CN104784105A - Gel compound of monoclonal antibody drugs - Google Patents

Abstract

The invention relates to a gel composition of a monoclonal antibody drug, which can be clinically used by patients for intradermal injection, peritumoral injection or intratumoral injection, and belongs to the technical field of pharmaceutical preparations. The gel composition of the monoclonal antibody drug includes a monoclonal antibody, at least one extracellular matrix degrading enzyme, such as collagenase or hyaluronidase, a temperature-sensitive gel material and a solvent. The monoclonal antibody gel composition may further include buffers, stabilizers, nonionic surfactants, and osmo-regulators. The invention also provides a method for preparing the monoclonal antibody gel composition and its application. The monoclonal antibody gel composition involved in the present invention can not only prolong the release of the monoclonal antibody, but also promote the tissue penetration of the monoclonal antibody or penetrate into the tumor, and has the advantages of reducing the number of administrations and enhancing the therapeutic effect.

Description

A kind of gel composition of monoclonal antibody medicine

technical field

The invention relates to a gel composition containing a monoclonal antibody, which belongs to the technical field of pharmaceutical preparations.

Background technique

With the development of biotechnology, biopharmaceuticals represented by monoclonal antibodies have gradually entered people's field of vision, and have increasingly become the mainstream in the treatment of malignant tumors and autoimmune diseases. Monoclonal antibody drugs can overcome the disadvantages of poor selectivity and high toxicity of common chemotherapy drugs because of their high specificity in their effects.

In the treatment of malignant tumors and autoimmune diseases, monoclonal antibody drugs specifically bind to the corresponding antigen on the cell surface, with high affinity and complex mechanism of action. Single or multiple pathways work together to achieve the purpose of killing tumor cells. These include 1. Direct effects of antibodies: receptor blockade, agonist inhibition, and mediation of autophagy. 2. Immune system-mediated killing: complement-dependent cytotoxicity, antibody-dependent cytotoxicity. 3. Specific effect on blood vessels and matrix.

Based on the above characteristics, in the field of malignant tumors, compared with common chemotherapy drugs, monoclonal antibody drugs can kill tumor cells more accurately, greatly improve the treatment effect, and reduce adverse reactions, thereby prolonging the survival time of patients and improving the quality of life .

Nowadays, while making great progress, monoclonal antibody drugs also have unavoidable shortcomings. Because of the nature of protein drugs, monoclonal antibody drugs are more difficult to extract and purify, and have poor stability during storage and use, and their biological activity is easily affected, resulting in high cost for patients. Monoclonal antibody Access to medicines is low. Due to the nature of the protein, monoclonal antibody drugs are easily degraded by proteases in the gastrointestinal tract during oral administration. In addition, their biomacromolecule (molecular weight up to 150,000 Da) characteristics make it difficult to cross biological barriers such as small intestinal epithelium, making monoclonal antibody drugs Not for oral use. In the course of clinical use, monoclonal antibody drug administration is mostly a single intravenous infusion, which may be accompanied by severe "infusion reactions" during the infusion process, and it needs to be used for a long time, for example: in Her-2 positive breast cancer During the cancer treatment process, the patient needs intravenous infusion once every week or every three weeks, and the treatment time is as long as 52 weeks, which brings great pain to the patient and poor compliance.

Pharmaceutical companies are also exploring new delivery routes for monoclonal antibody drugs as much as possible. The U.S. patent with the publication number US 20110044977 discloses a method of intradermal administration of a monoclonal antibody for the treatment of Her-2 positive breast cancer. The specific reversible degradation of hyaluronic acid in the skin allows trastuzumab to enter the systemic circulation smoothly, play a therapeutic role, and reduce the toxic and side effects of systemic administration. Trastuzumab and rituximab for transdermal administration have been approved by the European Medicines Agency using this combined drug delivery technology.

