KR100362025B1 - Tumor-angiogenesis inhibitor and pharmaceutical composition - Google Patents

Abstract

It is an object of the present invention to provide a pharmaceutical composition capable of administering shark cartilage, which is a tumor angiogenesis inhibitor, as an anticancer agent.

The present invention relates to a tumor angiogenesis inhibitor, which comprises a finely divided shark cartilage embedded in a fat or oil matrix, wherein the surface of the matrix is further coated with a lipid different in melting point from the fat or oil .

Description

[0001] TUMOR ANGIOGENESIS INHIBITOR AND PHARMACEUTICAL COMPOSITION [0002]

The present invention relates to a tumor angiogenesis inhibitor having excellent anticancer activity and analgesic activity, a pharmaceutical composition containing the same, and a method of treating cancer using the same.

Sharks are fish belonging to the cartilaginous fish found on the coast. His skeleton is cartilage, and shark cartilage, prepared by slaughtering shark and separating cartilage, has anticancer activity and is known to be effective for cancer treatment. For example, cartilage, a cartilage product of American shark, is marketed as an anti-cancer agent.

When the tumor grows to a size of about 1 to 2 mm ³ , the tumor produces a vascular growth factor so that it can be supplied with the essential oil and oxygen necessary for its growth. Since inhibition of the proliferation of these new blood vessels is effective in inhibiting the growth of tumor cells and is effective in the treatment of cancer, various studies have been conducted to find substances having inhibitory activity against angiogenesis, Formation inhibitory activity.

However, oral administration is difficult in most cases because an effective daily dose of shark cartilage for inhibiting angiogenesis is considerable at 50 to 60 g, and its taste and aroma are unpleasant. Since shark cartilage is a mixture of mucopolysaccharides, other appropriate routes of administration other than oral administration can not be used, and when oral administration, shark cartilage is left undissolved on strong acidic conditions, but it is required to dissolve in the intestinal tract do. For very weak cancer patients, oral administration is often difficult. Without solving such flavors, aromas and administration problems, shark cartilage can not be used as an effective anti-cancer agent that can be used extensively.

Thus, there has been a strong desire for a technique that allows shark cartilage to be substantially used as an effective tumor angiogenesis inhibitor.

Interleukin 12 (IL-12), on the other hand, is a type of cytokine having an NK (natural killer) cell-activating effect. Subsequent studies have shown that the interleukin stimulates and activates the growth of T cells (killer T cells) that have specific cytotoxic activity against tumor cells, and further induces the production of interferon gamma (IFN-y) that activates killer T cells . Therefore, it has attracted much attention as a useful substance for the treatment of cancer patients.

Recently, it has become possible to produce IL-12 in large quantities in the United States using genetic engineering technology (recombinant IL-12 (rt-IL-12)).

Attempts have already been made to directly introduce this interleukin 12 gene coding into cancer cells using gene transfer technology that allows IL-12 to be directly administered to cancer cells.

The method of inhibiting or exterminating tumor growth by the proper use of IL-12 involves administering IL-12 to a living organism from the outside, but another method is to induce autologous IL-12 in the organism. Such autologous IL-12 is advantageous because it does not pose a risk of causing an abnormal immune response. In addition, it is highly sensitive. Therefore, it is expected to exhibit a remarkable tumor destruction or eradication effect.

In view of the above-mentioned prior art, an object of the present invention is to provide a pharmaceutical composition capable of administering shark cartilage, which is a tumor angiogenesis inhibitor, as an anticancer agent.

It is another object of the present invention to provide a highly useful anticancer composition in which such a tumor angiogenesis inhibitor and an IL-12 inducer are used in combination.

The present invention relates to a tumor angiogenesis inhibitor containing finely divided shark cartilage embedded in a fat or oil matrix wherein the surface of the matrix is further coated with another lipid having a different melting point from the fat or oil.

The present invention will now be described in detail.

