WO2012019423A1

WO2012019423A1 - Angiogenesis inhibitor, method for purifying thereof and pharmaceutical composition comprising thereof

Info

Publication number: WO2012019423A1
Application number: PCT/CN2011/001000
Authority: WO
Inventors: 李小羿; 戴向荣; 杨中强; 张国辉; 方丽; 钱芳; 刘娟娟
Original assignee: 兆科药业(香港)有限公司
Priority date: 2010-08-13
Filing date: 2011-06-16
Publication date: 2012-02-16
Also published as: CN102372770B; CN102372770A

Abstract

The invention discloses an angiogenesis inhibitor, which has an amino acid sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2, and a molecular weight of 25000-35000 daltons as determined by non-reducing SDS-PAGE. The invention further discloses a purification method of the angiogenesis inhibitor by anion-exchange chromatography and immunoaffinity chromatography, and a pharmaceutical composition and an injection comprising the angiogenesis inhibitor.

Description

Angiopoietin, purification method thereof and pharmaceutical composition containing same

Technical field

The present invention relates to the field of biomedicine, and in particular to an inhibitor of angiogenesis, and more particularly, to an angiopoietin, a method for purifying the same, and a pharmaceutical composition containing the same. Background technique

Tumor angiogenesis (ang iogenes i s) refers to the complex biological process by which vascular endothelial cells differentiate and migrate from existing vascular systems to form new microvessels. Adult endovascular cells are basically at rest, and new blood vessels are formed under physiological stimulation such as wound healing, tissue repair, female fertility and menstrual period, fetal development, which is physiological angiogenesis. At this time, angiogenesis is under the strict control and coordination of stimulating factors and inhibitors. In an orderly physiological process within a limited time, the proliferating endothelial cells quickly return to a normal resting state. When the angiogenic regulation mechanism is out of control and angiogenesis is excessive, it becomes a causative factor, leading to the occurrence and development of angiogenesis-dependent diseases such as rheumatoid arthritis, diabetic or macular degeneration retinopathy, infantile hemangiomas, and malignant tumors. As early as 1971, Folkman clarified the importance of tumor angiogenesis in tumor development, metastasis and spread, and suggested that inhibitors of angiogenesis may become a new and valuable treatment for cancer. Currently, disrupting angiogenesis plays an important role in cancer research. Endogenous and exogenous tumor angiogenesis inhibitors provide a new clinical drug orientation for the treatment of such diseases.

Snake venom is a venom secreted from the venom gland of snakes. It mainly contains proteins, peptides and some enzymes, and has a wide range of biological activities. At present, many components of snake venom have been studied intensively, and they have a good effect on antithrombotic, hemostasis, analgesic and anti-tumor. Since the separation and purification of snake venom in 1965, it has been possible to find specific anti-tumor components that have entered cell and molecular levels, many of which have been found to have varying degrees of in vivo or in vitro anticancer activity. . Persistent angiogenesis is a feature of tumor growth. Angiogenesis is not only necessary for tumor growth, but the contact of tumor cells with the neovascular system is also responsible for distant metastasis. In recent years, the treatment of tumors has mainly relied on the development of new drugs and gene therapy. Such as the Chinese patent entitled "angiogenesis inhibitory peptide and its preparation method and application"

2QQ61Q (929S. 2 discloses a polypeptide obtained by modifying the amino acids 6 to 49 of endostatin, which has stronger in vivo antitumor activity and tumor targeting than endostatin. Although the E. coli expression system used in the invention has a high expression level, the obtained inclusion body protein is difficult to dissolve and difficult to reconstitute, which is easy to cause inconvenience; even if the protein is refolded and soluble, the process will lose a large amount of protein, resulting in low utilization rate. In addition, endostatin has a short half-life in vivo, and the preparation process of the angiogenesis-inhibiting polypeptide is complicated and relatively high in cost, which is not conducive to large-scale production. Therefore, some malignant degrees are high, the surgical cure rate is low, and It is very important to actively seek new safe and effective anti-tumor drugs for tumors that are not sensitive to radiotherapy and chemotherapy.

The present invention aims to solve at least one of the technical problems existing in the prior art. Accordingly, it is an object of the present invention to provide an angiopoietin which achieves an antitumor effect by inhibiting tumor angiogenesis.

Another object of the present invention is to provide a method for purifying the angiopoietin. It is still another object of the present invention to provide a pharmaceutical composition for inhibiting tumor angiogenesis to achieve an antitumor effect.

In order to achieve the above object, the present invention provides an angiopoietin having an amino acid sequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, having non-reducing SDS-PAGE (non-reducing polyacrylamide gelation) The molecular weight between 25,000 ~ 35 t) D0 Daltons determined by gel electrophoresis) has anti-angiogenic activity.

The angiopoietin according to the present invention can effectively inhibit unwanted angiogenesis, particularly angiogenesis associated with tumor growth, and is more effective in antitumor and tumor metastasis.