In the tissue structure of malignant tumors and other diseases, extracellular matrix cells have played a huge role. Among them, in the extracellular matrix, collagen and hyaluronic acid are the two most important and most abundant components. In malignant tumors, the two fibrous proteins can increase the compactness of the tumor matrix and increase the interstitial pressure of the tumor tissue, thereby blocking the therapeutic drug from entering the deep tumor from the outside of the tumor and destroying the therapeutic effect of the drug. The specific degradation of the tumor matrix can effectively promote the therapeutic drug to enter the tumor and further improve the therapeutic effect of the drug. Mako Kato et al. reported that intravenous injection of collagenase can reduce tumor interstitial pressure and increase the accumulation of cationic liposomes in tumors (Kato et al, Collagenase-1 injection improved tumor distribution and gene expression of cationic lipoplex, Int J Pharm.2012 Feb 28; 423(2):428-34).

Temperature-sensitive gels are gel systems composed of amphiphilic polymers. At low temperature (lower than the critical gelling temperature), the distance between the polymer polymers is large and the force is small, and the force between the hydrophilic end and the water molecule is the main force, making it in a liquid form. At high temperature (higher than the critical gelling temperature), the distance between the polymer polymers decreases, the amphiphilic block takes the form of micelles, the micelles are further compressed, and the force between the hydrophobic cores of the micelles is sharply strengthened, making it appear solid form. The characteristics of polymer polymers create their phase transition characteristics at different temperatures.

After the temperature-sensitive gel injection is injected into the human body in liquid form, a phase transition occurs rapidly at the injection site to form a solid gel. After the drug is entrapped, it can achieve local drug delivery, prolong the residence time of the drug at the drug site, and achieve good relief. Release effect, to achieve the effect of one injection, long-term treatment. The Chinese patent with publication number CN 103239389 discloses a progesterone temperature-sensitive gel based on poloxamer 407, which is used as a treatment for threatened abortion.

Based on the above technical background, the object of the present invention is to provide a gel composition containing monoclonal antibody drugs and extracellular matrix degrading enzymes, which can specifically degrade matrix tissue, enhance the deep penetration of drugs, enhance the therapeutic effect, and single administration , long-term onset of therapeutic effect.

Contents of the invention

The object of the present invention is to provide a monoclonal antibody gel composition.

The present invention realizes above-mentioned object through following technical scheme:

A monoclonal antibody gel composition, the monoclonal antibody gel composition comprises: monoclonal antibody, temperature sensitive material, extracellular matrix degrading enzyme and solvent.

In the monoclonal antibody gel composition provided by the present invention, the extracellular matrix degrading enzyme includes one or more of collagenase, hyaluronidase, and dispase.

The monoclonal antibody gel composition provided by the present invention, wherein the temperature-sensitive material includes poloxamer, polylactic acid-polyethylene glycol-polylactic acid, polyglycolide lactide-polyethylene glycol- Polyglycolide lactide, polyethylene glycol-polylactic acid-polyethylene glycol, polyethylene glycol-polyglycolide lactide-polyethylene glycol, polycaprolactone-polyethylene glycol-polyethylene glycol Caprolactone, N-isopropylacrylamide polymer and chitosan and its derivatives neutralize one or more.

The monoclonal antibody gel composition provided by the present invention, wherein the monoclonal antibody includes: trastuzumab, cetuximab, bevacizumab, rituximab, pertuzumab One or more of , nimotuzumab, infliximab, adalimumab, gemmumab, ipilimumab.

In the monoclonal antibody gel composition provided by the present invention, further preferably, the monoclonal antibody is trastuzumab.

In the monoclonal antibody gel composition provided by the present invention, it is further preferred that the temperature-sensitive material is polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide, wherein polyglycolide-lactide The molar ratio of lactide to polyethylene glycol is 3:1, the molecular weight of polyethylene glycol is 1500Da, and the total molecular weight of polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide is 5000Da.

In the monoclonal antibody gel composition provided by the present invention, it is further preferred that the temperature-sensitive material is poloxamer 407 or poloxamer 188 or a mixture thereof.

