JP2006008715A - Phospholipase a2 inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new highly safe medicine for inhibiting phospholipase A<SB>2</SB>(PLA<SB>2</SB>), and to provide a therapeutic agent for diseases, such as inflammatory diseases and cardiovascular diseases, in which PLA<SB>2</SB>is estimated to participate. <P>SOLUTION: The PLA<SB>2</SB>inhibitor and the therapeutic agent for diseases in which PLA<SB>2</SB>is estimated to participate, each contains astaxanthin and/or its ester as an active ingredient. The PLA<SB>2</SB>inhibitor is useful for treating various diseases in which PLA<SB>2</SB>is estimated to participate. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to phospholipase _{A 2} inhibitor. More specifically, the present invention relates to a phospholipase A ₂ inhibitor containing astaxanthin and / or an ester thereof as an active ingredient, and a therapeutic agent for diseases involving phospholipase A ₂ such as cardiovascular disease.

Phospholipase A ₂ (PLA ₂ ) is known as an enzyme that hydrolyzes the ester bond at the C2 position of 1,2-diacylphosphoglyceride, a biological membrane constituent. There are various types of PLA ₂ , such as type I exocrine as a digestive enzyme from the pancreas and endocrine type II present in cells and involved in the initial process of arachidonic acid metabolism. This type II is the rate-determining enzyme of the arachidonic acid cascade and is known to be associated with normal inflammation through its metabolites, prostaglandins and leukotrienes. Therefore, PLA ₂ inhibitors that can suppress the biosynthesis of these inflammatory substances are used as anti-inflammatory agents.

Currently, PLA ₂ inhibitors that are actually used as anti-inflammatory agents include steroidal drugs. Steroidal drugs indirectly inhibit PLA ₂ by the following mechanism: binding to cytoplasmic receptors to form a complex, then translocating into the nucleus, and promoting protein transcription such as lipocortin Is expressed, and PLA ₂ is inhibited by these expressed proteins (Non-patent Documents 1 and 2). Thus, since steroidal drugs affect transcription, there are problems with various side effects such as immunosuppression and other effects on metabolism (for example, protein catabolism and calcium metabolism abnormality). .

Indirect PLA ₂ inhibition involves various problems of side effects as described above, and various studies have been conducted on substances that directly inhibit PLA ₂ activity. For example, various chemically synthesized substances such as benzoic acid derivatives (Patent Document 1) and indole compounds (Patent Document 2) have been reported as PLA ₂ inhibitors. However, since these PLA ₂ inhibitors are synthetic products, strong toxicity and side effects are considered, and none of them has been put into practical use. Alternatively, there are antibodies such as an anti-mouse PLA ₂ antibody (Patent Document 3), but it is still difficult to put an antibody drug into practical use.

PLA ₂ is deeply involved not only in the above-mentioned inflammatory action but also in the generation and development of arteriosclerosis via lipid chemical mediators such as prostaglandins, thromboxanes, and leukotrienes (Non-patent Document 3). . Therefore, PLA ₂ inhibitors are expected to be applied to the treatment of cardiovascular diseases such as myocardial infarction, which is an arteriosclerotic disease.

Carotenoids (carotenoids) are fat-soluble biological pigments that are widely distributed in animals, plants, and microorganisms, and have about 600 to yellow to orange to red colors. Astaxanthin, which is one type, is contained in crustaceans such as krill, shrimp and crabs, salmon and trout muscles and eggs (such as salmon roe), and body surfaces such as Thai, carp and goldfish. Astaxanthin is known not only to be provitamin A and to have a remarkable antioxidant effect, but also to have an anti-inflammatory effect (for example, Patent Documents 4 and 5). The mechanism of action has been reported to be due to inhibition of expression of inflammatory cytokines and chemokines (Patent Document 6), suppression of histamine release (Patent Document 7), and the like. However, there is no report about the influence on the activity of PLA ₂ as described above.
JP-A-8-325154 Special table 2003-505372 JP-A-8-188600 JP 7-300421 A JP 2004-331512 A Special table 2003-528139 gazette US Pat. No. 5,886,053 Flower, RJ and Blackwell, GJ, “Nature”, 1979, 278, pp. 456-459 Di Rosa, M. et al., "Prostaglandins", 1984, 28, pp.441-442 Kugiyama K. et al., “Cardiovasc Res.”, 2000, 47, pp.159-65

An object of the present invention is to provide a new highly safe drug that inhibits phospholipase A ₂ (PLA ₂ ). The present invention further aims to PLA ₂ provides a therapeutic agent for diseases such as inflammatory diseases and cardiovascular diseases thought to be involved.

As a result of various studies on astaxanthin known as an anti-inflammatory agent, it was found that astaxanthin has a PLA ₂ inhibitory action, and the present invention was completed.

The present invention provides a phospholipase A ₂ inhibitor containing astaxanthin and / or an ester thereof as an active ingredient.

In one embodiment, the phospholipase A ₂ inhibitors, directly inhibit the activity of phospholipase A _2.

The present invention also contains astaxanthin and / or its ester as an active ingredient, phospholipase A ₂ to provide a therapeutic agent for a disease associated.