According to an embodiment of the present invention, the angiopoietin is reduced by SDS-PAGE (also The original polyacrylamide gel electrophoresis method showed two bands, called CX chain and β chain, respectively, and the molecular weights were 12000 ~ 22000 Daltons and 9000 ~ 199000 Daltons respectively. The amino acid sequence of the α chain was as follows. The amino acid sequence of the β chain is shown in SEQ ID NO.

According to an embodiment of the present invention, the amino acid sequence is substituted, deleted, or added by one or several amino acid residues and has a protein derived from angiogenic activity.

The angiopoietin of the present invention can improve the amino acid sequence under the condition that the angiogenesis activity is not affected, for example, mutating, deleting or adding one or several amino acids to one or several amino acids respectively does not affect the activity of the protein. Even some amino acid changes may optimize the properties of angiogenesis inhibition, which can be achieved by standard procedures by those skilled in the art.

According to an embodiment of the present invention, the angiopoietin is produced by two-dimensional electrophoresis to produce three isomers corresponding to the α chain and the β chain, respectively.

After two-dimensional electrophoresis, i.e., isoelectric aggregation electrophoresis and reduction SDS-PAGE electrophoresis, the hematopoietic tuberogen can obtain six bands, wherein the o chain and the β chain each have three bands. The identification of the six bands by mass spectrometry indicates that they are isoforms of the two subunits α and β, respectively, that is, the protein will produce isomers due to its structural specificity under isoelectric aggregation conditions. As a result, six bands appear in the two-dimensional electrophoresis, and there is no single isoelectric point.

According to an embodiment of the present invention, the angiopoietin specifically binds to the secreted monoclonal antibody by hybridoma cell line 1D91D9, accession number CCTCC C201036, and the main resistance of the monoclonal antibody secreted by the hybridoma cell line is Angiopoietin specifically binds. The hybridoma was deposited on April 14, 2010 at the China Center for Type Culture Collection of Wushan University, Wuhan, Wuchang, Hubei Province, under the name Hybridoma Cell Line 1D91D9, and the accession number is CCTCC C201036.

According to an embodiment of the present invention, the angiopoietin is capable of inhibiting tumor growth, wherein the tumor is selected from the group consisting of human melanoma, He la cells, and K562 cells. The snake venoms related to anti-tumor effects reported in domestic and foreign literatures are the most cobra and python, and the golden snake, the scorpion snake, and the rattlesnake venom. At present, the source of snake venom is mainly to take snakes or snakes to extract snake venom. However, snake venoms are different in species, different in origin, and even snakes of the same species. The composition of the venom is different during the collection period. Therefore, the purification process of snake venom is difficult to fix. The barramundi is the only one of the genus Aphididae, also known as the snake, which is widely distributed in China. Among them, the Wuyishan Mountain Area and the Weinan Mountain Area have the most storage. Living in mountainous or hilly woods and lush places, the vertical distribution range is 100 - 1350 meters above sea level. The Agkistrodon acutus venom used in the present invention is purchased from Huizhou Institute of Venom and Venom in Huangshan City.

In order to obtain high-purity angiopoietin, after repeated studies, the present invention adopts the principle of modern immunology, combines antigen-antibody specific reaction and affinity chromatography technology, and separates and purifies angiopoietin from the crude venom of sharp python. . The method first extracts a crude solution of angiopoietin from the crude venom of Agkistrodon acutus, and then obtains the crude product of angiogenin by cation exchange chromatography and gel chromatography respectively; The primary purified substance is an antigen, and a specific monoclonal antibody specific thereto is prepared; and then coupled to an appropriate affinity chromatography carrier to prepare an antibody affinity chromatography column; finally, the antibody affinity chromatography obtained by coupling is used. The column can be subjected to immunoaffinity chromatography on a crude solution of angiopoietin having an inhibitory activity on angiogenesis to prepare a high-purity angiopoietin.

Preferably, the method for purifying the angiopoietin of the present invention comprises the following steps:

(a) Dissolving the crude venom of Agkistrodon acutus in a buffer, wherein the volume ratio of snake venom to buffer is 1~2:10, and the supernatant is obtained by centrifugation;

(b) the resulting supernatant is subjected to DEAE (diethylamine ethyl) anion exchange chromatography to obtain a crude solution of angiopoietin;

(c) the hybridoma cell line 1D91D9, the accession number CCTCC C201036, the secreted monoclonal antibody is conjugated to the affinity chromatography carrier to prepare an immunoaffinity chromatography column;

(d) Purifying step (b) The obtained crude angiogenic solution is eluted with the obtained immunoaffinity chromatography column, and eluted with an elution buffer to collect the angiopoietin.

Wherein, the buffer in the step (a) is a 0.01 to 0.1 lmol/L Tris-HCl buffer solution having a pH of 7.0 to 9.0. The Agkistrodon acutus was centrifuged twice for centrifugation at a rate of 4000 r/min and centrifuged for 15 min each time.