In the monoclonal antibody gel composition provided by the present invention, it is further preferred that the extracellular matrix degrading enzyme is I-collagenase.

In the monoclonal antibody gel composition provided by the present invention, the lysozyme is one of water, physiological saline, and 5% glucose aqueous solution.

The monoclonal antibody gel composition provided by the present invention, wherein the composition further comprises a buffer, and the buffer is one of phosphate buffer and histidine hydrochloride buffer, and its pH is 5.5-7.0 , is weakly acidic, and is more preferably a histidine hydrochloride buffer.

The monoclonal antibody gel composition provided by the present invention, wherein the composition further comprises a stabilizer, and the stabilizer is one of lactose, trehalose, and galactose, more preferably trehalose.

The monoclonal antibody gel composition provided by the present invention, wherein the composition further comprises and a nonionic surfactant, the nonionic surfactant is one of Tween 20 or Tween 80, more preferably Tween Wen 20.

Monoclonal antibody gel composition provided by the present invention, wherein the weight ratio of monoclonal antibody and extracellular matrix degrading enzyme is 1:40-40:1, and the weight ratio of temperature-sensitive material and solvent is 1:10-2: 1.

Monoclonal antibody gel composition provided by the present invention, wherein the weight ratio of monoclonal antibody and extracellular matrix degrading enzyme is 1:20-20:1, and the weight ratio of temperature-sensitive material and solvent is 1:10-1: 1.

Further preferably, the monoclonal antibody drug gel composition provided by the present invention, wherein the weight percentage of the monoclonal antibody drug and extracellular matrix degrading enzyme in the temperature-sensitive gel composition is:

A. 0.01%-5% monoclonal antibody drugs,

B. 0.01%-5% extracellular matrix degrading enzymes.

In the monoclonal antibody drug gel composition provided by the invention, the contents of various components are:

Wherein, said percentage is weight percentage.

Further preferably, the content of various ingredients is

In the monoclonal antibody drug gel composition provided by the invention, the contents of various components are:

The monoclonal antibody gel composition of the present invention, its preparation method is as follows:

A. Select solvent and temperature sensitive material to prepare blank temperature sensitive gel;

B. Fully mix the blank temperature-sensitive gel with the monoclonal antibody and the extracellular matrix degrading enzyme, and dissolve to obtain the monoclonal antibody gel composition.

Preferably, the preparation method of the monoclonal antibody gel composition of the present invention is as follows:

A. Weigh 250 mg of polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide, add 1000 mg of water, and stir slowly under ice bath to obtain a blank temperature-sensitive gel.

B. Weigh 20 mg of collagenase and 7.5 mg of trastuzumab, add a blank temperature-sensitive gel, and stir slowly under an ice bath to obtain a monoclonal antibody drug and a tumor matrix-specific drug gel preparation.

The gel preparation comprising monoclonal antibody drug and tumor stroma-specific drug provided by the present invention can be used for intratumoral injection, peritumoral injection and intradermal injection.

A kind of monoclonal antibody pharmaceutical gel composition provided by the present invention has the following advantages:

1. Reduce the frequency of use of monoclonal antibody drugs. Compared with clinical intravenous infusion, the preparation stays in the body for a long time. Once administered, it will take effect for a long time and improve patient compliance.

2. To enhance the curative effect of monoclonal antibody drugs, compared with intravenous injection of monoclonal antibody drugs, temperature-sensitive gels of monoclonal antibody drugs alone, and temperature-sensitive gels of extracellular matrix degrading enzymes alone, containing monoclonal antibody drugs and extracellular matrix Gel formulations of degrading enzymes can significantly enhance the therapeutic effect of monoclonal antibodies.

After research, we found that at this stage, there are few researches on pharmaceutical preparations for monoclonal antibody drugs, and the biodegradability and safety of temperature-sensitive gels represented by PNIPAAm limit their applications.

