Preparation inhibiting malignant tumour development and containing dis-a-fibrin

FIELD: medicine.

SUBSTANCE: preparation inhibiting malignant tumour development contains dis-A-fibrin and may inhibit malignant tumours by inhibiting distribution and migration of malignant tumour cells thereby may inhibit malignant tumour development.

EFFECT: inhibition of tumour invasion and dissemination.

8 cl, 4 ex, 2 tbl, 9 dwg

 

The SCOPE of the INVENTION

The present invention relates to the preparation inhibitory to the development of malignant tumors and including des-A-fibrin.

PREREQUISITES

In recent years therapy of malignant tumors is constantly being improved, increasing the percentage of successful removal of the primary tumor by surgery, radiotherapy or chemotherapy. However, even if the primary cancer is completely removed, often death associated with cancer metastasis.

In particular, melanoma, lung cancer, liver cancer, pancreatic cancer and other tumors with high malignancy is difficult to detect in the early stages, therefore, at the time of diagnosis of a malignant tumor primary tumor and metastases may already exist simultaneously, and in many cases surgical treatment is impossible. Radiotherapy does not give satisfactory results in the treatment of such malignancies. In addition, the majority of chemotherapeutic drugs that are in clinical use, such as adriamycin, operate by a direct attack tumour cells, but as they both focused on normal cells, that have strong side effects that preclude their clinical use. Thus, C the last decade, there has been discovered a fundamentally new drugs. To overcome this situation, it is expected the emergence of new drugs for treatment of malignant tumors.

In this context, recently, an assumption was made on the basis of a large number of basic and clinical research on hard dependencies between malignant tumors and blood coagulation and fibrinolysis systems. For example, it is known that patients with malignant tumors microcirculation damage due to elevated levels of fibrinogen in plasma, increased viscosity of the blood, abnormal blood rheology and other disorders of coagulation and fibrinolysis systems. It is also reported that the actual elevated levels of fibrinogen in plasma or secretion of fibrinogen cells of malignant tumors cause the deposition of fibrinogen or fibrin in the extracellular matrix of malignant tumor tissues, and these factors act as part of the extracellular matrix, causing the proliferation, invasion and metastasis of tumour cells (see, for example,Cancer Research60:2033-2039(2000);Ann. NY Acad. Sci.936:406-425(2001); andBlood96:3302-3309(2000)).

Focusing on the above relationship between malignant tumors and blood coagulation and fibrinolysis systems, it was shown that the treatment of the malignant cells of the human whole fibrin in vitro, there is an increase in the impact of fibrin on experimental metastasis of tumour cells into the lungs (see, for example,Clin. Exp. Metastasis17:723-730(1999)). Fibrin (also called des-AB-fibrin or fibrin II) is a substance obtained by the action of thrombin on fibrinogen, causing the release of fibrinopeptide A (FPA) and fibrinopeptide B (FPB) from fibrinogen (see, for example,Nature275:501-505(1978) andBiochemistry35:4417-4426(1996)).

In addition, focusing on the relationship between the concentrations of plasma fibrinogen and the growth and metastasis of malignant tumors, report was made that the introduction combinatory enzymes batroxobin and ancrod that have defibrinate action, decreases the level of fibrinogen and inhibits the growth of malignant tumors and metastasis (see, for example,Eur. J. Cancer16:919-923(1980); andActa Haematol. Jpn.44:739-743(1981)). Batroxobin, combinatoria serine protease derived from the venom of the snakeBothrops atrox moojeniis a glycoprotein enzyme that releases from fibrinogen only FPA producing des-A-fibrin (also called fibrin I) (see, for example,Thromb. Haemost36:9-13(1976) andThromb. Diath. Haemorrh.45 (Suppl.):63-68 (1971)).

Based on the assumption that the fibrinogen acts as a barrier that protects cells PLN the quality of tumors from the attacks of the immune system, in the above links were attempted using techniques to inhibit the growth and metastasis of malignant tumors by reducing levels of fibrinogen combinatorily enzymes, thus facilitating the attack of the immune system on the cells of malignant tumors.

On the other hand, it is also known that cells of a malignant tumor characterized by the ability to spread and migration. In this case, the “spread” of tumour cells means that the rounded tumor cells form pseudopodia in response to some signal, that is the biological behavior of tumor cells and tumor growth, invasion and metastasis.

In addition, the migration of tumour cells indicates that the tumor cells leave the original place of their location through repeated binding and dissociation of ligands and cell adhesion molecules in the cell membrane in response to some signal, which is also the biological behavior and the basis for invasion and metastasis of tumors.

Therefore, by inhibiting the proliferation and migration of tumour cells can inhibit the invasion and metastasis of tumors and, thus, prevent the development of malignant tumor is.

However, nothing was reported about the drug that can inhibit the proliferation and migration of tumour cells for effective inhibition of the development of malignant tumors.

In addition, there were no reports of dependence between des-a-fibrin (the product of the decomposition of fibrinogen) and distribution, and migration of tumour cells.