Step (b) Linear gradient elution with 0.01-0.05 mol/L Tris-HCl buffer and 0.05-0.6 mol/L NaCl solution at pH 6.0-9.0 as eluent. The affinity chromatography column carrier of the step (c) is any one of a 4B agarose gel, a diethylaminodextran gel, and a carboxymethyldextran gel.

The elution buffer of the step (d) is pH 2. 0 ~ 4. 0, 0. 01 0. 03 mol / L G ly-HCl buffer, the elution time is 480 min, and the elution flow rate is 5 ml /mi.n.

The present invention also provides a pharmaceutical composition for inhibiting tumor angiogenesis to achieve an antitumor effect. The pharmaceutical composition contains angiopoietin and contains a pharmaceutically acceptable carrier. The present invention also provides an injection comprising angiostatin and a pharmaceutically acceptable excipient, a stabilizer.

According to an embodiment of the present invention, at least 1 mg of the angiogenin raw material is contained per ml of the injection, and then a pharmaceutically acceptable excipient, a stabilizer is directly or indirectly added by conventional processing to prepare an injection.

According to an embodiment of the present invention, the injection contains at least 1 mg of angiopoietin, 32 - 48 mg of dextran, and 1.6 to 2.4 mg of trehalose per ml.

The angiopoietin of the present invention has the following advantages: First, the specificity is strong. Cancer treatment currently uses surgery, radiotherapy, chemotherapy, biological therapy and combination therapy. The common features of various anticancer drugs that can be used for chemotherapy are the inhibition of the proliferation of all rapidly dividing cells, but the lack of anti-cancer cell proliferation. The specificity, while resisting the proliferation of cancer cells, has great toxic and side effects on the rapidly dividing cells in the gastrointestinal and other tissues and organs of the bone marrow. The anti-angiogenin of the invention has strong specificity, and can effectively inhibit tumor growth and metastasis by inhibiting unwanted angiogenesis, particularly angiogenesis related to tumor growth; secondly, high utilization rate, relatively precise preparation process Simple. Compared with the current preparation of angiogenesis inhibitors, the preparation process of the angiopoietin of the invention is simple, can overcome the shortcomings of short half-life of endogenous angiogenesis inhibitors, and avoids the cause of recombination or transgenesis. The problems of low transfection rate, low protein utilization rate and high cost, and high reproducibility and low cost are beneficial to industrial production. In addition, snake venom has anti-thrombotic, hemostasis, analgesic and its anti-tumor effect. It has received wide attention worldwide. China's snake venom resources are abundant and have broad research and application prospects. DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

The hybridoma cell line established according to the present invention was deposited on April 14, 2010 at the China Center for Type Culture Collection (CCTCC) of Wuhan University, Wuchang, Wuhan, Hubei Province, under the accession number C201036, and the classification is Hybrid. Tumor cell line 1D91D9.

Figure 1 is a flow chart showing the preparation process of angiopoietin according to the present invention;

2 is a UV absorption (280 nm) map of anion exchange column chromatography in an angiopoietin preparation process according to an embodiment of the present invention;

3 is a UV absorption (280 nm) map of cation exchange column chromatography in the preparation of angiopoietin according to an embodiment of the present invention;

Figure 4 is a diagram showing the ultraviolet absorption (280 nm) of gel S-100 column chromatography in the preparation process of angiopoietin according to an embodiment of the present invention;

Figure 5 is a chromatographic ultraviolet absorption (280 nm) map of an affinity chromatography column in the preparation of angiopoietin according to an embodiment of the present invention;

Figure 6 is a non-reducing SDS-PAGE electropherogram of angiopoietin according to an embodiment of the present invention;

Figure 7 is a two-dimensional electropherogram of angiopoietin according to one embodiment of the present invention (28-1, 28-2, 28-3, 28-4, 28-5 represent five band spots, respectively);

Figure 8 is a graph showing the inhibitory effect of angiopoietin on chicken embryo angiogenesis in a chicken chorioallantoic membrane model in accordance with one embodiment of the present invention;

Figure 9 is a graph showing changes in body weight of mice after 4 weeks of administration of an inhibitor of angiogenin against transgenic mouse colon cancer in accordance with one embodiment of the present invention;

Figure 10 is a graph showing the effect of angiopoietin on tumor mass in an in vivo pharmacodynamic test against angiogenic colon cancer in accordance with one embodiment of the present invention;

Figure 11 is a graph showing the effect of angiopoietin on the number of tumors of different sizes in an in vivo pharmacodynamic test against angiogenic murine colon cancer in accordance with one embodiment of the present invention. detailed description