The present invention aims at the disadvantage of poor safety of the existing gel preparation, and adopts a gel material with good safety and biocompatibility to ensure its safety. Combined with extracellular matrix degrading enzymes to enhance its therapeutic effect.

The tumor extracellular matrix mainly composed of collagen can increase the compactness of the tumor matrix, making it difficult for therapeutic drugs to cross the matrix and enter the deep part of the tumor, reducing the therapeutic effect. The tumor extracellular matrix degrading enzyme of the present invention is used in combination with the monoclonal antibody, the tumor extracellular matrix degrading enzyme specifically degrades the tumor extracellular matrix, increases the dose of the monoclonal antibody entering the tumor, enhances its therapeutic effect, and produces a good synergistic effect .

In the present invention, the proportion of monoclonal antibody drugs is controlled at 0.01%-5%, which can minimize the use of expensive monoclonal antibodies and reduce the burden on patients under the premise of ensuring enhanced therapeutic effect.

Description of drawings:

Figure 1 Example 14 contains the in vitro release curve of bovine serum albumin temperature-sensitive gel.

Fig. 2 The tumor growth curve of the in vivo pharmacodynamic study of the gel preparation containing the monoclonal antibody drug and the extracellular matrix degrading enzyme in Example 15.

Body weight change curve in vivo pharmacodynamics study of the gel formulation comprising monoclonal antibody drug and extracellular matrix degrading enzyme in Fig. 3 Example 15.

Fig. 4 is a graph of fluorescence intensity of apoptosis in tumor slices in the in vivo pharmacodynamics study of the gel preparation containing monoclonal antibody drug and extracellular matrix degrading enzyme in Example 15.

Fig. 5 is a diagram of the in vivo pharmacodynamics study of the gel preparation containing the monoclonal antibody drug and the extracellular matrix degrading enzyme in Example 15, and the collagen fluorescence intensity graph of tumor slices.

Detailed ways:

The present invention is further illustrated and explained by specific examples below, but not as a limitation of the present invention.

Example 1. Preparation of temperature-sensitive gel comprising trastuzumab and I-collagenase

1. Preparation of blank temperature-sensitive gel: Accurately weigh 500 mg of polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide, add 2000 mg of deionized water, stir slowly under ice bath for 48 hours, and wait for complete dissolution After that, a blank temperature-sensitive gel was obtained.

2. Preparation of temperature-sensitive gel containing trastuzumab and Ⅰ-collagenase: Accurately weigh 15 mg of trastuzumab and 40 mg of Ⅰ-collagenase (201u/mg), mix slowly by shaking, and add the blank prepared above For the temperature-sensitive gel, stir slowly for 10 minutes under an ice bath, and mix evenly to obtain a temperature-sensitive gel preparation containing trastuzumab and collagenase.

Example 2, Preparation of Temperature Sensitive Gel Containing Trastuzumab and I-Collagenase

1. Preparation of blank temperature-sensitive gel: Precisely weigh 407,500 mg of poloxamer, add 2,000 mg of deionized water, and stir slowly for 48 hours in an ice bath. After complete dissolution, a blank temperature-sensitive gel is obtained.

2. Preparation of temperature-sensitive gel containing trastuzumab and Ⅰ-collagenase: Accurately weigh 15 mg of trastuzumab and 40 mg of Ⅰ-collagenase (201u/mg), mix slowly by shaking, and add the blank prepared above For the temperature-sensitive gel, stir slowly for 10 minutes under an ice bath, and mix evenly to obtain a temperature-sensitive gel preparation containing trastuzumab and I-collagenase.

Example 3, Preparation of Temperature Sensitive Gel Containing Trastuzumab and I-Collagenase

1. Preparation of blank temperature-sensitive gel: Accurately weigh 500 mg of polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide, add 2000 mg of deionized water, stir slowly under ice bath for 48 hours, and wait for complete dissolution After that, a blank temperature-sensitive gel was obtained.