Description of the INVENTION

Thus, the aim of the present invention is a drug that will inhibit the development of malignant tumors and containing a new active ingredient, which is a drug that will inhibit the development of malignant tumors.

To solve the above problems by the inventors, it has been assumed that the effect of des-a-fibrin on the cells of malignant tumors may be different from the action of fibrin, and after extensive research on the effect of des-a-fibrin on the distribution and migration of tumour cells they discovered that des-A-fibrin acts by inhibiting the proliferation and migration of tumour cells. The present invention based on this discovery.

Thus, the present invention relates to the preparation inhibitory to the development of malignant tumors containing des-A-fibrin.

BRIEF DESCRIPTION of DRAWINGS

On Phi is. 1 presents a photograph showing electrophoregram fibrinogen, des-a-fibrin and fibrin.

In Fig. 2 presents a graph showing electrophoregram Fig. 1, calculated using a system of image analysis.

In Fig. 3 shows a micrograph of melanoma cells, which were cultured in the wells and was treated with PBS (A), fibrinogen (B), batroxobin (C), thrombin (D), des-a-fibrin (E) and fibrin (F).

In Fig. 4 shows a graph of the degree of proliferation of melanoma cells, which were cultured in the wells and was treated with PBS (A), fibrinogen (B), batroxobin (C), thrombin (D), des-a-fibrin (E) and fibrin (F).

In Fig. 5 shows a micrograph of breast cancer cells, which were cultured in the wells and was treated with PBS(A), fibrinogen (B), batroxobin (C), thrombin (D), des-a-fibrin (E) and fibrin (F).

In Fig. 6 shows a graph of the degree of proliferation of breast cancer cells, which were cultured in the wells and was treated with PBS (A), fibrinogen (B), batroxobin (C), thrombin (D), des-a-fibrin (E) and fibrin (F).

In Fig. 7 shows a micrograph of cells fibrosarcoma, which were cultured in the wells and was treated with PBS (A), fibrinogen (B), batroxobin (C), thrombin (D), des-a-fibrin (E) and fibrin (F).

In Fig. 8 shows a graph of the degree distribution of cells fibrosarcoma is, which were cultured in the wells and was treated with PBS (A), fibrinogen (B), batroxobin (C), thrombin (D), des-a-fibrin (E) and fibrin (F).

In Fig. 9 presents a graph showing the effect of the number of des-a-fibrin on the proliferation of melanoma cells.

The BEST WAYS of carrying out the INVENTION

The present invention is explained in detail below.

A drug that will inhibit the development of malignant tumors of the present invention, contains des-A-fibrin as an active ingredient.

Des-A-fibrin is a substance, resulting from the release of FPA from fibrinogen.

Fibrinogen is a dimeric glycoprotein (AαBβγ)2formed through disulfide linkage of two subunits (AαBβγ), each of which consists of three circuits: circuit-Aα, chain-β and chain-γ, which are connected by disulfide bonds. As the chain-Aα consists of 610 amino acids (68 kDa), chain-Bβ of 461 amino acids (54 kDa) and chain-γ of 411 amino acids (48 kDa), the molecular weight of fibrinogen is a 340 kDa (seeThromb. Res.83:1-75(1996) and Blood Coagulation, Fibrinolysis and Kinin, Aoki, A. and Iwanaga, S. (Eds), Chugai Igaku Co., Tokyo, 1979, pp 59-71). It is also known that the chain-Bβ and chain-γ also have diabetes relationship, whereas in the circuit-Aα such a relationship is absent (seeInt. J. Biochem. & Cell Biol.31:741-746(1999)).

FPA represents a peptide corresponding to 16 Amin the acids (NH 2-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg) (Seq. ID No.1) at the amino end of the chain-fibrinogen Aα.

Therefore, des-A-fibrin is the remainder [(αBβγ)2], resulting from the release of FPA from fibrinogen (seeThromb. Haemost.36:9-13(1976) andThromb. Diath. Haemorrh.45 (Suppl.):63-68(1971)). Since the molecular mass of the FPA is 1536 Yes, the calculated molecular mass of des-a-fibrin is approximately 337 kDa.

For the release of FPA from fibrinogen can be used combinatoria enzymes. Trombonology the enzyme is a kind of serine protease. Commonly used enzymes derived from snake venom. Specific examples include batroxobin (Tobishi Pharmaceutical Co., Ltd. and Beijing Tobishi Pharmaceutical Co., Ltd., Beijing, China, a subsidiary of Tobishi Pharmaceutical Co., Ltd.), which is extracted and purified from the venom ofBothrops atrox moojeniand ancrod and other combinatoria enzymes (e.g., catalase), which are derived from snake venom, and the like. These combinatoria enzymes may be natural products or products of genetic recombination.