The invention adopts the principle of modern immunology, combines antigen-antibody specific reaction and affinity chromatography technology to separate and purify angiopoietin from the crude venom of Agkistrodon acutus.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a flow chart showing the preparation process of angiopoietin according to the present invention. As shown in Figure 1: First, the crude venom of Agkistrodon acutus is dissolved in the buffer for a certain period of time, and then the supernatant is centrifuged to obtain a solution of Agkistrodon acutus venom; the resulting snake venom solution is anion exchange chromatography to inhibit angiogenesis. a crude solution of the prime, followed by taking an appropriate amount of the crude solution through cation exchange chromatography and gel chromatography to prepare an initial product of angiopoietin; and then, using the pure product as an antigen, preparing a specific single for the same The antibody is cloned; then, coupled with a suitable affinity chromatography carrier to prepare an antibody affinity chromatography column; finally, the previously prepared antibody affinity chromatography column is used to inhibit the angiogenic activity. The crude angiogenin solution is subjected to immunoaffinity chromatography to prepare a high-purity angiopoietin.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention in any way. Certain changes and modifications made by those skilled in the art within the scope of the claims are also considered to be within the scope of the invention.

Example 1 Preparation of High Purity Angiopoietin

1, the treatment of snake venom

First, accurately weigh 5g of Agkistrodon acutus venom from the Huizhou Institute of Venoms in Huangshan City, and dissolve it in a 4°C water tank with 0.02mol/L pH 8.0 Tris-HCl buffer. The dissolution time is 6~ After 12 h, it was centrifuged at 4000 r/min, and centrifuged twice, each time for 15 min, and the supernatant was collected for use.

2. Preliminary purification of the antigen used to prepare the monoclonal antibody

(1) Anion exchange chromatography

First, the obtained supernatant was applied to an equilibrated anion exchange chromatography DEAE column at a flow rate of 3.0 ml/min. After loading, it was first eluted with 0.02 mol/L pH 8.5 Tris-HCl buffer. The heterogeneous protein adsorbed by the medium was eluted for 360 min, and the elution flow rate was 3 ml/min. Then, 0.5 mol/L NaCl was added to the 0.02 mol/L pH 6.5 Tris-HCl buffer. Gradient elution was performed, the gradient was 0-100%, the elution time was 1440 min, and the elution flow rate was 5 ml/min; finally, the components having the activity of inhibiting angiogenesis were collected. The protein detection was carried out by a purification system (AKTA prime plus) with a UV detector at 280 nm. The UV absorption spectrum is shown in Figure 2. The collection was collected by the system's fraction collector. The collection was set to 4 min/tube. A crude solution of angiopoietin.

(2) Cation exchange chromatography

First, the collected crude anti-angiogenin solution is ultrafiltered with a membrane having a molecular weight cut off of 10,000, concentrated to a volume of 30-80 ml, and transferred to a cooked dialysis bag, and placed in a refrigerator at 4 ° C. Internal dialysis for 6~12h. Then, the sample obtained after dialysis was applied to an equilibrated cation exchange chromatography CM (carboxylated cellulose) column at a flow rate of 3.0 ml/min, and then loaded with 0.02 mol/L Tris-pH 6.5. The HCl buffer eluted the heteroprotein that could not be adsorbed by the chromatographic medium, the elution time was 360 min, the elution flow rate was 3 ml/min, and then 0.5 mol/L was added to the 0.02 mol/L Tris-HCl buffer at pH 8.5. The gradient elution (0-100%) was carried out with NaCl, the elution time was 1440 min, and the elution flow rate was 5 ml/min. Finally, components having an activity of inhibiting angiogenesis are collected. The protein detection was carried out by using the purification system AKTA prime plus with a UV detector at 280 nm. The UV absorption spectrum is shown in Figure 3. The collection was collected by the system's partial collector, and the collection was set to 4 min/tube. The first product of pheromone.

(3) Gel chromatography

First, the collected anti-angiogenin initial solution is ultrafiltered with a filter having a molecular weight cut off of 10,000, concentrated to a volume of 30-80 ml, and then subjected to a millipore ultra series centrifugal ultrafiltration tube at 4 ° C for low temperature. Concentrate by centrifugation ultrafiltration and concentrate the volume to 5~15ml. Then, the sample obtained after concentration was applied to an equilibrated gel chromatography S-100 column at a flow rate of 1.0 ml/min, and 0.15 mol/L was added to a 0.02 mol/L pH 7.6 Tris-HCl buffer. The NaCl was eluted with an elution time of 960 min and an elution flow rate of 0.3 ml/min. Finally, components having inhibitory angiogenic activity are collected. The detection method is the same as above. The protein detection is detected by the purification system AKTA prime plus with UV detector at .28Gnm. The UV absorption spectrum is shown in Figure 4. The collection is collected by the system with the fraction collector, and the collection is set to lOmin/tube. The obtained sample is an initial pure product of angiopoietin. 3. Preparation of angiopoietin-specific monoclonal antibody

(1) Identification of antigen: The obtained pure product of angiopoietin was first identified by SDS-PAGE electrophoresis, and its purity was not less than 85%, which showed a main band, and the activity detection inhibited ristocetin induction. After platelet aggregation, it can be used to prepare monoclonal antibodies.

