JPH0853351A - Enzyme-inhibiting agent, arterialization-inhibiting agent, and cancer metastasis-inhibiting agent - Google Patents

Abstract

PURPOSE:To obtain the subject medicine containing docosahexaenoic acid (derivative) as an active ingredient, exhibiting excellent enzyme-inhibiting, arterialization-inhibiting and cancer metastasis-inhibiting activities, and useful for inhibiting the cancer metastasis, etc. CONSTITUTION:This medicine contains decosahexaenoic acid (derivative) [e.g. docosahexaenoic acid, its salt, ester, glyceride, its phospholipid derivative, choline compound thereof or ascorbate thereof] as an active ingredient. The medicine is prepared in the form of preparations such as powder, granules, capsules, tablets, syrup and elixir in the case of oral administration agents and in the form of injections in the case of parenteral administration agents. The docosahexaenoic acid (derivative) is compounded in an amount of 1-90wt.%, especially 10-80wt.%, in the preparations. The medicine is preferably administered in a daily dope of 0.1-5g, especially 1-2.5g for an adult based on the amount of active ingredient.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzyme inhibitor, an angiogenesis inhibitor and a cancer metastasis inhibitor characterized by containing docosahexaenoic acid and / or its derivative as an active ingredient.

[0002]

2. Description of the Related Art In Japan, the percentage of deaths due to cancer among adult deaths tends to increase year by year. Although research on cancer treatment and metastasis mechanism has made rapid progress in recent years, no epoch-making treatment has been discovered yet, and treatment with chemotherapeutic agents has not been sufficiently effective. . Most cancer deaths are due to metastasis, and suppression of metastasis is considered a good measure to reduce cancer deaths. Conventionally, ovostatin, matristatin, aprotinin and the like have been known as cancer metastasis inhibitors, but their effects have not been sufficient yet.

On the other hand, docosahexaenoic acid (DHA) is an n-3 type unsaturated fatty acid contained in excitable membranes such as the brain and retina in a large amount, and improves memory and learning ability and suppresses deterioration of visual acuity.
It is said to have pharmacological actions such as antitumor action and immunosuppressive action. It also inhibits macrophage activity in fish oil containing a large amount of DHA (Dustin LB, et al., J. IMMUNOL, 14
4,488-4897 (1990)), LTB ₄ and LTC ₄ production inhibition (L
okesh BR, et al., Biochem Biophys Acta, 958,99-1
07, (1988)), which suppresses the production of TNF (Endres S., et al., N. Eng. J. Med., 320, 265-271,
(1989)) and reported that the immunosuppressive effect was enhanced when combined with CyA in organ transplantation (Kelley VE, e
al., Transplantation, 48, 98-102, (1989)).
However, there is no report on the enzyme inhibitory action, the angiogenesis inhibitory action, and further the cancer metastasis inhibitory action.

[0004]

Cancer metastasis is a very complex phenomenon consisting of multiple stages. Cancer cells are detached from the primary lesion, invaded into blood vessels, adhered to vascular endothelial cells, and adhering to vascular basement membrane. The process involves destruction, invasion of extracellular matrix, and proliferation. Various enzymes are involved in these processes, and their inhibitors are attracting attention as new cancer metastasis inhibitors. Further, cancer cells induce angiogenesis because they supply nutrients and enzymes necessary for expressing the abnormal growth that is a characteristic of cancer cells, or excrete metabolic waste products. Therefore, by inhibiting angiogenesis, the growth of cancer cells can be suppressed and the metastasis of cancer can be suppressed.

[0005]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to develop a new cancer metastasis inhibitor, and as a result, docosahexaenoic acid and / or its derivative inhibits the enzyme,
In addition, they found that the formation of blood vessels was suppressed, and completed the present invention.

That is, the present invention provides an enzyme inhibitor containing docosahexaenoic acid and / or its derivative as an active ingredient. The present invention also relates to docosahexaenoic acid and / or
Alternatively, an angiogenesis inhibitor containing the derivative thereof as an active ingredient is provided. Furthermore, the present invention relates to docosahexaenoic acid and / or
Alternatively, a cancer metastasis inhibitor containing the derivative thereof as an active ingredient is provided.