The shark cartilage, which is a major component of the tumor angiogenesis inhibitor of the present invention, must be in finely divided form. As used herein, the term " finely divided form " means that the particle size is from 1 to 90 μm. In order to produce such finely divided shark cartilage, it is desirable to finely crush the commercial shark cartilage. When heated at a temperature exceeding 60 ° C, the proteins and other active ingredients contained in the shark cartilage are thermally denatured and lose their effects, so it is necessary to finely grind them at a low temperature. The fine grinding at a low temperature is carried out, for example, by instantly freezing the shark cartilage with liquid nitrogen at -196 DEG C, roughly crushing the frozen material with a cutter-type grinder such as an Orient mill and roughly crushing it in a frozen state with a high speed rotor type grinder such as an impeller mill And then further pulverizing the lumps obtained with an air-jet type pulverizer such as a jet mill.

When preparing the tumor angiogenesis inhibitors of the present invention, the above-mentioned finely divided shark cartilage is incorporated into the fat or oil matrix.

The fat or oil constituting the fat or oil matrix is not limited to any particular kind as long as it can be generally used for pharmaceutical purposes. Thus, it may be suitably selected from among hardened vegetable oils such as soybean oil, rapeseed oil, palm oil, corn oil and cottonseed oil, hardened animal oils such as bovine oil, lard and fish oil, and mixtures thereof. Of these, fats and oils having a melting point of 42 to 56 ° C are preferred. Antioxidants such as vitamin E and catechins may be used in combination if needed.

The method of incorporating the finely divided shark cartilage in the fat or oil as a matrix is not critical. For example, the impregnation can be achieved by granulating a stirred mixture of the fat or oil and the finely divided shark cartilage in the liquefied state in a high-speed agitator.

The fat or oil matrix containing the finely divided shark cartilage embedded therein, such as that produced by the method described above, is then additionally surface coated with another lipid having a different melting point from the fat or oil. The lipids used in the present invention can be selected from those having a different melting point from the fat or oil used in the preparation of the fat or oil matrix in the preceding stage, and generally have a high melting point. Specific examples include waxes such as carnauba wax, rice wax, wax, hardened animal or vegetable oils, fatty acids such as stearic acid and palmitic acid, natural resins such as shellac, long chain alcohols such as palmityl alcohol and stearyl alcohol . When carrying out the coating, for example, a powder coating technique may be used.

Other methods that can be used in the preparation of the tumor angiogenesis inhibitor of the present invention include, for example, dispersing the finely divided shark cartilage in advance in molten fat or oil, spray-cooling the dispersion, And coating the lipid in the pellet with a lipid different in melting point from the fat or oil.

The thus prepared tumor angiogenesis inhibitor of the present invention contains shark cartilage as a main active ingredient, and its taste and flavor are completely shielded, so that difficulties in oral administration can be completely solved. In addition, manufactured tumor angiogenesis inhibitors are well suited for oral administration because they exhibit reduced surface friction, improved fluidity and reduced adhesion. Moreover, it is insoluble under the above acidic conditions and can be designed to be soluble in the intestine, which is its absorption site.

In the finely divided shark cartilage used in the present invention, particles having a particle size of 32 탆 or less correspond to at least 99.5% by weight. As described in detail in the following examples, the particle size of the finely divided shark cartilage has a great influence on the effect of the present invention. If the particle size is too large, the tumor angiogenesis inhibitory activity is reduced. As a method for producing shark cartilage particles of uniform size, for example, there is a method of passing finely divided shark cartilage through a screen having a desired mesh.

The shark as a raw material of shark cartilage used in the present invention is not limited to a particular kind, but shark cartilage obtained from Prionace glauca is most preferable. The cartilage of the medullary fish contains a large amount of active ingredient protein and copolysaccharide and is well suited for use. Since the American product cartilage is made from a mixture of many shark types, the quality of the product may be uneven. Another disadvantage is that the effect is somewhat low.

It was confirmed that the tumor angiogenesis inhibitor of the present invention has not only inhibitory activity on tumor angiogenesis but also reliable analgesic activity. Patients with cancer, especially those with terminal cancer, are generally suffering from extreme pain. Accordingly, when the tumor angiogenesis inhibitor of the present invention is administered to a cancer patient suffering from severe pain as an anticancer agent, the anticancer activity and the analgesic activity are synergistically effected, and a highly efficient therapeutic effect can be obtained.

It was investigated how the tumor angiogenesis inhibitor of the present invention exhibited tumor angiogenesis inhibitory activity and it was suggested that apoptosis is involved as described in detail in the following examples.