(2) Animal immunization of antigen: 6 to 12 weeks old Ba lb/c mice were used to immunize the antigen and divided into three immunizations. For the first time, each mouse was injected with 0. lmg (about 0. 2ml) plus an equal amount of Freund's complete adjuvant for intraperitoneal immunization; after 2-4 weeks, boost the immunization, reduce the amount by half, and switch to incomplete adjuvant. The third impact immunization was performed 3 days before the fusion, and each mouse was 0.03 mg. The successful fusion was marked by the spleen providing a specific B cell in a proliferative state during fusion, and indirect ELISA (indirect enzyme coupling) Immunosorbent assay) detects antibody titers in serum.

(3) Establishment of hybridoma cell lines: First, the spleen cell suspension and myeloma cells of the immunized mice were fused in a conventional ratio by a ratio of 1:5 under the action of polyethylene glycol. Then, the hybridoma cell culture supernatant was detected by indirect ELISA, and the positive clone was screened, then subcloned, and expanded and frozen. Subsequently, after three times of limiting dilution cloning, the hybridoma cell line secreting the specific antibody was extensively expanded and frozen, and after long-term subculture, the same method was used to clone the sputum. The established hybridoma cell line was deposited on April 14, 2010 at the China Center for Type Culture Collection, Wuhan University, Wuchang, Wuhan, Hubei Province.

(CCTCC), the accession number is C201036, and the classification is named hybridoma cell line 1D91D9.

(4) Preparation and purification of monoclonal antibody: The hybridoma cell strain obtained above is cultured in vitro in a serum-free medium, and the obtained culture solution is subjected to Prote in A, recombinant Staphylococcus aureus protein A, coupled. The affinity chromatography column is purified by affinity chromatography to obtain a specific monoclonal antibody against angiopoietin.

4. Preparation of angiopoietin-specific monoclonal antibody affinity chromatography column

First, the purified anti-angiogen monoclonal antibody was dialyzed against 0. lmol/L sodium citrate buffer at 4 ° C overnight, and then added with 10 ml of CNBr-Sepharose 4B, cyanogen bromide-conjugated agarose. Gel 4B, to 10 ml of antibody solution, was placed on a shaker or shaker and gently shaken overnight at 4 °C. Then, 1 ml of a 2 mol/L ethanolamine solution was added to block the residual active group of CNBr-Sepharose 4B, and shaking was continued for 1 hour at 4 °C. Subsequently, the antibody-conjugated Sepharose 4B (Sepharose 4B) column was loaded onto an appropriate column. The column was washed with 0. lmol/L of sodium citrate buffer, followed by elution with twice column volume of 0.1 mol/L, pH 6.5, NaCl sodium citrate buffer containing 1 mol/L. Finally, elute with twice the column volume of PBS (phosphate buffer). In addition, the gel should always be kept below the liquid level during use and storage, and should not be dried.

5. Preparation of high purity angiopoietin

First, the crude solution of the angiopoietin obtained by the anion exchange chromatography in step 2 (1) is subjected to ultrafiltration and concentration, and then the affinity buffer is used to equilibrate the buffer, specifically 0.02 mol / L of Tr i s-HCl. I. dialysis, pH 7.8, containing 0.05 mol / L of NaCl. The antibody is then affinity-column and equilibrated with the affinity chromatography equilibration buffer described above until the eluent elution profile is shown as baseline after UV detection. Finally, the activity peak was collected by elution with Gly-HCl buffer at pH 4.0, 0.02 mol/ml, glycine-hydrochloric acid buffer as an affinity chromatography eluate. The detection method is the same as above. The protein detection is detected by the purification system AKTA pr ime p lus with UV detector at 280mn. The UV absorption spectrum is shown in Figure 5. The collection is collected by the system's branch collector. The collection is set to 1⁄2in/ Tube, the obtained sample is a high-purity angiopoietin. The above preparation method was repeated 10 times, numbered 1-10 groups.

Example 2 Study on the quality of the angiopoietin of the present invention

1. Purity and structure identification

Purity identification: a group of high-purity angiopoietin group 3 samples obtained in Example 1 were randomly selected for non-reducing SDS-PAGE electrophoresis (polyacrylamide gel electrophoresis), as shown in Fig. 6, showing a band , the molecular weight is 30,000 35,000 Daltons.

Structural identification: The third group of samples extracted above were subjected to two-dimensional electrophoresis, ie, isoelectric aggregation electrophoresis and reduction SDS-PAGE electrophoresis. As shown in FIG. 7, the angiopoietin can obtain six bands after two-dimensional electrophoresis. Wherein the alpha chain and the beta chain each have three bands. The identification of six bands by mass spectrometry showed that they are isoforms of two subunits, that is, the isoforms of the protein due to its structural specificity under isoelectric aggregation conditions, resulting in Six strips appear in two-dimensional electrophoresis and do not have a single isoelectric point.