2. Preparation of temperature-sensitive gel containing trastuzumab and Ⅰ-collagenase: Accurately weigh 1 mg of trastuzumab monoclonal antibody and 40 mg of Ⅰ-collagenase (201u/mg), shake slowly to mix, add the above-prepared The blank temperature-sensitive gel was slowly stirred for 10 minutes under an ice bath, and mixed evenly to obtain a temperature-sensitive gel preparation containing trastuzumab and Ⅰ-collagenase.

Example 4, Preparation of Temperature Sensitive Gel Containing Trastuzumab and I-Collagenase

1. Preparation of blank temperature-sensitive gel: Accurately weigh 500 mg of polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide, add 2000 mg of deionized water, stir slowly under ice bath for 48 hours, and wait for complete dissolution After that, a blank temperature-sensitive gel was obtained.

2. Preparation of temperature-sensitive gel containing trastuzumab and Ⅰ-collagenase: Accurately weigh 1600 mg of trastuzumab and 40 mg of Ⅰ-collagenase (201u/mg), mix slowly by shaking, and add the blank prepared above For the temperature-sensitive gel, stir slowly for 10 minutes under an ice bath, and mix evenly to obtain a temperature-sensitive gel preparation containing trastuzumab and I-collagenase.

Embodiment 5, the preparation that comprises trastuzumab and hyaluronidase temperature-sensitive gel

1. Preparation of blank temperature-sensitive gel: Accurately weigh 500 mg of polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide, add 2000 mg of deionized water, stir slowly under ice bath for 48 hours, and wait for complete dissolution After that, a blank temperature-sensitive gel was obtained.

2. Preparation of temperature-sensitive gel containing trastuzumab and hyaluronidase: Accurately weigh 15 mg of trastuzumab and 40 mg of hyaluronidase (205u/mg), mix slowly by shaking, and add the blank prepared above For the temperature-sensitive gel, stir slowly for 10 minutes under an ice bath, and mix evenly to obtain a temperature-sensitive gel preparation containing trastuzumab and I-collagenase.

Example 6, Preparation of Temperature Sensitive Gel Containing Rituximab and I-Collagenase

1. Preparation of blank temperature-sensitive gel: Accurately weigh 500 mg of polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide, add 2000 mg of deionized water, stir slowly under ice bath for 48 hours, and wait for complete dissolution After that, a blank temperature-sensitive gel was obtained.

2. Preparation of temperature-sensitive gel containing cetuximab and collagenase: Accurately weigh 15 mg of rituximab and 40 mg of Ⅰ-collagenase (201u/mg), shake slowly and mix, add the blank temperature prepared above For the sensitive gel, stir slowly for 10 minutes under an ice bath, and mix well to obtain a temperature-sensitive gel preparation containing rituximab and I-collagenase.

Example 7, Preparation of Temperature Sensitive Gel Containing Pertuzumab and I-Collagenase

1. Preparation of blank temperature-sensitive gel: Accurately weigh 500 mg of polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide, add 2000 mg of deionized water, stir slowly under ice bath for 48 hours, and wait for complete dissolution After that, a blank temperature-sensitive gel was obtained.

2. Preparation of temperature-sensitive gel containing bevacizumab and collagenase: Accurately weigh 15 mg of Pertuzumab and 40 mg of I-collagenase (201u/mg), shake slowly and mix, add the blank temperature-sensitive gel prepared above For the gel, stir slowly for 10 minutes under an ice bath, and mix well to obtain a temperature-sensitive gel preparation containing Pertuzumab and I-collagenase.

Example 8, Preparation of Temperature Sensitive Gel Containing Nimotuzumab and I-Collagenase

1. Preparation of blank temperature-sensitive gel: Accurately weigh 500 mg of polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide, add 2000 mg of deionized water, stir slowly under ice bath for 48 hours, and wait for complete dissolution After that, a blank temperature-sensitive gel was obtained.

