffers.

Then a series of imiquimod and hydrochloric acid buffer ratios were investigated, and the best ratio of imiquimod and hydrochloric acid buffer was determined. The following table shows the difference in water solubility and acidic residue of imiquimod freeze-dried powder after acidification with different concentrations of hydrochloric acid buffer. It can be seen from the table below that when the concentration of hydrochloric acid is lower than 0.1 mole per liter, the acidified imiquimod freeze-dried powder has poor water solubility. When the concentration of hydrochloric acid is between 0.1 and 2 moles per liter, the acidified imiquimod lyophilized powder has good water solubility and stability. The concentration of hydrochloric acid is between 2 and 12 moles per liter. Imiquimod freeze-dried powder after strong acid acidification has high residual hydrochloric acid. The aqueous solution is acidic and cannot be injected into the human body, which will cause obvious toxic and side effects.

This table reflects the differences in water solubility and acidic residues of imiquimod freeze-dried powder after acidification with different concentrations of hydrochloric acid buffer

The final preparation process is as follows: weigh 50-100mg of imiquimod in a 50mL serum bottle, add 1M dilute hydrochloric acid to fully dissolve imiquimod until it is colorless and transparent, and dilute with deionized water to make the final concentration of imiquimod At 2.5-5 mg/ml, lyophilize to obtain imiquimod hydrochloride lyophilized powder.

Example 2

Preparation of immune adjuvant emulsion (taking Imiquimod as an example):

Using a 50mL ball milling tank, zirconia ball milling, adding imiquimod and deionized water ball milling for 3 cycles to obtain imiquimod emulsion. The detailed process is as follows.

Preparation of immune adjuvant emulsion (taking imiquimod as an example): In addition to acidifying imiquimod to dissolve in water, ball milling the imiquimod emulsion to reduce its particle size to the micron level can also make Imiquimod has good water dispersibility. In this example, the imiquimod emulsion with different ball milling time was studied. The following table shows the changes in particle size, water dispersibility and stability of the imiquimod emulsion after ball milling. As can be seen from the table below, after ball milling for more than 15 minutes, the imiquimod emulsion showed good dispersibility.

Use a 50Ml zirconia ball mill tank, zirconia ball mill, add imiquimod R837 and deionized water ball mill for 3 cycles for about 3 hours to obtain a uniformly dispersed imiquimod emulsion.

球磨时间(分钟)Ball milling time (minutes)	水分散性Water dispersibility	稳定性stability	粒径(微米)Particle size (micron)
11	不均匀Uneven	不稳定Unstable	2-52-5
1515	均匀Evenly	稳定stable	2-32-3
6060	均匀Evenly	稳定stable	1-21-2
180180	均匀Evenly	稳定stable	0.3-10.3-1

This table reflects the changes in particle size, water dispersibility and stability of imiquimod emulsion after ball milling

Example 3

Preparation of gel complex containing immune adjuvant (the gel-forming matrix is mixed with fibrinogen and thrombin as an example, and the immune adjuvant is imiquimod as an example):

Weigh 40 mg of fibrinogen in a 5 mL serum bottle A and add deionized water to dissolve it to a final concentration of 10-200 mg/ml. Weigh 100 IU of thrombin in a 5 mL serum bottle B, and add calcium ion buffer to make the final concentration of thrombin 20-100 IU/mL. Add the immune adjuvant emulsion to bottle A or bottle B, mix well, and load bottle A liquid and bottle B liquid into a dual-mixer for use.

Example 4

Preparation of gel complex containing chemotherapeutic drugs (the gel-forming matrix is mixed with fibrinogen and thrombin as an example, and the chemotherapeutic drugs are oxaliplatin, gemcitabine, doxorubicin (DOX), pentafluorouracil (5 for short) -FU) as an example):

Weigh 40 mg of fibrinogen in a 5 mL serum bottle A and add deionized water to dissolve it to a final concentration of 10-200 mg/ml. Weigh 100 IU of thrombin in a 5 mL serum bottle B, and add calcium ion buffer to make the final concentration of thrombin 20-100 IU/mL. Add chemotherapeutic drugs to bottle A or bottle B, mix them, and load bottle A liquid and bottle B liquid into a dual-mixer for use. Take a photo to observe the process of changing from a solution to a gel. As shown in Figure 1, the clear solution gradually becomes a translucent jelly-like solid.