The tumor angiogenesis inhibitor of the present invention, which is very useful as an anticancer agent, but also has analgesic effect, can be used in other pharmacological fields. Thus, in a second aspect, the present invention provides a pharmaceutical composition comprising a tumor angiogenesis inhibitor of the present invention.

Furthermore, the method for treating cancer wherein the tumor angiostatic agent of the present invention is administered at a dosage sufficient to inhibit tumor angiogenesis is also within the scope of the present invention.

The pharmaceutical composition of the present invention is characterized by containing a tumor angiogenesis inhibitor and an interleukin 12 derivative. The pharmaceutical composition reflects the second aspect of the present invention. The second aspect of the invention will now be described in detail below.

In the specification, the term "interleukin 12 inducer" means not only IL-12 itself, IL-12 transduced cancer cells, rt- IL-12 and, in summary, all substances capable of inducing the production of IL-12 in vivo without any particular limitation.

Examples thereof include active hemicellulose compounds (AHCC) and the like. AHCC is already known as a physiologically active substance obtainable by enzymatic treatment of vegetable fibers contained in the cell wall of mushroom mycelium, and is known as β- (1 → 3) D-glucan, β- (1 → 6) D- - (1 → 4) D-glucan, peptide glucan, proteoglucan, lecithin, nucleic acid, non-digestible polysaccharide and the like.

As the IL-12 inducing factor used in the present invention, mention may be made of the above-mentioned AHCCs and mushroom mycelium components. The mushroom mycelium component is not limited to a specific kind, but PSK, which is a mycelial component of polyporus berscht color used as an anticancer agent, SPG, which is a mycelial component of the Schizophyllum commune, and lentinan, .

As such mycelial component of mycelia, there is also a mycelial component of agaricus, such as Ganoderma lucidum, Albatrellus lupulus, Agaricus blazei, Tricoloma thalium, Inonotus obculus, Grapolar frondosa, Hericum erina And the like, such as Phellinus ostreatus, Ganoderma marcidium, Panellus serotinus, Felinus, Rengizthes Bertulinus, Tricoloma matsutake, Perennialia proxinacea, .

The IL-12 inducer used in the present invention further contains cell components of streptococcus pyogenes or hemoriticus. Such cellular components may be referred to as anticancer agents such as OK-432 (Pichibanil).

They are already known as biological response modifiers (BRMs).

The above-mentioned IL-12 inducing factor has an effect of enhancing killer T cell activity. Thus, killer T cells that occur in vivo are activated by the IL-12 inducing factor.

On the other hand, the tumor angiogenesis inhibitors described in detail above inhibit the proliferation of new blood vessels caused by cancer cells (tumor cells). When new blood vessel proliferation is inhibited, tumor cells express adhesion agents such as Fas antigen, CD 80 and CD 86 on their surfaces due to blood supply disturbances. Killer T cells can recognize other attachment factors such as Fas antigen, CD80, CD86 and antigen. Thus, the thus expressed adherence factor is in a condition that can be easily recognized by killer T cells, and this easily modified tumor cell is now easily attacked by killer T cells.

In such a state, the above-mentioned IL-12 can attack the tumor cells very significantly compared to the case where only the IL-12 inducer is used. The synergistic effect of the tumor angiogenesis inhibitor of the present invention and the IL-12 inducing factor is based on the above-mentioned mechanism and exhibits a strong anti-cancer effect, leading to cell necrosis in which tumor cells are destroyed. This discovery was first made by the present inventors.

Dosage forms in which the pharmaceutical compositions of the present invention are administered to humans or other animals include, but are not limited to, those common to various medicaments and may be carrier-supported dosage forms.

As such a carrier, at least one of solid, semi-solid, liquid diluent, filler, and other agent auxiliary is used in a proportion of, for example, 0.1 to 99.5%, preferably 0.5 to 90%. The pharmaceutical composition of the present invention can be applied fully orally or parenterally. Parenteral routes include, for example, local (intra-tissue), subcutaneous, intramuscular, intraarterial or intravenous, and rectal administration. Dosage forms suitable for such route of administration may be prepared using conventional techniques.