Docosahexaenoic acid and its derivatives (hereinafter referred to as DHAs) used in the present invention include free acids (that is, DHA), their salts (for example, sodium salt, potassium salt, calcium salt, ammonium salt) and esters. (For example, methyl ester, ethyl ester, propyl ester), glyceride (mono-, di-, tri-), phospholipid, choline compound, ascorbic acid compound, amino acid compound and the like. Docosahexaenoic acid is fish oil extracted from blue-backed fish such as sardines, mackerel, horse mackerel, salmon, saury, fish oil derived from orbital fat of large marine fish such as tuna and bonito, oil and fat derived from microorganisms and seaweeds, krill oil, cod and squid. It can be obtained by isolation and purification from seafood-derived oils and fats extracted from the liver according to known methods.

The toxicity of DHA is weak, and no sign of toxicity was observed when the ethyl ester was orally administered to mice at 2000 mg / kg.

The dose of these DHAs varies depending on the type of target disease, age, sex, body weight, symptom of patient, or administration form, but in general, about 0.1 to 0.1 per day for an adult.
It is 5 g, preferably 1 to 2.5 g, and it is suitable to administer the drug once or in several divided doses.

The drug of the present invention can be administered orally or parenterally for therapeutic purposes. As the orally-administered agent, solid preparations such as powder, granules, capsules and tablets, or liquid preparations such as syrups and elixirs can be used. Moreover, an injection can be prepared as a parenteral preparation.

These preparations are manufactured according to a conventional method by adding pharmacologically and pharmaceutically acceptable manufacturing aids to the active ingredient. Further, it is also possible to prepare a sustained-release preparation by a known technique. When using the manufacturing aid, D
The content of HAs (as free acid) is usually 1 to 90% by weight, preferably 10 to 80% by weight.

[0012] The above-mentioned production aids include oral preparations (oral preparations), injection lumber (injection preparations), mucosal administration preparations (buccal,
Troches, suppositories, etc.), external preparations (ointments, patches, etc.), and other suitable ingredients for formulation depending on the route of administration.

For example, in the case of oral agents and mucosal agents, excipients (eg, starch, lactose, crystalline cellulose, calcium lactose, magnesium aluminometasilicate, anhydrous silicic acid), disintegrating agents (eg, carboxymethyl) Cellulose, carboxymethylcellulose calcium), lubricant (eg:
Pharmaceutical ingredients such as magnesium stearate, talc), co-tenting agents (eg hydroxyethyl cellulose, sucrose, hydroxypropyl cellulose, polyvinylpyrrolidone, corn protein), flavoring agents and the like are used.

To produce granules, wet or dry granulation may be carried out, and to produce tablets, these powders and granules may be tableted as they are or by adding a lubricant such as magnesium stearate or talc. . These granules or tablets are coated with an enteric base such as hydroxypropylmethyl cellulose phthalate, methacrylic acid, and methyl methacrylate copolymer to form an enteric preparation, or coated with ethyl cellulose, carnauba wax, hardened oil, etc. to form a sustained-release preparation. You can also do it. Further, in order to produce a capsule, a powder or granules can be filled in a hard capsule, or the active ingredient can be dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc. and then coated with a gelatin film to give a soft capsule. . In the case of capsules, the content may be 100% by weight of DHAs.

To prepare a liquid preparation for oral administration, an active ingredient and a sweetener such as sucrose, sorbitol and glycerin are dissolved in water to prepare a transparent syrup, and essential oil, ethanol and the like are added to form an elixir. Or gum arabic,
An emulsion or suspension may be prepared by adding tragacanth, polysorbate 80, sodium carboxymethyl cellulose and the like. If desired, flavoring agents, coloring agents, preservatives and the like may be added to these liquid preparations.

In the case of injectable preparations, solubilizers or solubilizing agents (eg, distilled water for injection, physiological saline, propylene glycol), suspending agents (eg, polysorbate) capable of forming an aqueous injectable preparation. -Pharmaceutical ingredients such as surfactants such as 80), pH adjusters (eg, organic acids or metal salts thereof), stabilizers, etc. are used. In order to produce an injection, the active ingredient is adjusted as necessary with hydrochloric acid, sodium hydroxide, emulsion, sodium lactate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc. pH adjusting agents, sodium chloride, glucose, etc. It may be dissolved in distilled water for injection with the agent, aseptically filtered and filled in an ampoule, or mannitol, dextrin, cyclodextrin, gelatin, etc. may be added and freeze-dried under vacuum to give a solution-injectable injection at the time of use. Alternatively, lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, etc. may be added to the active ingredient and emulsified in water to give an emulsion for injection.