2. Preparation of temperature-sensitive gel containing rituximab and collagenase: Accurately weigh 15 mg of Nimotuzumab and 40 mg of I-collagenase (201u/mg), shake slowly and mix, add the blank temperature-sensitive gel prepared above The gel was stirred slowly for 10 minutes under an ice bath, and mixed evenly to obtain a temperature-sensitive gel preparation containing Nimotuzumab and I-collagenase.

Example 9, Preparation of Temperature Sensitive Gel Containing Infliximab and I-Collagenase

1. Preparation of blank temperature-sensitive gel: Accurately weigh 500 mg of polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide, add 2000 mg of deionized water, stir slowly under ice bath for 48 hours, and wait for complete dissolution After that, a blank temperature-sensitive gel was obtained.

2. Preparation of temperature-sensitive gel containing Pertuzumab and collagenase: Accurately weigh 15 mg of Infliximab and 40 mg of I-collagenase (201u/mg), shake slowly and mix, add the blank temperature-sensitive gel prepared above Gel, stirred slowly for 10 minutes under an ice bath, and mixed evenly to obtain a temperature-sensitive gel preparation containing infliximab and Ⅰ-collagenase.

Example 10, Preparation of Temperature Sensitive Gel Containing Adalimumab and I-Collagenase

1. Preparation of blank temperature-sensitive gel: Accurately weigh 500 mg of polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide, add 2000 mg of deionized water, stir slowly under ice bath for 48 hours, and wait for complete dissolution After that, a blank temperature-sensitive gel was obtained.

2. Preparation of temperature-sensitive gel containing nimotuzumab and collagenase: Accurately weigh 15 mg of adalimumab and 40 mg of Ⅰ-collagenase (201u/mg), shake slowly to mix, add the blank temperature-sensitive gel prepared above Gel, stirred slowly for 10 minutes under an ice bath, and mixed evenly to obtain a temperature-sensitive gel preparation containing adalimumab and Ⅰ-collagenase.

Example 11, Preparation of Temperature Sensitive Gel Containing Gemumab and I-Collagenase

1. Preparation of blank temperature-sensitive gel: Accurately weigh 500 mg of polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide, add 2000 mg of deionized water, stir slowly under ice bath for 48 hours, and wait for complete dissolution After that, a blank temperature-sensitive gel was obtained.

2. Preparation of temperature-sensitive gel containing gemtuzumab and collagenase: Precisely weigh 15 mg of pertuzumab and 40 mg of Ⅰ-collagenase (201u/mg), shake slowly and mix, add the blank temperature-sensitive gel prepared above , stirred slowly in an ice bath for 10 minutes, and mixed evenly to obtain a temperature-sensitive gel preparation containing gemmumab and Ⅰ-collagenase.

Example 12, Preparation of Temperature Sensitive Gel Containing Ipilimumab and I-Collagenase

1. Preparation of blank temperature-sensitive gel: Accurately weigh 500 mg of polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide, add 2000 mg of deionized water, stir slowly under ice bath for 48 hours, and wait for complete dissolution After that, a blank temperature-sensitive gel was obtained.

2. Preparation of temperature-sensitive gel containing ipilimumab and collagenase: accurately weigh 15 mg of ipilimumab and 40 mg of I-collagenase (201u/mg), shake slowly and mix, add the blank temperature-sensitive gel prepared above , stirred slowly for 10 minutes in an ice bath, and mixed evenly to obtain a temperature-sensitive gel preparation containing ipilimumab and Ⅰ-collagenase.

Embodiment 13, the preparation that comprises bovine serum albumin temperature-sensitive gel

1. Preparation of blank temperature-sensitive gel: Accurately weigh 500 mg of polyglycolide-lactide-polyethylene glycol-polyglycolide-lactide, add 2000 mg of deionized water, stir slowly under ice bath for 48 hours, and wait for complete dissolution After that, a blank temperature-sensitive gel was obtained.