  In a preferred embodiment, the diseases are inflammatory diseases and cardiovascular diseases.

In one embodiment, the therapeutic agent is directly inhibit the activity of phospholipase A _2.

According to the present invention, a new PLA ₂ inhibitor is provided. This PLA ₂ inhibitor can be used as a therapeutic agent for various diseases such as cardiovascular diseases that are thought to involve PLA ₂ . Since the PLA ₂ inhibitor of the present invention directly inhibits the activity of PLA ₂ , there are few side effects. Furthermore, astaxanthin and / or its ester, which is an active ingredient of the PLA ₂ inhibitor of the present invention, is highly safe because it has a long diet and very low toxicity.

Astaxanthin and / or its ester, which is an active ingredient of the PLA ₂ inhibitor of the present invention, has the following formula:

(Here, R ¹ and R ² are each independently a hydrogen atom or a fatty acid residue). The ester of astaxanthin is not particularly limited. For example, saturated fatty acids such as palmitic acid and stearic acid, or oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, bishomo-γ-linolenic acid, arachidonic acid, Examples thereof include monoesters or diesters of unsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. These can be used alone or in appropriate combination. Astaxanthin has a structure having extra oxo groups and hydroxy groups at both ends of the skeleton of β-carotene, and therefore has low molecular stability unlike β-carotene. On the other hand, the ester body (for example, krill extract) in which the hydroxyl groups at both ends are esterified with unsaturated fatty acid or the like is more stable.

  Astaxanthin and / or its ester used in the present invention may be either chemically synthesized or derived from natural products. Examples of the latter natural products include red yeast containing astaxanthin and / or an ester thereof; shellfish shells such as tigliopath (red daphnia) and krill; and microalgae such as green algae. In the present invention, an extract containing astaxanthin and / or its ester produced by any method can be used as long as the characteristics of astaxanthin and / or its ester can be utilized. In general, extracts from these natural products are used, and the extract may be in the form of an extract or may be appropriately purified as necessary. In the present invention, such a crude extract or crushed powder containing astaxanthin and / or an ester thereof, or an appropriately purified or chemically synthesized product containing astaxanthin and / or its ester may be used alone or in appropriate combination. Can do. Considering the stability in the body, an ester form is preferably used.

The PLA ₂ inhibitors of the present invention may be useful for treating or preventing diseases or conditions involving PLA ₂ . Such diseases or symptoms include pancreatitis, pancreatic cancer, chronic hepatitis, cirrhosis, cholecystitis, rheumatism, allergy, bronchial asthma, arthritis, dermatitis, gout, multiple sclerosis, trauma-induced inflammation, etc .; Cardiovascular diseases such as arteriosclerosis, myocardial infarction, angina pectoris, cerebral infarction; ulcer caused by microbial infection, etc .; psoriasis; diabetes and its complications;

The therapeutic agent for a disease involving PLA ₂ of the present invention contains astaxanthin and / or an ester thereof as an active ingredient, similarly to the PLA ₂ inhibitor of the present invention. It is particularly useful for the treatment of inflammatory diseases and cardiovascular diseases.

The administration route of the PLA ₂ inhibitor of the present invention or the therapeutic agent for diseases involving PLA ₂ may be either oral administration or parenteral administration. The dosage form is appropriately selected depending on the administration route. For example, injections, infusions, powders, granules, tablets, capsules, pills, intestinal solvents, troches, liquids for internal use, suspensions, emulsions, syrups, liquids for external use, poultices, nasal drops, ear drops Agents, eye drops, inhalants, ointments, lotions, suppositories, enteral nutrients and the like. This can be used alone or in combination depending on the symptoms. In these preparations, auxiliary agents usually used in the pharmaceutical preparation technical field such as excipients, binders, preservatives, oxidation stabilizers, disintegrants, lubricants, and corrigents are used as necessary. .

The dosage of the PLA ₂ inhibitor of the present invention or the therapeutic agent for a disease involving PLA ₂ varies depending on the purpose of administration and the situation (sex, age, body weight, etc.) of the administration subject. Usually, for adults, 0.1 mg to 2 g per day, preferably 4 mg to 500 mg per day in the case of oral administration, in terms of astaxanthin free body, while 0.01 mg to 1 g per day for parenteral administration, Preferably it can be administered at 0.1 mg to 500 mg.

The PLA ₂ inhibitor of the present invention can be used not only as a pharmaceutical as described above, but also as a quasi-drug, cosmetic, functional food, nutritional supplement, food and drink, and the like. When used as a quasi-drug or cosmetic, it can be used together with various adjuvants commonly used in the technical field such as quasi-drug or cosmetic, if necessary. Or, when used as a functional food, nutritional supplement, or food and drink, it is usually used in foods such as sweeteners, spices, seasonings, preservatives, preservatives, bactericides, and antioxidants as necessary. You may use with the additive used. Further, it may be used in a desired shape such as solution, suspension, syrup, granule, cream, paste, jelly, etc., or as necessary. The ratio contained in these is not specifically limited, It can select suitably according to a use purpose, a usage form, and a usage-amount.