In addition, the drug of the present invention having the above-mentioned constitution can be manufactured by a known manufacturing method, for example, the method described in the Japanese Pharmacopoeia 10th Edition General Rules for Preparation or a method with appropriate modification.

The present invention will be described in detail below with reference to examples.

[0019]

【Example】

Example 1. Enzyme Inhibitory Activity Test Polyunsaturated fatty acids [(Ethyl ester of each of DHA, eicosapentaenoic acid (EPA), eicosatetraenoic acid (ETA)] were SPF-Balb / fed in F1-fish meal for 2 weeks before administration. c AnNCrj mouse (6 weeks old, male) was dissolved in a 5% aqueous solution of gum arabic at a dose of 50 mg / kg / day / once and orally administered by a sonde for 10 days.
24 hours after the final administration, the animals were sacrificed under anesthesia and the spleen was extracted. The extracted spleen is cold PBS (phosphate buffer, phosphat
e buffered saline) was used for homogenization using a histocol (Nippon Medical and Scientific Instruments, Tokyo). 3,000g
After centrifuging at 20 minutes, the enzyme activity of the supernatant was measured.

[0020]

[Table 1] Note: ^* P <0.05, ^** P <0.01, ^*** P <0.001

As described above, DHA showed a stronger enzyme inhibitory activity than EPA.

Example 2. Anti-angiogenic activity Chorioallantoic membrane (Chorioallantoic)
Membrane CAM) Method On the 3rd day of fertilization, the top and sides of the egg were sterilized with 70% ethanol. Next, a hole was made in the upper and side surfaces with a hole, about 2 ml of egg white was suctioned and removed from the side hole, and a small amount of air was removed from the upper hole using a dropper. Optics (Smith & Nephew Medical Limited, USA) were pasted on the side holes to close the holes, and then the upper shell was peeled off in a draft, and a cap made of Teflon was put on, and the plate was incubated at 37 ° C for 1 day. Then CAM
Place a silicon cap on top, and in the ring 10 μl of sample suspended in physiological saline containing 1% methylcellulose
Was injected. Six eggs were used in each group. Furthermore, 2
After culturing at 37 ° C. for one day, the shell was opened wide, intralipos was injected into the CAM, and the inhibitory effect was determined and a photograph was taken.

[0023]

[Table 2] Table 2. Angiogenesis inhibition test of polyunsaturated fatty acids ────────────────────── Sample dose inhibition rate (%) ²⁾ Effect ³⁾ ───── ───────────────── DHA 0.2mg / CAM 66.7 2.5 EPA 0.2mg / CAM 50.0 1.5 ETA 0.2mg / CAM 33.3 1.0 ──────────────────────

1) Administration of each ethyl ester compound 2) Representation of the percentage of eggs in which angiogenesis is inhibited, based on the whole eggs. 3) The strength of inhibition of angiogenesis is-, ±, +, ++, +++
It is evaluated on the basis of 5 grades, a score of 0, 1, 2, 3, 4 is given to each, and the integrated value is divided by n.

As a result, it was revealed that DHA significantly suppressed the neovascularization as compared with EPA and ETA.

[0026]

EFFECTS OF THE INVENTION Docosahexaenoic acid and its derivatives inhibit enzymes and suppress angiogenesis. Therefore, docosahexaenoic acid and / or its derivatives are
It can be effectively used as an angiogenesis inhibitor, and further as a cancer metastasis inhibitor.

Claims (3)

[Claims] 1. An enzyme inhibitor comprising docosahexaenoic acid and / or its derivative as an active ingredient. 2. An angiogenesis inhibitor containing docosahexaenoic acid and / or its derivative as an active ingredient. 3. A cancer metastasis inhibitor containing docosahexaenoic acid and / or its derivative as an active ingredient.