When the composition is used, for example, as an anticancer agent, its administration is preferably determined in consideration of the patient's age, weight, route of administration, disease, degree thereof and other factors. However, the oral dose of an adult, which is generally expressed as an active ingredient, is 1 to 10 g, preferably 5 to 6 g per day. The parenteral dose which can vary widely depending on the route of administration is 100 to 10,000 mg, preferably 2 to 5 g per day. In some cases, smaller dosages are sufficient, but conversely, larger doses may be required in certain cases. Each daily dose can be administered in two to four divided doses.

Oral administration can be carried out using solid or liquid unit dosage forms such as bulk powders, powders, granules, tablets, capsules, syrups, emulsions, suspensions and the like.

Bulk powders can be prepared by powdering the active ingredient with a suitable powder. The powder may be prepared by powdering the active ingredient with a suitable powder and mixing it in the same manner with a finely divided pharmaceutical carrier, such as edible carbohydrates such as starch or mannitol or other suitable excipients. If necessary, a flavoring agent, preservative, dispersant, coloring agent, perfume or the like may be added.

Capsules can be obtained by preparing granules from tablets or the bulk powder or powder obtained above and filling the resulting granules into capsule shells such as gelatin capsules. Prior to filling, a lubricant and / or a flow improver such as colloidal silica, talc, magnesium stearate, calcium stearate, or solid polyethylene glycol may optionally be added. In the case of capsules, the efficacy of the drug is determined by the addition of a disintegrant and a solubilizing agent such as carboxymethylcellulose, carboxymethylcellulose calcium, low substituted hydroxypropylcellulose, croscarmellose sodium, sodium carboxy starch, calcium carbonate, .

The soft capsule can be prepared by suspending the finely divided active substance in a vegetable oil, polyethylene glycol, glycerol or a surfactant, and enclosing the obtained dispersion with a gelatin sheet.

The granules may be prepared by mixing the active substance in powder form and the above-mentioned excipients with a disintegrant and adding a wetting agent (e.g., syrup, starch paste, gum arabic, cellulose solution, polymer solution) The resulting mixture is kneaded, the mixture is passed through a sieve, and the mixture is kneaded, and the mixture is kneaded, . In addition to such powder granulation methods, granules can also be prepared by first kneading the mixture into a purifier and pulverizing the resulting lump of incomplete form.

(For example, quaternary salt) or an absorbent (e.g., bentonite, kaolin, dicalcium phosphate) may be added in advance.

The tablets can be prepared by adding a lubricant such as stearic acid, stearic acid, talc, mineral oil and the like to granules obtained by the above method, and then tableting the resulting mixture. The tablets thus prepared can be coated with a film coating or a sugar coating.

Tablet manufacture may also be accomplished by mixing the active ingredient of the present invention with a flowable inert carrier followed by direct tableting without a granulating step or lump forming step as mentioned above. Transparent or translucent protective coatings made of confidential cell coatings, coatings made of sugar or polymeric materials, and glossy coatings made of waxes may also be used.

Other oral dosage forms, such as syrups, emulsions and suspensions, may be obtained in unit dosage form in which a specified amount thereof contains a certain amount of the active substance. Syrups may be prepared by dissolving the active substance in an appropriately flavored aqueous solution. Ilyics is manufactured using non-toxic alcohol-based carriers. Suspensions are formulated by dispersing the active substance in a non-toxic carrier. A suspending agent or an emulsifying agent (for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester), an antiseptic agent, a flavoring agent (for example, peppermint oil, saccharine) and the like may optionally be added.

Unit dosage forms for oral administration may be microencapsulated, if desired. The active period may be extended by further coating such formulation or by incorporating the active material into the polymer or wax, and sustained release may be obtained.

Subcutaneous, intramuscular, intraarterial or intravenous administration can be achieved by preparing injectable compositions in liquid unit dosage forms such as solutions or suspensions. They can be prepared by dissolving or suspending a certain amount of the active substance in a non-toxic liquid carrier suitable for injection purposes, such as an aqueous or oily solvent, and sterilizing the resulting solution or suspension. It is also possible to put a certain amount of the active substance into a glass bottle in powder or lyophilized form, and sterilize and seal the glass bottle and its contents. In this case, preliminary vials and carriers for dissolving or mixing may be prepared immediately before administration. A non-toxic salt or salt solution may be added to make the injectable composition an isotonic solution. In addition, stabilizers, preservatives, suspending agents, emulsifiers, etc. may additionally be used.