(Preparation Example 1: Preparation of astaxanthin monoester)
Astaxanthin monoester was prepared as follows. The Haematococcus pulvialis strain K0084 was cultured at 25 ° C. under irradiating gas containing 3% CO ₂ under light irradiation conditions, under nutrient stress (nitrogen source deficiency), and cysted. The cysted cells were crushed by means commonly used by those skilled in the art, and the oily fraction was extracted with ethanol. The extract contained lipids such as triglycerides in addition to astaxanthins. The extract was subjected to column chromatography using a synthetic resin adsorbent to obtain a purified product containing an astaxanthin monoester. This purified product was analyzed by HPLC, and it was confirmed that this purified astaxanthin monoester contained a monoester having a molecular weight of 858 as a main component, did not contain free and diester forms of astaxanthin, and contained a slight amount of diglyceride. .

(Example 1: Effect on the activity of PLA ₂ )
Regarding the astaxanthin monoester obtained in Preparation Example 1, the effect on the activity of PLA ₂ was examined. For PLA ₂ , purified porcine pancreas type I (PLA ₂ -I) and snake pancreas type II (PLA ₂ -II) were used. Astaxanthin monoester was dissolved in dimethyl sulfoxide (DMSO), added to the assay system shown in Table 1 below, and incubated at 250, 25, 2.5, and 0.25 μM, and the inhibition rate was measured. .

The 50% inhibitory concentration (IC ₅₀ ) by astaxanthin monoester was 53.1 μM for PLA ₂ -I and 67.3 μM for PLA ₂ -II. The inhibition rate of enzyme activity at 250 μM was 79% and 77%, respectively. Thus, astaxanthin monoester showed a relatively low inhibitory concentration and a high inhibition rate for any PLA ₂ .

(Reference Example 1: Measurement of 50% lethal concentration against HUVEC)
Human umbilical vein endothelial cells (HUVEC) (ATCC CRL-1730) were obtained from American Type Culture Collection and endothelial cell growth containing 10% fetal bovine serum supplemented with 1% Antibiotic-Antimycotic (GIBCO BRL, USA) Medium (CELL APPLICATIONS, USA)) was pre-cultured at 37 ° C. in a 5% CO ₂ atmosphere.

  Matrigel matrix (BD Biosciences, USA) was thawed and kept on ice at 4 ° C., and 50 μL of matrix was transferred to each well of a 96-well tissue culture plate. The plate was incubated at 37 ° C. for at least 1 hour to solidify the matrix solution.

  On the other hand, the astaxanthin monoester obtained in Preparation Example 1 above was dissolved in dimethyl sulfoxide (DMSO) and then diluted with distilled water to obtain 25000, 2500, 250, 25, and 40% in 40 (v / v)% DMSO. A stock test solution containing 2.5 μM astaxanthin monoester was prepared.

100 μL of HUVEC suspension (approximately 2.5 × 10 ³ cells / well) was placed in a 96-well Matrigel plate at 37 ° C. in a 5% CO ₂ atmosphere. After 24 hours, 100 μL of growth medium and 2 μL of each of the above stock test solutions or vehicles (40 (v / v)% DMSO) were added to each of the two wells and incubated for a further 72 hours. The final concentrations of DMSO and astaxanthin monoester were 250, 25, 2.5, 0.25, and 0.025 μM.

  At the end of the incubation, 20 μL of 90% alamarBlue reagent was added to each well and incubated for an additional 6 hours. Next, the fluorescence intensity of each well was measured at an excitation wavelength of 530 nm and an emission wavelength of 590 nm using a Spectrafluor Plus plate reader, and the number of viable cells was counted. This is based on the ability of viable cells to change alamarBlue from a non-fluorescent oxidized form (blue) to a fluorescent reduced form (red). The 50% lethal concentration was calculated as the concentration at which 50% of the number of cells at the start of the experiment.

As a result, the 50% lethal concentration (LC ₅₀ ) of astaxanthin monoester with respect to HUVEC was 250 μM (maximum dissolution concentration in DMSO), indicating that the toxicity was low.

According to the present invention, a new PLA ₂ inhibitor is provided. This PLA ₂ inhibitor can be used as a therapeutic agent for various diseases such as inflammatory diseases and cardiovascular diseases that are thought to involve PLA ₂ . Since the PLA ₂ inhibitor of the present invention directly inhibits the activity of PLA ₂ , there are few side effects. Furthermore, astaxanthin and / or an ester thereof, which is an active ingredient of the PLA ₂ inhibitor of the present invention, has a long diet and very low toxicity, so it is extremely safe. Therefore, it can be used not only as a medicine but also on a daily basis as a health food.

Claims (5)

As an active ingredient astaxanthin and / or its esters, phospholipase A ₂ inhibitors. Directly inhibit the activity of phospholipase A _2, phospholipase A ₂ inhibitors according to claim 1. Containing astaxanthin and / or its ester as an active ingredient, therapeutic agents for diseases involving phospholipase A _2.   The therapeutic agent according to claim 3, wherein the diseases are inflammatory diseases and cardiovascular diseases. Phospholipase A directly inhibits the _second active therapeutic agent according to claim 3 or 4.