According to the corresponding detection method of the drug described in the Pharmacopoeia, the drug release curve in the gel at different time points was measured by ultraviolet or HPLC. The results are shown in Figure 2, and the drug is gradually released from the gel over time.

Example 5

Preparation of gel complex containing antibody (the gel-forming matrix is mixed with fibrinogen and thrombin as an example, and the antibody is a-PDL1 as an example):

Weigh 40 mg of fibrinogen in a 5 mL serum bottle A and add deionized water to dissolve it to a final concentration of 10-200 mg/ml. Weigh 100 IU of thrombin in a 5 mL serum bottle B, and add calcium ion buffer to make the final concentration of thrombin 20-100 IU/mL. Add antibody to bottle A or bottle B, mix well, and load bottle A liquid and bottle B liquid into the dual-mixer for use.

Example 6

Preparation of gel complex containing chemotherapeutics, adjuvants, and antibodies (the gel-forming matrix is mixed with fibrinogen and thrombin as an example, the chemotherapeutic agent is adriamycin as an example, and the immune adjuvant is imiquimod as an example For example, the antibody takes a-PDL1 as an example):

The gel-forming matrix can be adjusted as follows: the first solution is a fibrinogen solution with a concentration of 1-400 mg per ml, the second solution is a thrombin solution with a concentration of 5-200 IU per ml, and the activity is added to the first solution or the second solution Ingredients, mixing the first solution and the second solution to form a gel sustained-release pharmaceutical composition. Preferably, the fibrinogen solution of the first solution can be adjusted to 10-200 mg/ml, and the concentration of the thrombin solution in the second solution is 20-100 IU/ml. Generally speaking, the higher the concentration, the faster the gelation, and the stronger the gel formed. The concentration is the preferred concentration, and the gel can be formed within seconds or minutes.

Weigh 40 mg of fibrinogen in a 5 mL serum bottle A, add deionized water to dissolve it, and make the final concentration of 10-200 mg/ml. Weigh 100 IU of thrombin in a 5 mL serum bottle B, and add calcium ion buffer to make the final concentration of thrombin 20-100 IU/mL. Add doxorubicin, imiquimod, and a-PDL1 to bottle A or bottle B, mix well, and load bottle A liquid and bottle B liquid into a dual-mixer for use. Figure 3 is a scanning electron microscope picture after mixing into glue.

Example 7

The rheological properties of the gel complex containing chemotherapeutics, adjuvants and antibodies (the gel-forming matrix is mixed with fibrinogen and thrombin as an example, the chemotherapeutic agent is adriamycin as an example, and the immune adjuvant is imiquine Take Mott as an example, the antibody takes a-PDL1 as an example):

Prepare DOX, R837, a-PDL1 protein glue complex, and examine the rheological properties of the gel formed by mixing 40 mg/ml fibrinogen solution and 25 IU/mL to 100 IU/mL thrombin complex solution. It can be seen from Figure 4 that the storage modulus (G') of the compound is smaller than the loss modulus (G") when the compound is just mixed, showing the behavior of a fluid. When mixed for a certain period of time, its storage modulus (G') ) Is greater than the loss modulus (G"), showing the behavior of a gel, which proves that the composite gel will form a colloid after mixing. The gelation time is related to the concentration of the gelation matrix, the higher the concentration, the shorter the gelation time.