For example, by blending a hydrophobic or hydrophilic suppository base with, for example, polyethylene glycol, cacao butter, a high-grade ester (for example, myristyl palmitate) or a mixture thereof and an active substance, and kneading the resulting mixture.

As described above, the tumor angiogenesis inhibitor of the present invention can be conveniently administered by oral administration because it has no unpleasant taste and odor of shark cartilage, is resistant to acid, and only dissolves in the intestine. In addition, it has reliable anticancer activity and analgesic activity and is very useful as a pharmaceutical composition.

The combined use of the tumor angiogenesis inhibitor of the present invention and an IL-12 inducer exhibits excellent anticancer activity and is thus very useful for preparing pharmaceutical compositions.

Best Mode for Carrying Out the Invention

The following preparations and examples illustrate the invention in great detail. However, the scope of the present invention is not limited thereto.

Production Example 1

Preparation of Coating Formulations of Oil or Fat Matrix Containing Shark Cartilage

60 parts by weight of finely divided shark cartilage having an average particle size of 27 占 퐉 was filled in a high-speed agitating granulator (high-speed mixer, manufactured by Goochogyo Co., Ltd.), the internal temperature of the granulator was maintained at 10 占 폚, 10 parts by weight of hydrogenated curing oil (melting point 48 DEG C) derived from the small-scale oil is injected while stirring. Small droplets of molten oil absorb the finely divided shark cartilage and form a matrix. However, at this stage, the shark cartilage is partially exposed on the surface, resulting in insufficient coating performance. Thus, 30 parts by weight of finely divided hardened rapeseed oil having a mean particle size of 5 mu m selected as lipids with different melting points (melting point 68 DEG C) were charged to the same stirring granulator and the mixture was stirred at a speed of about 1000 rpm for 30 minutes , The oil is disposed and spread on the primary matrix surface so that the surface of each primary matrix is coated with the oil. Thus, the most satisfactory fat matrix-coated formulation with excellent acid resistance and sustained release properties is obtained.

Example 1

Test for inhibiting tumor angiogenesis in mice

(Evaluation according to shark cartilage particle size)

The test was performed by the dorsal air sac method. A Millipore filter was applied to both sides of a plastic ring having a diameter of 5 mm, and a chamber was prepared by injecting 1 x 10 ⁶ MH-134 tumor cells and a culture medium into the chamber. Six mice were used as the group 1, and the chamber was transplanted subcutaneously into each mouse.

Shark cartilage described below was orally administered to group 4 rats at a dose of 1,000 mg / kg for 4 days. On the fifth day, the inhibition of angiogenesis was evaluated. In the additional group 1 (control), the degree of inhibition was evaluated for 5 days in the same manner without administration of shark cartilage.

The release of the angiogenic enhancer from the tumor via the millipore filter results in the formation of a new blood vessel, formopm, on the surface in contact with the chamber.

New blood vessels induced by these tumors are spiral. These angiogenesis inhibition levels were evaluated according to the following evaluation criteria.

(++) 3 points: Significant angiogenesis

(+) 2 points: Limited angiogenesis

(±) 1 point: slight angiogenesis

(-) 0 point: No blood vessel formation

The percent inhibition was expressed as a percentage reduction in the rating compared to the control.

Shark Cartilage (1): Cartierade, a US shark cartilage product, was used as a raw material for shark cartilage. The product was instantaneously frozen in liquid nitrogen (-196 DEG C) and roughly ground in a cutter mill. Thereafter, the pulverized material still in a frozen state was further pulverized with a high-speed rotor type mill (impeller mill) and finally pulverized in a final air-jet mill (jet mill). The thus obtained product was used as it was.

Shark cartilage (2): The above shark cartilage (1) was subjected to a 32 탆 sieve and a portion above the sieve was used.

Shark Cartilage (3): The above-mentioned shark cartilage (1) was passed through a sieve after sieving through a 32 탆 sieve.

The results are shown in Table 1.