Des-A-fibrin can be obtained, for example, by the methods described in paragraphs (1)to(6) below, using fibrinogen and combinatory enzyme:

(1) the way in which get fibrinogen and attached by adhesion to the surface of the plastic vessel for culturing, the glass with the court for cultivation, subject glass vessel made of stainless steel or vessel for artificial blood artificial hard material EPTFE, or the like, and add combinatory enzyme with the receipt of des-a-fibrin;

(2) the manner in which the fibrinogen and combinatory enzyme added simultaneously to the above solid substance, receiving des-A-fibrin;

(3) the manner in which the fibrinogen and the enzyme type thrombin interact in a liquid reaction system in the presence of 0.1-15 N urea and/or anticoagulant peptide (Gly-Pro-Arg-Pro-amide, GPRP-NH2(SEQ. ID No:2)), receiving des-A-fibrin, thus preventing the agglutination of monomers des-a-fibrin (Clin. Exp. Metastasis17: 723-730 (1999));

(4) the manner in which the fibrinogen is attached to the column, and poured into the column a solution containing combinatory enzyme, receiving des-A-fibrin;

(5) the way in which terminology the enzyme attached to the column, and poured into the column a solution containing fibrinogen, receiving des-A-fibrin; and

(6) the way in which terminology enzyme is administered intravenously, intraperitoneally, subcutaneously, intramuscularly or the like, to affect fibrinogen in the body, giving des-A-fibrinin vivo(seeActa Haematol. Jpn.44:706-711 (1981)).

Fibrinogen and combinatory enzyme used to obtain the des-a-fibrin in the present the invention, themselves are known substances, which are commercially available or which can be easily obtained. Actually des-A-fibrin is also a known substance which can be obtained by the above methods.

A drug that will inhibit the development of malignant tumors of the present invention, targets malignant tumors. Depending on the source location of the tissue malignant tumors usually can be classified into epithelial malignant tumors and non-epithelial malignant tumors. It is believed that about 90% of tumors are epithelial. Non-epithelial malignant tumors can be further classified into malignant tumors derived from mesenchymal tissue, malignant tumors derived from the neural tissue, and malignant tumors originating from undifferentiated cells. Specific examples of malignant tumors of each type is given below.

Epithelial malignant tumors

Adenocarcinoma (cancer originating in glandular epithelium that occur anywhere on the body, including the stomach, intestines, pancreas, trachea, lungs, mammary glands, ovaries, body of the uterus, prostate gland, and the like, it is believed that they constitute 70-80% of cases placecast the military neoplasm), squamous cell carcinoma (cancer originating from stratified squamous epithelium found in the epithelial tissue of the epidermis, lips, tongue, throat, esophagus, anus, vulva, cervix, and the like, and pulmonary blaskapelle cancers are classified as non-small cell lung cancer, basal cell carcinoma, originating from the basal cells of the skin and appendages, transitional cell carcinoma (originating from the transitional epithelium, such as bladder cancer, carcinoma of the liver cells (originating from hepatocytes), renal cell carcinoma (originating from the renal epithelium), cholangiocarcinoma (originating from the bile ducts) and choriocarcinoma (originating from the placental epithelium).

Non-epithelial malignant tumor

Malignant tumors derived from mesenchymal tissue

Fibrosarcoma (originating from connective tissue and fibrous tissue), liposarcomas (originating from connective tissue and adipose tissue), chondrosarcoma (originating from connective tissue and cartilage), osteosarcoma (originating from connective tissue and bone), angiosarcoma (originating from blood vessels), lymphangiosarcoma (originating from lymphatic ducts), myelogenous leukemia (originating from hemopet the ical cells), monocytic leukemia (originating from hematopoietic cells), malignant lymphoma (originating from lymphoid tissue), lymphocytic leukemia (originating from lymphoid tissue), plasmacytoma (multiple myeloma, originating from lymphoid tissue), jackinsky cell (originating from lymphoid tissue), leiomyosarcoma (originating from smooth muscle), rhabdomyosarcoma (originating from striated muscle).

Malignant tumors derived from the neural tissue

Neuroblastoma (originating from neuroblastoma), medulloblastoma (originating from medulloblastoma), malignant astrocytoma (originating from astrocytes), retinoblastoma (originating from retinoblastoma), glioblastoma (originating from glioblastoma), malignant neurilemoma (originating from Swarovski cell), melanoma (originating from neuroectoderm).

A malignant tumor originating from undifferentiated cells

Malignant teratoma (originating from totipotent cells), nephroblastoma (originating from nephroblastoma), hepatoblastoma (originating from hepatoblasts), mixed tumor (originating from different cell types).

From the above malignant tumors of the drug that will inhibit the development of malignant tumors, according to the present invention, can be vsokoeffectiven the m against epithelial malignant tumors and against non-epithelial malignant tumors, derived from the neural tissue and mesenchymal tissue, especially melanoma, breast cancer and fibrosarcoma.

A drug that will inhibit the development of malignant tumors, according to the present invention, may inhibit the invasion and metastasis of tumors by inhibiting the proliferation and migration of tumour cells and thereby inhibiting the development of malignant tumors.