2, neurotoxicity, hemorrhagic toxicity, endotoxin testing (1) Test criteria for angiopoietin hemorrhagic toxicity: The three groups (Group 1, Group 4, Group 9) obtained in Example 1 were randomly selected to obtain high-purity angiopoietin, respectively, and sodium chloride injection was used respectively. Prepare a solution containing 20 units per ml; then select 15 mice with a body weight of 18-22 g, 5 samples per group, subcutaneously inject 0.2 ml of the solution; then, sacrifice the animals 24 hours after the injection. , peeling observation, no bleeding on the back of the mouse.

After testing, the high-purity angiopoietin samples tested were in compliance with the regulations.

(2) Anti-angiogenin neurotoxicity test criteria: Take the appropriate amount of high-purity angiopoietin in group 1, group 4, and group 9 above, and prepare 20 units per milliliter with sodium chloride injection. Solution; then select 9 pigeons with a body weight of 300 ~ 500g, 3 samples per group; the dose used is 0.5 ml per kg injection, intravenously, for 24 hours, the animals shall not have convulsions or death. For example, in each of the three groups, one of the three patients had convulsions or death, and another five retests were performed, and no death occurred.

(3) Detection standard of angiopoietin endotoxin: Take the appropriate amount of high-purity angiopoietin in the first group, the fourth group, and the fifth group, and check according to the XIE of the Chinese Pharmacopoeia 2005 edition.

After testing, the endotoxin content per unit sample is not higher than 20EU.

Example 3 High-purity angiopoietin drug substance content detection method

In order to detect the content of anti-angiogenin in serum for pharmacokinetic experiments, a method for detecting the content of angiopoietin drug substance by double antibody sandwich method was established, and the sensitivity of the method was 1 ng/ml.

The specific detection methods are as follows:

1. Coated with a monoclonal antibody: firstly dilute the monoclonal antibody prepared in Example 1 with a 0.05% molar solution of pH 9.6 in carbonate buffer, and react in each polystyrene plate. Add 0.1 ml to the wells at 4 ° C overnight or for 18-24 h. On the next day, the solution in the well was discarded and washed 3 times with a washing buffer for 3 minutes each time (abbreviated as washing, the same below).

2. Loading: Add the sample to be tested or make a standard reference for proper dilution in the reaction well, and make blank, negative and positive well controls. Incubate at 37 ° C for 1 hour and wash. 3. Add another monoclonal antibody with HRP labeling, ie horseradish peroxidase labeling: Add another self-prepared angiopoietin monoclonal antibody that has been properly diluted to the ELISA plate. , 0. 1ml per well, incubate at 37 °C for 30 ~ 60min, wash.

4, the color of the substrate is added to the above reaction wells to temporarily prepare 0PD (o-phenylenediamine) substrate reaction solution 0.1 ml, reaction at 37 ° C for 10 ~ 30min, plus 2mol / L 0. 05ml H ₂ S0 ₄ to terminate the reaction.

6. Judgment of results: The above-mentioned ELISA plate was placed on a microplate reader to measure the 0D value at 450 mn. A standard curve is drawn based on the relationship between the 0D value and the concentration of the corresponding known concentration of the reference substance. Then, the 0D value measured by the sample to be tested is substituted into the standard curve to determine the concentration of angiopoietin in the corresponding sample. The detection sensitivity of this method can reach lng/ml.

Example 4 Preparation method of angiopoietin for injection

1000 ± 20mg

20 ± 4g

1 ± 0. 2g

500ml

Packed in accordance with 0.5 ml / branch, a total of 1000 pieces.

Firstly, in the local 100-level clean zone, the angiostatin prepared according to the first embodiment of the present invention and qualified by the test is used as a raw material medicine according to the formula, and the auxiliary materials such as dextran are accurately weighed in the liquid-dispensing container, and the injection is added. The mixture was sufficiently dissolved or diluted with water to make the concentration of dextran 1%, and the mixture was stirred and mixed to obtain a preparation intermediate. Then, sterile filtration, in which the integrity of the sterilization filtration system should be tested before and after filtration. Subsequently, the filtered intermediates are loaded into the vials according to the loading, and at least 4 loadings should be checked during the dispensing process to ensure that the amount of the drug loaded in each bottle is accurate. Finally, the vials filled with the drug are lyophilized, vacuumed, and slowly warmed to 35 to 40 ° C under vacuum, and then kept down to room temperature for a certain period of time and taken out, and the finished product is pressed and rolled.

After the finished product has been inspected and meets the anti-angiogenin shield standard for injection, it is put into stock. Example 5 Pharmacodynamic evaluation of angiopoietin for injection

The injection angiopoietin prepared in Example 4 was used in the following experiment.