++ (3 points) + (2 points) ± (1 point) - (0 point) grade control(%) Control group Four Two 0 grains 0 grains 2.7 ± 0.5 - Shark cartilage (1) Two Three One 0 grains 2.2 ± 0.8 18.5 Shark cartilage (2) Two Four 0 grains 0 grains 2.3 ± 0.5 14.8 Shark cartilage (3) One Four One 0 grains 2.0 ± 0.8 25.9 * * P < 0.05

The above results showed that the finely divided shark cartilage having passed through the sieve of 32 mu m among the tested shark cartilage powder had a significantly high anti-angiogenic activity.

Example 2

Test for the inhibition of tumor angiogenesis in mice (Cartierade vs. Vetasak)

The vertebral air sac method was used to compare cartilage, an American shark cartilage, and cartilage (Bettersak) derived from a blue shark, in the same manner as in Example 1. The results are shown in Table 2.

In the 100 mg / kg cartridade group, the inhibition rate (% reduction in the score compared with the control) was 3.6%, but in the 1000 mg / kg cartridade group, 21.4% with a significant difference (P <0.05). Conversely, the inhibition rate of 100 mg / kg group was 17.9% with a significant difference (P <0.05), and the inhibition rate was as high as 35.7% at 1,000 mg / kg group, and the difference was statistically significant &Lt; 0.002).

Significant differences were also found between Cartierade and Vettersak (P &lt; 0.05).

Dose (mg / kg) ++ (3 points) + (2 points) ± (1 point) - (0 point) grade control(%) Control group - 9 grains One 0 grains 0 grains 2.8 ± 0.4 - Cartierade 1,000 Three 6 grains One 0 grains 2.2 ± 0.8 21.4 * Cartierade 100 6 grains Four 0 grains 0 grains 2.7 ± 0.5 3.6 Vetasak 1,000 Three Two 5 grains 0 grains 1.8 ± 1.0 35.7 ** Vetasak 100 5 grains Four One 0 grains 2.3 ± 0.8 17.9 * * P &lt; 0.05, ** P &lt; 0.002

The results show that Vetasak has superior tumor angiogenesis inhibitory activity than cartileid.

Example 3

A mouse tumor growth inhibition test (an effective dose-response test of BETA Sark)

Tumor cells (Sarkoma 180) and 1 × 10 ⁶ mice are subcutaneously transplanted into each of three groups of mice (ICR mice, male 6-week-old) in groups of 10 mice. Group 1 was then dosed at a dose of 1,000 mg / kg once daily for 20 days, and the other group had a particle size adjusted to 32 μm or less, pH 2 Lt; / RTI &gt; for 3 hours) is administered at the same dose for the same period. In the third group, feed the animals for 20 days without any medication. After 25 days, all animals were sacrificed and the tumor weighed. The results are shown in Table 3. The percent inhibition was expressed as a percentage reduction in tumor weight compared to the control.

Here, in the cartilage group, the inhibition rate was 11.1% compared to the control group, and the inhibition rate of the betasac group was 47.0% with a significant difference (P <0.001). Comparing cartilage to bettersak, bettersak showed better antitumor activity (P &lt; 0.05).

Mean tumor weight (g) control(%) contrast 2.752 + 1.388 - Cartierade 2.446 ± 1.139 11.1 * Vetasak 1.457 ± 1.965 47.0 ** * P &lt; 0.05, ** P &lt; 0.001

The results show that Vetasak (particle size of less than 32 [mu] m) at daily doses of 20 g per body weight is effective enough in clinical outcomes.

Example 4

Analgesic effect test of mice

The acetic acid writhing method was used to evaluate the analgesic effect of Vetasak. ICR mice (male, 5 weeks old) were divided into 5 groups of 10 mice each, and each test material was administered to each group according to the schedule mentioned below. After 40 days, 2% acetic acid was administered intraperitoneally Lt; / RTI &gt; The wrist reflexes of each mouse were calculated over a period of 10 to 20 days after acetic acid administration.

After acetic acid administration, the test material was administered daily until the end of the struggle reflex measurement.