2. Preparation of temperature-sensitive gel containing bovine serum albumin: 40 mg of precision bovine serum albumin, shake slowly to mix, add the blank temperature-sensitive gel prepared above, stir slowly under ice bath for 10 minutes, mix well, and obtain bovine serum containing Temperature-sensitive gel formulations of albumin.

Example 14, In vitro Release Curve of Temperature-sensitive Gel Containing Bovine Serum Albumin

Get 2ml of the temperature-sensitive gel containing bovine serum albumin prepared in Example 13, and add 300 microliters of the temperature-sensitive gel liquid containing bovine serum albumin to the Transwell (Corning, 24 mm, 3 micron) chamber at 4 degrees quickly placed in an air bath constant temperature oscillator (ZHWY-100C, Shanghai Zhicheng Analytical Instruments) set to 37 degrees in advance to form a solid gel. Put the Transwell chamber containing the solid gel in a 50ml centrifuge tube (Corning) containing 10ml 37°C deionized water, place it in an air bath constant temperature shaker at 37°C, 100 rpm, take samples at intervals, and add the same volume of Blank release medium. According to the operating procedures of the BCA protein quantification kit (Dingguo Changsheng), the released release liquid was determined, and the cumulative drug release amount at each time point was calculated.

The release curve is shown in Figure 1. The results show that the cumulative release of bovine serum albumin of the temperature-sensitive gel involved in the present invention at 37 degrees for 9 days is less than 80%.

Example 15. In Vivo Pharmacodynamic Study of Gel Preparation Containing Monoclonal Antibody and Extracellular Matrix Degrading Enzyme

Select female Nu/nu nude mice (18-22 g, Beijing Weitong Lihua experimental animals), inoculate 1×10 ⁶ breast cancer BT474 cells in the right axillary skin, and administer 150 microliters 15 days after the inoculation, according to the specific examples The pharmaceutical composition was prepared in 1, and the administration components were divided into 6 groups, 7 in each group, respectively: control group, blank gel group, trastuzumab gel group, intravenous single-dose group, intravenous multiple-dose group, Collagenase trastuzumab gel set.

Control group: injection next to the tumor, single administration;

Blank gel group: paratumor injection, single administration;

Trastuzumab monoclonal antibody gel group: peritumoral injection, single administration, total dose 30mg/kg;

Intravenous single group: tail vein injection, single administration, total dose 30mg/kg;

Intravenous multiple times group: tail vein injection, administered once a week, four times in total, with a total dose of 30mg/kg;

Collagenase trastuzumab gel group: paratumor injection, single dose, total dose Herceptin 30mg/kg, collagenase 15mg/kg

(1) Tumor growth curve: use a vernier caliper to measure the long diameter (L) and short diameter (r) of the nude mouse tumors in each group every 5 days after administration, and calculate the tumor volume V (V=[L×(r) ² ]/2 ), draw the tumor growth curve, and the results are shown in Figure 3. The results in Figure 3 show that the blank gel has no tumor growth inhibitory effect, the collagenase trastuzumab gel group has the best drug effect, followed by trastuzumab gel, but better than the intravenous administration group.

(2) Body weight change curve: the body weight of the nude mice was weighed with a balance every 5 days after administration. The results shown in Figure 3 show that the body weight of the nude mice in each group did not change significantly, and there was no significant difference, indicating that the preliminary safety was good.

(3) Tumor tissue apoptosis experiment (TUNEL): After administration and observation, one nude mouse was selected from each group, and the tumor was cut out to prepare frozen sections, and paraffin sections were prepared from other tissues. The tumor frozen sections were stained with reference to the TUNEL kit for apoptosis, the fluorescence was observed with a confocal microscope, and the fluorescence intensity was counted. The results are shown in Figure 4. The greater the fluorescence intensity, the greater the number of apoptotic cells in the tumor tissue, and the better the efficacy of the drug from another aspect. The results in Figure 4 show that the collagenase trastuzumab gel group has the strongest fluorescence intensity, indicating that the cells undergo the most apoptosis, which is consistent with the drug efficacy results.