1. In vitro evaluation of chicken chorioallantoic membrane model

Taking the chicken chorioallantoic membrane as a model, the positive control drug VEGF (vascular endothelial growth factor) and the corresponding doses were mixed together as the experimental group. Three trials were performed, and the results showed that as shown in Figure 8: (1) VEGF The dose after mixing with the drug showed a dose relationship with the inhibitory effect on the angiogenesis of the chicken embryo. With the increase of the dose, the inhibitory effect was more obvious. The 10 ng drug did not significantly inhibit the blood vessel growth, and the 25 ng had a certain inhibitory effect, 50 and l O. Ong inhibition effect is more obvious, and in large doses, the 500ng dose still does not kill the chicken embryo, so the lethal dose exploration still needs to further increase the dosage. (2) In the case where the drug and its monoclonal antibody (excess) are fully combined and administered, the drug has no inhibition on the growth of chicken embryo blood vessels, and is as strong as the blood vessel growth in the positive control group, indicating that the blood vessel growth is inhibited by the action of the drug. . At the same time, when the drug and its monoclonal antibody (excess) were fully combined and administered, the drug did not substantially inhibit the growth of chicken embryo blood vessels, and the blood growth of the positive control group was as strong.

2, cytology test '

The effects of drugs on proliferation, differentiation, migration, adhesion, etc. of HUEVCs (human umbilical vein endothelial cells) were tested in vitro. The results showed that 5 y mo l /L samples were induced by FBS (fetal bovine serum) and VEGF. Cell proliferation has no obvious inhibitory effect, but it has a certain inhibitory effect on the tubular network structure induced by VEGF and FBS. Cell chemotaxis assay shows that angiopoietin induces FBS, FGF (fibroblast growth factor) and VEGF. The cell migration movement has a significant inhibitory effect, and at high doses, the migrated cells return to baseline levels. Angiopoietin significantly reduced the growth factor-induced HUVECs cell invasion through the matrigel surface, and this decrease was dose-dependent with the dose administered. In order to determine whether angiopoietin is an integrin receptor inhibitor, an adhesion test of HUEVCs cells was designed, using a known integrin inhibitor as a control group, and the results showed that the sample was integrin avb 3/ Avb5 mediated adhesion did not significantly affect.

The results of the above two cytological tests indicate that angiopoietin has a direct and significant inhibitory effect on endothelial cell differentiation, migration and invasion, but there is no proliferation and adhesion to endothelial cells. Significant impact. This suggests that the angiopoietin mechanism is not significantly associated with growth factor (VEGF and FGF2) or tyrosine protease receptor-related signaling pathways.

3, MTT (3- (4, 5-dimercaptothiazole_2)-2, 5-diphenyltetrazolium bromide; trade name: thiazole blue) method for detection of melanoma, Hela cells and K562 Inhibition.

The results of in vitro inhibition of angiopoietin on melanoma, Hela cells and K562 cells indicate: relative avastin (Avastin, also known as bevacizumab, human vascular endothelial growth factor VEGF monoclonal antibody) and cisplatin, angiogenesis The pheromone has a significant inhibitory effect on melanoma A375 cells, and also has a significant inhibitory effect on Hela cells, and the inhibitory effect on K562 cells is not obvious.

4, anti-transgenic mouse colon cancer in vivo efficacy test

In the transgenic mice, colon cancer was implanted, and from the 13th week after the implantation of the tumor cells, to the 17th week, the inhibitory effect of the drug on the tumor cells was evaluated by observing the colon section. The mode of administration was intraperitoneal injection twice a week for 4 weeks. The experimental design was control group (normal saline, 3 rats), test group 1 (low dose group, 10 yg, 3 rats), test group 1 (high dose group, 45yg, 3 rats), results examination method ^ microscopy The main parameters of the method, detection and determination include: mouse body weight change, number of tumor nodules, effective solid tumor size.

The results showed that: (1) As shown in Fig. 9, there was no significant difference between the control group and the test group, indicating that the low-dose and high-dose test drugs had little toxicity to mice. (2) As shown in Figure 10, both doses of the experimental group were able to significantly and effectively inhibit the total number of tumor nodules. The inhibition rate of the low dose group was 16.22%, and the high dose inhibition rate was 28.64%. (3) As shown in Fig. 11, both dose groups in the experimental group can significantly and effectively inhibit solid tumors of size l_2mm. The above results indicate that the drug can effectively inhibit the growth of colon cancer in the colon cancer model of transgenic mice.

Example 6 Evaluation of Acute Toxicity of Angiopoietin for Injection

The maximum tolerated or median lethal dose (LD ₅ ) was initially estimated by observing the acute toxicity of a single intravenous injection of angiopoietin into Kunming mice.

Eight Kunming mice were used for the experiment, and four dose groups were set up, two in each group, half male and half female. Intravenous administration, the administration volume is 0.25mL/10g, and the administration dose is 14.5, 10.88, 8.16, 6. 12 mg/kg body weight, continuous observation for 4 days after drug administration.