In the cartilage group, cartilage was orally administered at a daily dose of 1,000 mg / kg, in the Vetasak group, orally in a daily dose of 1,000 mg / kg, and in the indacin group, Indomethacin, nonsteroidal analgesics, and commercial products) were orally administered at a dose of 25 mg / kg, chondroitin sulphate (commercially available) was orally administered at a dose of 333 mg / kg in the chondroitin sulfate group, , The animals were fed with no medication. The percent inhibition was expressed as a percentage reduction in the number of reflexes compared to the control. The results are shown in Table 4. Likewise, for the analgesic effect, the superiority of Veta Sark was proven.

Number of struggle reflexes control(%) contrast 31.2 ± 9.6 - Cartierade 24.3 ± 3.9 23.6 * Vetasak 21.2 + - 10.4 32.1 * Indah Shin 10.3 ± 5.6 67.0 ** Chondroitin sulfate 23.4 ± 4.5 25.0 * * P &lt; 0.05, ** P &lt; 0.001

Example 5

Expression of immunologic specific antigen in the clinical case of Vetasak administration

An 80-year-old man who complained of an unpleasant sensation in the upper abdomen area was the subject. Based on gastroscopy, the patient was diagnosed with a type IIc advanced gastric cancer (signet ring cell carcinoma) in the upper part of the incisura angularis. Biopsy specimens were used for electron microscopy and biopsy (immunoassay). The patient was unable to operate because of myocardial infarction.

The shark cartilage 3 (hereinafter referred to as " Veta Sark " in Example 5) used in Example 1 was administered to the patient daily at a daily dose of 20 g.

Electron microscopy and immunological specific staining (primary cirrhosis) before the administration of Vetasak revealed only normal cancer cells with some cell necrosis. No adherence factors (fluorescent antibody technology) and Fas antibodies (special staining) were observed, and no HSP 60 (stress protein, heat shock protein, fluorescent antigen technology) was observed. Two and a half months after the primary cervical examination, the cervical examination revealed that the external carcinoma was clearly covered with normal mucosa, and the peripheral carcinoma protrusion tended to be flattened. Electron microscopic observations of the second biopsy showed no specific changes in cell membrane and cytoplasm, but shrinkage and destruction of cancer cell nuclei were observed, and cell carcasses were observed.

Thus, a positive result indicating cell necrosis of cancer cells appeared.

At this time, immunological specific coloring was performed with a biopsy specimen. Cancer cells were examined using anti-HSP60 antibody, anti-CD80 antibody, anti-CD86 antibody and also anti-Fas antibody. As a result of administration of Vetasak, the Fas antigen was positive, and the HSP 60 antigen and the attachment factors CD 80 and CD 86 were positive.

At the third cystoscopy (about 5 months after the first cystoscopy), the carcinoma of the upper upper corner completely disappeared. Biopsy specimens of the above sites in which gastric carcinoma was found were all negative for cancer cells.

The results are summarized as follows. The Fas antigen, HSP 60 antigen, and the attachment factors CD80 and CD86, which were not observed before the administration of the Vetasak preparation, were all positive two and a half months after administration of the Vetasak preparation. After that, the third cystoscopy showed that the carcinoma had completely disappeared. This phenomenon can be explained as follows. As a result, the blood which was supplied to the tumor was inhibited by the administration of Vetasak. As a result, the stress protein (HSP60) was expressed and the Fas antigen, CD80 and CD86 were positive. Thus, in the third cirrhosis, complete regression of the tumor was confirmed.

Immunological studies of tumors have shown that tumor degeneration is accompanied by desquamation and cell necrosis. Dacer is a phenomenon of extinction of tumors such as those caused by infection or other factors under the action of nonspecific immunity, where tumor cells are destroyed as a result of transformation in the order of cell membrane, cytoplasm, and nucleus. Histologically, in the case of sclerosis, especially penetration of neutrophils, macrophages and fibroblasts occurs, followed by calcification and fiber growth, followed by the disappearance of the cancer. As a result, traces remain on the site.

On the other hand, in the case of tumor necrosis caused by tumor necrosis, no infection occurs at all, and no trace remains, but normal cells are found to be substituted. In this case, it is known that killer T cells recognize and attach to Fas antigen on the surface of tumor cells and, at the same time, cooperate with adhesion factors such as CD80 and / or CD86 to induce cell necrosis. Nuclear degeneration (nucleation and formation of cell carcinoma) occurs first, followed by cellularization and cell membrane formation by macrophages.

Therefore, no trace remains.