(4) Collagen imaging of tumor tissue: After administration and observation, one nude mouse was selected from each group, and the tumor was cut out to prepare frozen sections, and paraffin sections were prepared from other tissues. The frozen sections of the tumor were labeled with collagen antibody, and the changes of collagen were observed by confocal microscopy. The results are shown in Figure 5. The greater the fluorescence intensity, the more collagen content in the tumor tissue, which shows that the drug effect is not ideal from another aspect. The results in Figure 5 show that the collagenase trastuzumab anti-gel group contains the least collagen, indicating that collagenase degrades the collagen in the tumor tissue and promotes the entry of the monoclonal antibody drug into the deep part of the tumor tissue, with the best drug effect.

Claims (10)

1. a monoclonal antibody gel combination, comprise: monoclonal antibody, temperature-sensitive material, extracellular matrix degrading enzymes and solvent, the weight ratio of monoclonal antibody and extracellular matrix degrading enzymes is 1:40-40:1, and the weight ratio of temperature-sensitive material and solvent is 1:10-2:1.

2. monoclonal antibody gel combination as claimed in claim 1, it is characterized in that, the weight ratio of monoclonal antibody and extracellular matrix degrading enzymes is 1:20-20:1, and the weight ratio of temperature-sensitive material and solvent is 1:10-1:1.

3. gel combination as claimed in claim 1, it is characterized in that, the percentage by weight that monoclonal antibody and extracellular matrix degrading enzymes account for monoclonal antibody gel combination is respectively: 0.01%-5% and 0.01%-5%.

4. gel combination as claimed in claim 1, it is characterized in that, the amount ratio of various composition is as follows:

5. monoclonal antibody gel combination as claimed in claim 1, it is characterized in that, described monoclonal antibody, comprising: one or more in Herceptin, Cetuximab, Avastin, Rituximab, handkerchief trastuzumab, Buddhist nun's trastuzumab, infliximab, adalimumab, gemtuzumab, her monoclonal antibody; Be preferably Herceptin;

Described temperature-sensitive material, comprise: poloxamer, PLA-PEG-PLA, poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide, polyethylene glycol-polylactic acid-Polyethylene Glycol, polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide-Polyethylene Glycol, PCL-PEG-PCL, NIPA polymer and Chitosan-phospholipid complex neutralize one or more, be preferably poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide, poloxamer188.

6. monoclonal antibody gel combination as claimed in claim 1, it is characterized in that, described extracellular matrix degrading enzymes, comprising: one or more in collagenase, hyaluronidase, Bacillus polymyxa Neutral proteinase, is preferably collagenase,

Described lyase is selected from: water, normal saline, the one in 5% D/W.

7. monoclonal antibody gel combination as claimed in claim 1, it is characterized in that, described compositions also comprises: buffer agent, stabilizing agent and non-ionic surface active agent.

8. the preparation method of monoclonal antibody gel combination according to claim 1, comprises the steps:

A. solvent and temperature-sensitive material is selected to prepare blank thermosensitive in situ gel;

B. blank thermosensitive in situ gel and monoclonal antibody and extracellular matrix degrading enzymes are fully mixed, dissolve, obtain monoclonal antibody gel combination.

9. the preparation method of monoclonal antibody gel combination as claimed in claim 1, it is characterized in that, step is as follows:

A. take 250mg poly (glycolide-co-lactide)-polyethylene glycol-Acetic acid, hydroxy-, bimol. cyclic ester lactide, add 1000mg water, under ice bath, be slowly stirred to abundant dissolving, obtain blank thermosensitive in situ gel;

B. take collagenase 20mg, Herceptin 7.5mg, join blank thermosensitive in situ gel respectively, under ice bath, slowly stir and collagenase and the appropriate pearl Dan Kekang of song are fully dissolved, obtain monoclonal antibody gel combination.

10. monoclonal antibody gel combination as claimed in claim 1, is characterized in that, in tumor, tumor week or intradermal injection.