“Distribution” of tumour cells in this case means that the rounded tumor cells form pseudopodia in response to some signal, that is the biological behavior of tumor cells and the growth of her tumor invasion and metastasis.

In addition, the migration of tumour cells indicates that the tumor cells leave the original place of their location through repeated binding and dissociation of ligands and cell adhesion molecules in the cell membrane in response to some signal, which is also the biological behavior and the basis for invasion and metastasis of tumors.

A drug that will inhibit the development of malignant tumors, according to the present invention may contain the actual des-A-fibrin or des-A-fibrin other active ingredients.

Examples of other active agents include antimetabolites such as FPO is uracil, antitumor antibiotics such as adriamycin, alkylating agents such as dacarbazine, anticancer drugs of vegetable nature, such as paclitaxel and the like.

For the preparation of a drug that inhibits the development of malignant tumors, according to the present invention can be applied to any division of the Japanese Pharmacopoeia, General Rules for Preparations. Examples of drugs drugs inhibiting the development of malignant tumors, according to the present invention include injections for direct use in the body (including suspensions and emulsions); ointment (including fatty ointment, emulsion ointments (creams), water-soluble ointments and the like), means for inhalation, liquid (including ophthalmic solutions, solutions for irrigation of the nasal cavity and the like), suppositories, plasters, lotions, lotions and other preparations for external use; and the preparations for internal use, including tablets (including tablets with sugar, and film the gelatinous shell), liquids, capsules, granules, powders (including grains), pills, syrups, lozenges and the like. These drugs can be obtained by methods described in the Japanese Pharmacopoeia, General Rules for Preparations.

A drug that will inhibit the development of malignant tumors, according to the present invention may also include Farmak is logically acceptable solid or liquid carriers or bases for interventional therapy. Examples of pharmacologically acceptable solid or liquid carriers include solvents, stabilizers, preservatives, agents that promote dissolution, emulsifiers, suspendresume agents, buffering agents, isotonicity agents for tinted agents, bases, thickeners, fillers, lubricants, binders, disintegrating agents, covering agents, corrective agents and the like.

Specific examples include water, lactose, sucrose, fructose, glucose, mannitol, sorbitol and other sugars and sugar alcohols, crystalline cellulose, methylcellulose, ethylcellulose, hydroxypropylcellulose, nitrosamino hydroxypropylcellulose, hypromellose, phthalate of hydroxypropylmethylcellulose, acetylsuccinate hydroxypropylmethylcellulose, carmellose, calcium carmellose, the sodium carmelose, the sodium croscarmelose, carboximetilzellulozu, acetated cellulose and other cellulose and related derivatives, corn starch, wheat starch, rice starch, potato starch, dextrin, pre gelatinising starch, partially reptitiously starch, hydroxypropylmethyl, natrocarbonatite, cyclodextrin, pullulan and other starches and related derivatives, agar, sodium alginate, Arabic gum, gelatin, collagen, shellac, tragakant, can the new resin and other natural polymers (seaweed, vegetable glues, proteins and the like), polyvinylpyrrolidone, aminoalkylsilanes copolymer, methacrylic acid copolymer, carboxyvinyl polymer, polyvinyl alcohol, dimethylpolysiloxane and other synthetic polymers, olive oil, cocoa butter, Carnauba wax, beef tallow, gidrirovannoe oil, soybean oil, sesame oil, Camellia oil, paraffin, vaseline oil, yellow beeswax, white petrolatum, coconut oil, microcrystalline wax and other oils and fats, stearic acid, aluminum stearate, calcium stearate, magnesium stearate, triethylcitrate, triacetin, triglyceride fatty acids medium chain tallow, isopropylmyristate and other fatty acids and their derivatives, glycerin, stearyl alcohol, cetanol, propylene glycol, macrogol, and other alcohols and polyvalent alcohols, zinc oxide, dibasic calcium phosphate, precipitated calcium carbonate, synthetic aluminosilicate, silicic anhydride (silica), kaolin, dried gel, aluminum hydroxide, synthetic hydrotalcite, titanium oxide, talc, bentonite, aluminum silicate, magnesium aluminum potassium sulfate, subgallate bismuth subsalicylate bismuth, calcium lactate, sodium bicarbonate and other inorganic substances and compounds in the form of metal salts, esters of sucrose and fatty acids, polyxystra,polyoxyethylene gidrirovannoe castor oil, polyoxyethyleneglycol, servicesquot, sorbifolia, carpetmuncher, carbamidomethylated, arbitrageurs, Polysorbate, glycerylmonostearate, nutriceuticals, lauromacrogol and other surfactants, dyes, perfumes and the like.

Examples of bases for surgical intervention include implants vessels, artificial blood vessels and the like.

The number of des-a-fibrin contained in the product, inhibiting the development of malignant tumors, according to the present invention is applied depending on the composition. Examples include from 0.01 to 900 mg per 1 g in the case of the drug for domestic use, from 0.01 to 500 mg per 1 ml in the case of injections, or from 0.01 to 500 mg per 1 g in the case of the preparation for external use.