Example 8

The therapeutic effect of the gel complex containing chemotherapeutics, adjuvants and antibodies (the gel-forming matrix is mixed with fibrinogen and thrombin as an example, the chemotherapeutic drug is adriamycin (DOX) as an example, and the immune adjuvant is Imiquimod (abbreviated as R837) as an example, antibody a-PDL1 as an example):

Divide subcutaneous breast cancer mice into 3 groups, with six mice in each group for treatment experiments. After surgical removal of most of the subcutaneous tumors (preserving the skin and muscles adjacent to the cancer), the following methods were taken:

1) No processing,

2) Form DOX, R837 protein glue complex after injection,

3) Formation of DOX, R837, a-PDL1 protein glue complex after injection.

The treatment effect is judged by observing the metastasis and recurrence of the tumor after surgery. Fluorescence in 5-1 describes the tumor signal in mice, and 5-2 describes the survival curve of mice. It can be seen from Figure 5 that the mice in the second and third groups showed a good effect of inhibiting tumor metastasis and recurrence, which confirmed the good effect of the coordination and synergy of DOX, R837, and a-PDL1 protein glue complex.

Example 9

Preparation of gel complex containing chemotherapeutics, adjuvants and antibodies (the gel-forming matrix is mixed with dexamethasone sodium phosphate and calcium ion solution to form a gel as an example, the chemotherapeutic drug is adriamycin as an example, and the immune adjuvant is imiquine Take Mott as an example, the antibody takes a-PDL1 as an example):

The gel-forming matrix includes, the first solution is a dexamethasone sodium phosphate solution with a concentration of 5-200 mg/ml, and the second solution is a calcium ion buffer solution with a calcium ion concentration of 0.4-16 mg/ml. Active ingredients are added to the solution or the second solution, and the first solution and the second solution are mixed to form a gel sustained-release pharmaceutical composition.

Specifically, the dexamethasone sodium phosphate solution (with a concentration of 5-200 mg/mL) containing DOX, R837, and a-PDL1 and a calcium ion buffer (with a calcium ion concentration of 0.4-16 mg/mL) were loaded into the dual mix Medicine device use. Figure 6 is an electron microscope picture after mixing into glue.

Example 10

Preparation of DOX, R837, a-PDL1 gel compound, detected with 1.00 × 10 ^-1 M calcium ion solution mixed synthetic rheological and mechanical properties of 1.00 × 10 ^-1 M dexamethasone sodium phosphate solution. It can be seen from Figure 7 that when mixed, the storage modulus (G') is greater than the loss modulus (G"), showing gel behavior, which proves that the composite gel will form a colloid after mixing. The gel time is related to the concentration of the gel-forming matrix, the higher the concentration, the shorter the gel-forming time.

Example 11

Preparation of gel complex containing chemotherapeutics (the gel-forming matrix is mixed with dexamethasone sodium phosphate and calcium ion solution to form a gel as an example, and the chemotherapeutic drugs are oxaliplatin, gemcitabine, and pentafluorouracil (5-FU) as examples) :

A gel complex of oxaliplatin, gemcitabine, and pentafluorouracil (5-FU) was prepared, and the release curve of the drug in the gel at different time points was determined by HPLC.

Figure 8 is the release curve of the drug in the gel complex in Example 11.

Example 12

Preparation of gel complex containing chemotherapeutic drugs (the chemotherapeutic drug is adriamycin DOX as an example). The following table shows that although the gelling raw materials can theoretically choose a variety of combinations, they can be combined with appropriate components, concentrations, and gelation conditions, so that they can be used with a variety of drugs in anti-tumor applications and stimulate the synergistic effect of tumor drugs. The working mechanism, and suitable for medical use, is more complicated.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

Claims

A multi-component gel sustained-release pharmaceutical composition for treating tumors, which is characterized in that it comprises active components and adjuvants, the active components including chemotherapeutics, or immune adjuvants, or immune adjuvants that cause immunogenic death of tumor cells, or Immune checkpoint inhibitors;

The auxiliary material includes a gel-forming matrix and a buffer;