The results showed that all animals were given a good mental state and normal activity after a single intravenous injection of angiopoietin for injection. No animal death occurred during the test, and gross anatomy revealed no abnormalities in the organs of each tissue.

Under the conditions of this experiment, the maximum tolerated dose (MTD) of Kunming mice administered by single intravenous injection of angiopoietin for injection was greater than or equal to 14. 5 mg/kg body weight.

The above pharmacodynamic evaluation of angiopoietin for injection showed that the dose of VEGF and the drug after mixing was dose-dependent with the inhibitory effect on the angiogenesis of the chicken embryo, and the inhibitory effect was more obvious as the dose was increased; Angiopoietin has direct and significant inhibitory effects on endothelial cell differentiation, migration and invasion, and has no significant effect on endothelial cell proliferation and adhesion. In addition, angiopoietin for injection has different degrees of inhibition on melanoma, Hela cells and K562, and can effectively inhibit colon cancer growth in transgenic mice implanted into colon cancer model.

The above technical solutions only exemplify the preferred technical solutions of the technical solutions of the present invention, and some variations that may be made by those skilled in the art to some of them may embodies the principles of the present invention and fall within the protection scope of the present invention.

Claims

Claim

An angiopoietin, characterized in that the amino acid sequence thereof is selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2, and having a non-reducing SDS-PAGE method between 25,000 and 35,000 daltons. Molecular weight, with anti-angiogenic activity.

The angiopoietin according to claim 1, which is characterized by reducing SDS-PAGE as two bands, respectively called α chain and β chain, each having a molecular weight of 12,000 to 22,000 Daltons, respectively. 9000 ~ 19000 Daltons, wherein the amino acid sequence of the α chain is shown in SEQ ID NO. 1, and the amino acid sequence of the β chain is shown in SEQ ID NO.

The angiopoietin according to claim 2, wherein three of the isomers corresponding to the α chain and the β chain are respectively produced by two-dimensional electrophoresis.

The angiopoietin according to claim 1, which specifically binds to the secreted monoclonal antibody secreted by the hybridoma cell line 1D91D9, accession number CCTCC C201036, and the monoclonal antibody secreted by the hybridoma cell line. The primary resistance is specific binding to angiopoietin.

The angiopoietin according to claim 1, which is capable of inhibiting tumor growth.

The angiopoietin according to claim 5, wherein the tumor is selected from human melanoma, He la cells, and Κ562 cells.

7. The method for purifying angiopoietin according to any one of claims 1 to 6, which comprises the steps of:

(a) dissolving the crude venom of the scorpion snake in a buffer, wherein the volume ratio of the snake venom to the buffer is 1 to 2:10, and the supernatant is obtained by centrifugation;

(b) the supernatant is subjected to chromatography on a diethylamine ethyl anion exchange chromatography column to obtain a crude solution of angiopoietin;

(c) hybridizing the hybridoma cell line 1D91D9, the accession number CCTCC C201036, and secreting the monoclonal antibody to the affinity chromatography carrier to prepare an immunoaffinity chromatography column; (d) Purifying the crude antiangiogenic solution obtained in the step (b) using the obtained immunoaffinity chromatography column, eluting with an elution buffer, and collecting the angiopoietin.

The method according to claim 7, wherein the buffer (b) is a buffer of 0.9 to 0.1 mol/L of Tris-HCl buffer of H 7.0 to 9.0.

9. The method according to claim 7, wherein the step (&> the scorpion python is centrifuged once for 1 time, the centrifugation speed is 4000 r/min, and each centrifugation is performed for 15 minutes.

The method according to claim 7, wherein the step (b) comprises 0.01 to 0.05 mol/L of Tris-HCl buffer and 0.05 to 0.6 mol/L of NaCl solution at a chromatography pH of 6.0 to 9.0. A linear gradient elution was performed as an eluent with an elution time of I440 min and an elution flow rate of 5 ml/min.

The method according to claim 7, wherein the affinity chromatography column carrier of the step (c) is a 4B agarose gel, a diethylaminodextran gel, or a carboxymethyl dextran condensate. Any of the glues.

12. The method according to claim 7, wherein the elution buffer in step (d) is a Gly-HCl buffer having a pH of 2.0 to 4.0 and 0.01 to 0.03 mol/L.

A pharmaceutical composition comprising the antiangiogenic agent of claim 1 and comprising a pharmaceutically acceptable carrier.

An injection comprising the angiopoietin of claim 1 and a pharmaceutically acceptable excipient and stabilizer.

The injection according to claim 14, wherein the injection contains at least 1 mg of angiopoietin per ml.

The injection according to any one of claims 14 or 15, wherein the injection contains at least 1 mg of angiopoietin, 32 to 48 mg of dextran, and 1.6 to 2.4 mg of trehalose per ml.