Injected dose of the drug that inhibits the development of malignant tumors, according to the present invention change depending on the weight of the patient, nature and severity of the disease and it is, for example, from 0.1 to 5000 mg or, preferably, from 100 to 2500 mg of des-a-fibrin per day for adults.

The present invention is described in detail below using examples, but is not limited to these examples.

[Example 1] Obtaining and identification of des-a-fibrin.

Des-A-fibrin get using combinatory enzyme and release of FPA from fibrinogen. Cu is IU, receiving des-a-fibrin set by comparing it with fibrinogen and fibrin.

(1) preparation of des-a-fibrin

Combinatory enzyme batroxobin (Tobarpin®, Beijing Tobishi Pharmaceutical Co., Ltd., Beijing, China) are added to a solution of human fibrinogen (F-4883, Sigma, MO, USA) with phosphate buffer (PBS), receiving the final reaction solution with a fibrinogen concentration of 3.0 mg/ml and the concentration batroxobin of 0.5 BU/ml, and incubated for 1 hour at 37°C with obtaining des-a-fibrin.

(2) Receiving fibrin

The PBS solution of thrombin (Sigma, MO, USA) are added to a PBS solution of human fibrinogen (F-4883, Sigma, MO, USA), obtaining the final reaction solution with a fibrinogen concentration of 3.0 mg/ml and the concentration of thrombin in 0.5 IU/ml, and incubated for 1 hour at 37°C with the receipt of fibrin.

(3) Obtaining fibrinogen

The PBS solution of fibrinogen with the final concentration of 3.0 mg/ml receive, using as control untreated human fibrinogen (F-4883, Sigma, MO, USA).

(4) Identification of the received des-a-fibrin

Production of des-a-fibrin in the above method (1) establish by electrophoresis. Specifically, it is determined that the fibrinogen is decomposed into chains of three types (chain-Aα, Bβ and γ) in the following processing, the receipt of the products assessed by electrophoresis after decomposition of des-a-fibrin on the chain is PEX types (chain-α, Bβ and γ) and decomposition of fibrin on the chain of three types (chain-α, β and γ).

Des-A-fibrin obtained in method (1), and fibrin obtained in method (2), three times washed with sterilized isotonic solution of sodium chloride, boiled for 5-6 minutes in a 0.5 ml solution of 2% SDS/2% beta mercaptoethanol/5M urea for the destruction of disulfide bonds and dissolve.

Fibrinogen obtained in method (3), is subjected to similar processing for the destruction of disulfide bonds, as described above, except the washing process, and dissolve by boiling.

To each sample solution (150 ál) add fifty microlitres electrophoretic buffer (4×2% SDS/0.1% bromcresol blue) and spend a 7.5% SDS-PAGE electrophoresis (Pagel®, Atto Corp., Tokyo, Japan).

The results are presented in Fig. 1 in the form of electrophoregram. In Fig. 1, lane 1 and lane 4 shows fibrinogen, lane 2 shows des-A-fibrin and lane 3 shows fibrin.

In each band, the lower band represents the chain-γ, the second lowest range is a chain-Bβ, and the third from the bottom of the range is a chain-Aα.

Evaluation of strip 2 (Des-A-fibrin) relative to band 1 and band 4 (fibrinogen) as the standard indicates that the position of the bottom third of the range (chain-Aα) on the strip 2 is shifted towards lower molecular weight). This indicates that the FPA is released from the chains of fibrinogen Aα when action is AI trombinoscope enzyme batroxobin. Provisions other ranges are consistent with the data of fibrinogen. Consequently, it is possible to bear in mind that in method (1) FPA released from fibrinogen and as a result discovered des-A-fibrin.

Evaluation of band 3 (fibrin) relative to band 1 and band 4 (fibrinogen) as standard indicates that the provisions of the second bottom range (chain-Bβ) and third from the bottom of the range (chain-Aα) on the strip 3 are shifted below (lower molecular weight). This shows that FPA and FPB are released from the chain-Aα chain and fibrinogen Bβ under the action of thrombin. Therefore, it is clear that in the method (2) is obtained fibrin.

These above results are consistent with previously published data (seeActa Haematol. Jpn.44:706-711(1981)).

The above electrophoregram calculated using image analysis (Furi Science & Technology Co., Ltd., Shanghai, China) and is shown in Fig. 2.

With regard to the three peaks in Fig. 2: the left peak shows the density range of the chain-Aα, average peak shows the density range of the chain-Bβ and right peak shows the density range of the chain-γ.

Evaluation of the left peak (chain-Aα) shows that the peaks of des-a-fibrin and fibrin match each other and are shifted towards lower molecular weight relative to the peak of fibrinogen. This means that in des-a-fibrin and fibrin FPA released from the chains of fibrinogen Aα./p>

Average peak (chain-Bβ) shows that the peaks of fibrinogen and des-a-fibrin match each other, whereas the peak of fibrin is shifted towards lower molecular weight. This means that in the fibrin FPB released from the chains of fibrinogen Bβ.