The gel-forming matrix includes serum albumin, fibrinogen, sodium alginate, calcium alginate, collagen, chitosan, butyltosan, gelatin, starch, hyaluronic acid, sodium carboxymethylcellulose, sodium glycerophosphate , Cyanoacrylate, acrylamide, polyoxyethylene, polyethylene glycol, polyvinyl alcohol, poloxamer, polyvinyl lactone, one or more of steroids.
The multi-component gel sustained-release pharmaceutical composition for treating tumors according to claim 1, wherein the gel-forming matrix comprises: the first solution has a concentration of 1-400 mg per milliliter of fibrinogen solution, and the second The solution is a thrombin solution with a concentration of 5-200IU per milliliter. Active ingredients are added to the first solution or the second solution, and the first solution and the second solution are mixed to form a gel sustained-release pharmaceutical composition;

Or the gel-forming matrix includes, the first solution is a dexamethasone sodium phosphate solution with a concentration of 5-200 mg/ml, and the second solution is a calcium ion buffer solution with a calcium ion concentration of 0.4-16 mg/ml. The active ingredient is added to the first solution or the second solution, and the first solution and the second solution are mixed to form a gel sustained-release pharmaceutical composition.
The multi-component gel sustained-release pharmaceutical composition for treating tumors according to claim 2, wherein the fibrinogen solution of the first solution is 10-200 mg/ml, and the concentration of the thrombin solution of the second solution is It is 20-100IU per milliliter.
The multi-component gel sustained-release pharmaceutical composition for treating tumors according to claim 1, wherein the buffer is a calcium ion buffer or a sodium ion buffer, and the ion concentration is 0.4-16 mg/ml; or The buffer is acetate buffer or hydrochloric acid buffer, with a concentration of 0.4-40 mg per milliliter.
The multi-component gel sustained-release pharmaceutical composition for treating tumors according to claim 1, wherein:

The multi-component gel sustained-release pharmaceutical composition is a multi-component gel sustained-release pharmaceutical composition for the treatment of breast cancer tumors, and the gel-forming matrix includes a first solution and a second solution, and the first solution is deionized water The final dissolved concentration is 10-200 mg/ml fibrinogen solution;

The second solution is a calcium ion buffer solution with a final concentration of 20-100IU/mL thrombin solution;

Add doxorubicin, imiquimod, and a-PDL1 to the first solution or the second solution, mix well, and mix the mixed first solution and the second solution to form a gel sustained-release pharmaceutical composition.
The multi-component gel sustained-release pharmaceutical composition for treating tumors according to claim 1, wherein:

The gel-forming matrix is a mixture of fibrinogen and thrombin to form a gel, the first solution is a fibrinogen solution with a concentration of 10-200 mg per milliliter, and the second solution is a final thrombin concentration of 20-100 IU/mL. The immune adjuvant emulsion is added to the first solution or the second solution, and mixed, the immune adjuvant emulsion is imiquimod emulsion, and imiquimod R837 and deionized water are ball milled to obtain a uniformly dispersed imiquimod Special emulsion, the imiquimod particles have a particle size of 20-300 microns, and the first solution containing the immune adjuvant emulsion is mixed with the second solution to form a gel.
The multi-component gel sustained-release pharmaceutical composition for treating tumors according to claim 1, wherein:

The gel-forming matrix is mixed with dexamethasone sodium phosphate and calcium ion solution to form a gel. The first solution is a dexamethasone sodium phosphate solution containing doxorubicin DOX, imiquimod R837, antibody a-PDL1, and dexamethasone The concentration of the sodium phosphate solution is 5-200 mg/ml, the second solution is a calcium ion buffer, and the concentration of the calcium ion solution is 0.4-16 mg/ml; the first solution is mixed with the second solution to form a gel.
The multi-component gel sustained-release pharmaceutical composition for treating tumors according to any one of claims 1-7, wherein the dosage form of the pharmaceutical composition is ointment, suppository, spray, Solution agent.