In these and other cases of Vetasak formulation administration, no signs of infection, such as calcification or fibrosis, have occurred in the cancer extinction site, and thus cancer extinction is thought to be due to cell death. It is evident that the cancer disappears in a very short period (one month to one month).

Example 6

Immuno-specificity test of mice

Sarkoma 180 tumor cells were transplanted to the back of each mouse. The tumor growth inhibitory effect was evaluated by intraperitoneal administration of 10 mg of TNP 470 (manufactured by Takeda Chemical Industries, Ltd.), oral administration of BETTERAK 1000 mg / kg, and angiostatin (chemotherapy and serum therapy research product) , And the control group in which no drug was administered. After 2 to 3 weeks, tumor growth was inhibited in all treatment groups with a significant difference (P &lt; 0.05) compared to the control. At this time, the tumor tissues were immunohistologically tested for the expression of HSP60, Fas antigen, CD80 and CD86, and the degree of expression of HSP60, Fas antigen, CD80 and CD86 was significantly increased.

Separately, the same test mentioned above was performed using T cell deficient mice, i.e., nude mice. Each formulation showed some tumor growth inhibitory effect, but there was no significant difference. Tumor tissues were immunohistochemically examined and the expression of HSP60, Fas antigen, CD80 and CD86 was positive. However, no findings indicating cell necrosis were obtained.

These results demonstrate that the antitumor activity of an angiogenesis inhibitor is not only a mere inhibition of blood supply to the tumor, but also a quantitative, quantitative, and quantitative analysis of stress protein expression, or expression of Fas antigen, CD80 and CD86 in the condition of inhibited blood supply to the tumor Or qualitative changes, and induces cell necrosis by exhibiting antitumor activity.

Examples 7 to 10

When AHCC and Vetasak are administered in combination

For the four cancer patients shown in Table 5, AHCC was administered daily at a daily dose of 3.0 g, and at the same time, Vetasac was orally administered daily at a daily dose of 20 g. Three months after administration, the treatment outcome was evaluated and NK activity, IL-12 content and CD4 / CD8 levels were measured. In each of Examples 7 to 10, tumors declined by more than 50%. However, NK activity remained in a normal state without any significant increase in activity. In each of Examples 7 to 10, the IL-12 content was significantly higher than the normal range. Thus, IL-12 is thought to be involved in tumor reduction.

In Table 5, CD4 is a helper T cell marker, CD8 is a killer T cell marker, and CD4 / CD8 levels reflect an increase in the number of killer T cells. A value less than 1 means an increase in the number of killer T cells. The normal range is 1.0 to 1.5.

Example patient Symptom NK activity Serum IL-12 Treatment Outcome CD4 / CD8 7 A 72 year old man Advanced gastric cancer 24% 240 ng / mL CR 0.34 8 A 57 year old man End stage of appendix cancer toxic peritonitis 62% 230 ng / mL CR 0.42 9 A 69 year-old male Stage of metastasis between stomach cancer 62% 103 ng / mL CR 0.62 10 A 71 year old woman End stage of liver cancer metastasis 68% 78 ng / mL PR 0.84 CR: full regression, PR: partial regression

As described above, the tumor angiogenesis inhibitor of the present invention can be conveniently administered by oral administration because it has no unpleasant taste and odor of shark cartilage, is resistant to acid, and only dissolves in the intestine. In addition, it has reliable anticancer activity and analgesic activity and is very useful as a pharmaceutical composition.

Claims (5)

Characterized in that it contains a shark cartilage which is divided into 1 to 90 占 퐉 embedded in a fat or oil matrix and wherein the surface of the matrix is further coated with another lipid which is different from the melting point of the fat or oil matrix . Wherein the shark cartilage contains finely divided shark cartilage, wherein the shark cartilage has finely divided shark cartilage having a particle size of 32 占 퐉 or less of 99.5% by weight or more. The tumor angiogenesis inhibitor according to claim 1 or 2, wherein the shark cartilage is derived from a medullary fish. An anticancer agent comprising the tumor angiogenesis inhibitor according to claim 1 or 2 as an essential ingredient. An anticancer agent comprising a tumor angiogenesis inhibitor according to claim 1 or 2 and an inducer of interleukin 12.