As indicated above, this means that the substance produced when the action batroxobin on fibrinogen ((AαBβγ)2), is a des-A-fibrin ((αBβγ)2), whereas the substance produced by the action of thrombin on fibrinogen, is a fibrin ((αβγ)2).

[Example 2] the Effect of des-a-fibrin on the proliferation of tumour cells.

To create ambient artificial extracellular matrixin vitrousing Matrigel® (BD Biosciences, NJ, USA) and to evaluate the effect of des-a-fibrin on the proliferation of tumour cells in this environment uses three types of cells of malignant tumors (melanoma, breast cancer and fibrosarcoma).

(1) Used cells from malignant tumors

As of melanoma cells will subcultured cells of malignant melanoma mice B16-BL6 (Academy of Chinese Medical Sciences, Beijing, China) in medium RPMI-1640 (Gibco, MD, USA)containing 10% fetal calf serum (FBS, HyClone, Utah, USA), and these cells are used in this experiment.

As breast cancer cells, subculture who are breast cancer cells mice MMT060562 (ATCC, VA, USA) in MEM medium (Gibco, MD, USA)containing 10% FBS and 1% solution of non-essential amino acids (Non-Essential Amino Acids Solution (Gibco, MD, USA), and these cells are used in this experiment.

As cells fibrosarcoma will subcultured cell fibrosarcoma human HT-1080 (Academy of Chinese Medical Sciences, Beijing, China) in MEM medium (Gibco, MD, USA)containing 10% FBS, and the cells used in this experiment.

(2) the Method of determining the distribution of cells

Two hundred fifteen microlitres Matrigel (Matrigel® to 7.5 mcg/ml, BD Biosciences, NJ, USA) is added to each well vosmiballnogo slides (Nunc, IL, USA) and incubated for 1 hour at room temperature to be applied on the glass cover of the Matrigel. Then in wells with Matrigel-coated type solutions A-F (0.25 ml), respectively.

A: Add saline phosphate buffer (PBS) (control and solvent).

B: Add fibrinogen (3 mg/ml).

C: Add batroxobin (0,5 BU/ml).

D: Adding thrombin (0,5 IU/ml).

E: Add des-A-fibrin (3 mg/ml fibrinogen and 0.5 BU/ml batroxobin).

F: Add fibrin (3 mg/ml fibrinogen and 0.5 U/ml thrombin)

In all the above cases is used to process the solvent is a PBS.

Used herein, the abbreviation BU (unit batroxobin) denotes the unit indicating the enzymatic activity batroxobin is; when activity 2 BU coagulation is achieved for 19,0±0.2 with, adding 0.1 ml of a solution batroxobin to 0.3 ml of standard human plasma, containing citric acid, at 37°C.

In cases A to D components added to the wells at the indicated final concentrations and incubated for 1 hour at room temperature, then remove the liquid and wash the wells PBS.

In cases E and F components are added to the wells at the indicated final concentrations, spend interaction 60 seconds, and remove the liquid before possible coagulation product (processing E: des-A-fibrin processing F: fibrin). This was followed by incubation for 1 hour at room temperature and washed by PBS. In the case of processing receive E des-A-fibrin under the action batroxobin on fibrinogen, despite the fact that in the case of treatment F get fibrin under the action of thrombin on fibrinogen.

Five thousand six hundred melanoma cells B16-BL6 suspended in medium RPMI-1640 containing no serum, were seeded in each treated well and incubate the slide with multiple cameras for 2 hours in CO2-incubator. Count the number of spreading cells (cells with pseudopodia) among approximately 150 cells, adherent to each treated well, using phase-contrast microscope, and counting the percentage of spreading cleto is how the degree distribution according to the following formula.

The degree distribution (%)=(the number of spreading cells/the number of adherent cells)×100

Similar methods apply to 5,600 breast cancer cells MMT060562, suspended in MEM medium containing no serum and containing 1% solution of non-essential amino acids, and 5600 cells fibrosarcoma HT-1080, suspended in MEM medium containing no serum, instead of 5600 melanoma cells B16-BL6, suspended in medium RPMI-1640 containing no serum.

(3) Inhibition of proliferation of melanoma cells

In Fig. 3 shows a micrograph (45-fold increase) of melanoma cells, cultured for 2 hours in each of the differently treated well. In Fig. 4 shows the extent of spread of melanoma cells cultured in each differently treated well. The majority of melanoma cells formed pseudopodia and they are distributed (Fig. 3) in the presence of PBS (A), fibrinogen (B), batroxobin (C), thrombin (D) and fibrin (F) with a prevalence of more than 80% in each case (Fig. 4).

On the other hand, in the presence of des-a-fibrin (E) the majority of treated melanoma cells remain rounded and even if pseudopodia and form, their length less than the length of the other of the above cases (Fig. 3). The degree distribution in the processing of E is of 12.53±6.69 per cent (Fig. 4).

So about what atom, it is shown that des-A-fibrin more other factors inhibits the proliferation of melanoma cells in the presence of Matrigel (Fig. 4, p<0,01).

As stated above, it is possible to have in mind that des-A-fibrin effectively inhibits the proliferation of melanoma cells.

(4) Inhibition of proliferation of breast cancer cells

In Fig. 5 shows a micrograph (45-fold increase) of breast cancer cells, cultured for 2 hours in each of the differently treated well. The extent of spread of breast cancer cells, cultivated in each different processed hole shown in Fig. 6. The majority of breast cancer cells develop pseudopodia and distributed (Fig. 5) in the presence of PBS (A), fibrinogen (B), batroxobin (C), thrombin (D) and fibrin (F) with a prevalence of over 60% in each case (Fig. 6).

On the other hand, in the presence of des-a-fibrin (E), the majority of treated breast cancer cells remain rounded and even if pseudopodia are formed, their length less than the length of the other of the above cases (Fig. 5). The degree distribution in the processing of E is 8.87±3,06% (Fig. 6).

Thus, it is shown that des-A-fibrin more other factors significantly inhibits the proliferation of breast cancer cells in the presence of Matrigel (Fig. 6, p<0,01).

As the criminal code is shown above, you can keep in mind that des-A-fibrin effectively inhibits the proliferation of breast cancer cells.

(5) Inhibition of proliferation of cells of sarcoma

In Fig. 7 shows a micrograph (45-fold increase) of sarcoma cells, cultured for 2 hours in each of the differently treated well. The extent of spread cells fibrosarcoma, cultivated in each different processed hole shown in Fig. 8. Most cells fibrosarcoma develop pseudopodia and distributed (Fig. 7) in the presence of PBS (A), fibrinogen (B), batroxobin (C), thrombin (D) and fibrin (F) with a prevalence of more than 70% in each case (Fig. 8).

On the other hand, in the presence of des-a-fibrin (E) the majority of treated cells fibrosarcoma remain rounded and even if form pseudopodia, their length less than the length of the other of the above cases (Fig. 7). The degree distribution in the processing of E is 34,19±6,55% (Fig. 8).

Thus, it is shown that des-A-fibrin more other factors significantly inhibits the proliferation of cells of sarcoma in the presence of Matrigel (Fig. 7, p<0,01).

As stated above, it is possible to have in mind that des-A-fibrin effectively suppresses the proliferation of cells of sarcoma.

[Example 3] the Influence of the number of des-a-fibrin on the distribution of cells of a malignant tumor is.

Suppose that the number of des-a-fibrin obtained by the action batroxobin on fibrinogen, increased with increasing amounts of fibrinogen and increased time interaction between fibrinogen and batroxobin. Therefore, when interacting with the same concentration batroxobin (0,5 BU/ml) with different concentrations of fibrinogen (0.1 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 6 mg/ml, 9 mg/ml, 12 mg/ml and 15 mg/ml) for 30 s, 60 s and 90 s, respectively receive different amounts of des-a-fibrin. Investigate these effects on the proliferation of tumour cells. Experimental methods similar to the processing of E, which is described in(2) the method of determining the distribution of cellsexample 2, except fibrinogen concentration and the interaction time. The results are presented in Fig. 9.

If the interaction time is 30 s (), an increase in the concentration of fibrinogen does not affect the decrease of the degree distribution, and the least attainable degree distribution is 76,23%. This is attributed to the fact that for 30 s interaction is not reproduced such amount of des-a-fibrin, which is sufficient to influence the distribution of cells.

When the interaction time is 60 s (O) and 90 (∆), see a strong decrease of the degree distribution, if the concentration of fibre is ogena reaches 3 mg/ml (60: 12.53 per cent, 90: 9,13%). If the fibrinogen concentration increases further, I almost didn't see the spreading cells. This indicates that the spread of tumour cells is inhibited even more while increasing the number of the produced des-a-fibrin.

On the other hand, if the concentration of fibrinogen greater than 9 mg/ml, the degree of proliferation of the cells begins to increase, and at a concentration of 15 mg/ml not see the effect of reducing the extent of spread cells. The effect of the fibrinogen attributed to the fact that due to the presence of high concentrations of fibrinogen, a large amount of fibrinogen covers the hole before the hole can be covered with a coating of the resulting des-a-fibrin (des-A-fibrin, not formed a coating on the hole, wash in the washing process).

As stated above, it is possible to have in mind that des-A-fibrin inhibits the proliferation of melanoma cells dependent on dose.

[Example 4] the Effect of des-a-fibrin on the migration of tumour cells.

Migration of tumour cells is a biological behavior that forms the basis for metastasis of the tumor. The migratory ability of malignant tumors with high malignancy is high. In this example, we evaluate the effect of des-a-fibrin on the migration locates the public two types of tumors (melanoma and breast cancer), using a study with the application of scratches (seeGynecol. Oncol.89:60-72(2003)).

(1) Used cells from malignant tumors

As of melanoma cells will subcultural malignant human melanoma cells in mice B16-BL6 (Academy of Chinese Medical Sciences, Beijing, China) in medium RPMI-1640 (Gibco, MD, USA)containing 10% fetal calf serum (FBS, HyClone, Utah, USA), and these cells are used in this experiment.

As breast cancer cells will subcultural of breast cancer cells mice MMT060562 (ATCC, VA, USA) in MEM medium (Gibco, MD, USA)containing 10% FBS and 1% solution of non-essential amino acids (Non-Essential Amino Acids Solution (Gibco, MD, USA), and these cells are used in this experiment.

(2) Determination of cell migration

To each well of a 6-cell tablet impose a cover glass (Corning, NY, USA), seeded in the wells of 2.5×104melanoma cells B16-BL6, suspended in 2 ml of 10% FBS-containing RPMI-1640 medium (2.5×104of breast cancer cells MMT060562, suspended in 2 ml of 10% FBS-containing MEM medium, and cultured for 24 hours in CO2the incubator.

After culturing using a plastic cell scraper for applying Carpediem 0.5 mm line on top of the glass, where there was a proliferation of cells. Cells that initially proliferated on the scratch is removed and the remaining cell fragm what you wash in PBS.

Add to holes cultivated with melanoma two milliliters of 0.05 mg/ml of fibrinogen, diluted with 10% FBS-containing medium RPMI-1640, or des-a-fibrin (obtained by adding 0.05 mg/ml fibrinogen and 2 BU/ml batroxobin), and incubated for 48 hours in CO2the incubator.

Add to the wells cultured with breast cancer two milliliters of 0.05 mg/ml of fibrinogen, diluted with 10% FBS MEM, or des-a-fibrin (obtained by adding 0.05 mg/ml fibrinogen and 2 BU/ml batroxobin), and incubated for 48 hours in CO2the incubator.

After culturing test is done with staining by Wright, using phase-contrast microscope, count the number of cells that migrated to the scratched line (Olympus, Tokyo, Japan), and represent the result as the number of migrated cells per square millimeter glass cover (number of cells/mm2).

In addition, because fibrinogen is present around the cells of malignant tumorsin vivoand handling of fibrinogen used as a control for treatment of des-a-fibrin.

(3) the Inhibition of migration of melanoma cells

The results are presented in table 1.

Table 1
Migration of melanoma cells
ProcessingThe number of migrated cells (cells/mm2)
Fibrinogen87±14
Des-A-fibrin14±4**
**p<0,01 compared with fibrinogen

In the processing of des-a-fibrin number of migrated cells (14±4

cells/mm2substantially less than the number of migrated cells treated with fibrinogen (87±14 cells/mm2) (p<0,01).

As stated above, it can be assumed that the des-A-fibrin inhibits migration of melanoma cells.

(4) Inhibition of migration of breast cancer cells

The results are presented in table 2.

Table 2
Migration of breast cancer cells
ProcessingThe number of migrated cells (cells/mm2)
Fibrinogen29±10
Des-A-fibrin12±3**
**p<0,01 compared to the national Academy of Sciences of the Yong

In the processing of des-a-fibrin number of migrated cells (12±3

cells/mm2substantially less than the number of migrated cells treated with fibrinogen (29±10 cells/mm2) (p<0,01).

As stated above, it is possible to have in mind that des-A-fibrin inhibits the migration of breast cancer cells.

INDUSTRIAL APPLICABILITY

As shown in the above examples, a drug that will inhibit the development of malignant tumors, according to the present invention can effectively inhibit the proliferation and migration of tumour cells. Therefore, the present invention can be effectively used for inhibiting the development of malignant tumors.

1. A drug that will inhibit the development of malignant tumors containing des-A-fibrin.

2. A drug that will inhibit the development of malignant tumors according to claim 1, in which des-A-fibrin is produced by exposure trombinoscope enzyme on fibrinogen.

3. A drug that will inhibit the development of malignant tumors, according to claim 2, and combinatory enzyme is a natural product or a product of genetic recombination.

4. A drug that will inhibit the development of malignant tumors, according to claim 2, and combinatory enzyme is batroxobin.

5. The drug, which inhibits p is sweetie malignant tumors, according to any one of claims 1 to 4, and a malignant tumor is a malignant epithelial tumor.

6. A drug that will inhibit the development of malignant tumors, according to any one of claims 1 to 4, and a malignant tumor is non-epithelial malignant tumor.

7. A drug that will inhibit the development of malignant tumors, according to any one of claims 1 to 4, and a malignant tumor is a malignant tumor derived from neural tissue, or malignant tumor originating from mesenchymal tissue.

8. A drug that will inhibit the development of malignant tumors, according to any one of claims 1 to 4, and a malignant tumor is melanoma, breast cancer or fibrosarcoma.



 

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