TW202005647A - COX-2 inhibitor, medicinal composition including the same and application thereof - Google Patents

Abstract

The present invention relates to a cyclooxygenase-2 (COX-2) inhibitor and a medicinal composition including the same. The COX-2 inhibitor includes 8-hydroxydaidzein (8-OHD) as an active ingredient. The medicinal composition including an effective dose of 8-OHD can beneficially inhibit COX-2 activity, for inhibiting or alleviating a disease or a syndrome associated with elevated COX-2 activity, thereby being applied on a use of preparation of the medical composition for inhibiting COX-2 activity.

Description

Cyclooxygenase-2 inhibitor and pharmaceutical composition containing the same and its application

The invention relates to a cyclooxygenase-2 inhibitor, in particular to an isoflavone derivative as a cyclooxygenase-2 inhibitor and a pharmaceutical composition containing the same.

General animal cells contain two types of cyclooxygenase (COX), namely COX-1 and COX-2, of which COX-2 can hardly detect its presence in normal cells, only when the cells are injured, inflammatory factors or Cytokines are only induced when stimulated. COX-2 can convert arachidonic acid to prostaglandin, which in turn causes vasodilation and inflammation such as redness, swelling, heat, and pain.

Non-steroidal anti-inflammatory analgesics (NSAIDs) mainly inhibit the conversion of arachidonic acid into prostaglandins by inhibiting COX, thereby producing anti-inflammatory and analgesic effects. However, each NSAID has different levels of anti-inflammatory and analgesic effects, and can cause various side effects, such as gastrointestinal discomfort, kidney damage, and inhibition of platelet aggregation. If you can selectively inhibit COX-2 without inhibiting COX-1, you can retain its anti-inflammatory ability and reduce its side effects.

Soybeans have been the main food source in Asia for thousands of years. Until the 1970s, soy foods were gradually paid attention to in the western mass diet due to their high-quality protein and low saturated fat. In addition to the above-mentioned influencing ingredients, soybean contains various isoflavones and is beneficial to health, and can be used as an early functional food. More and more studies have shown that isoflavones have the potential to prevent chronic diseases and play an important role in the inflammatory response.

Isoflavones are edible phytoestrogens, mainly found in legumes (including pod legumes and legumes) plants. There are 12 types of soybean isoflavones, which can be roughly divided into four categories: free type, glucoside type, acetoside type and diglycoside type. Genistein (4',5,7-trihydroxyisoflavone) and soybean isoflavone (4',7-dihydroxyisoflavone) are the two major aglycone isoflavones in soybean, and there are also glycitein ; 4',7-dihydroxy-6-methoxyisoflavone). Many clinical studies have shown that genistein and soy isoflavones are beneficial in preventing breast and prostate cancer, cardiovascular disease and osteoporosis, and alleviating the symptoms after menopause.

8-hydroxydaidzein (8-hydroxydaidzein; 8-OHD; also known as 4',7,8-trihydroxyisoflavone, 4',7,8-trihydroxyisoflavone) is a kind of fermented soybean food found in Isoflavone derivatives have been known to have strong antioxidant, anti-tumor, anti-melanogenesis, inhibition of aldose reductase and liver protection functions.

Although the geometric isomer of 8-OHD, genistein (Genistein; Gen; also known as 4,5,7-trihydroxyisoflavone, 4,5,7-trihydroxyisoflavone) has anti-inflammatory effects, but in the past research and The specific efficacy of 8-OHD has not been confirmed. In view of this, there is an urgent need to develop other new functions of 8-OHD to expand the application of 8-OHD.

Therefore, one aspect of the present invention is to provide a cyclooxygenase-2 (COX-2) inhibitor, which uses an isoflavone derivative as an active ingredient.

Another aspect of the present invention is to provide a pharmaceutical composition comprising an effective dose of 8-hydroxyisoflavone (8-OHD).

Another aspect of the present invention is to provide an isoflavone derivative and its use for preparing a pharmaceutical composition that inhibits cyclooxygenase-2 activity, wherein the aforementioned pharmaceutical composition includes an effective dose of 8-hydroxyisoflavone glycoside .

According to the above aspect of the present invention, a cyclooxygenase-2COX-2) inhibitor is proposed, comprising 8-hydroxydaidzein (8-hydroxydaidzein; 8-OHD) as an active ingredient.

According to another aspect of the present invention, a pharmaceutical composition is proposed that includes an effective dose of 8-hydroxyisoflavone (8-OHD).

In the above embodiment, the effective dose of the 8-hydroxyisoflavone glycoside in vitro to inhibit COX-2 enzyme activity may be, for example, 12.5 μM to 25 μM .

According to another aspect of the present invention, an isoflavone derivative and its use for preparing a pharmaceutical composition that inhibits cyclooxygenase-2 activity are proposed, wherein the aforementioned pharmaceutical composition includes an effective dose of 8-hydroxyisoflavone glycoside .

Application of the cyclooxygenase-2 inhibitor of the present invention and a pharmaceutical composition containing the same and its application, wherein the cyclooxygenase-2 inhibitor uses 8-hydroxyisoflavone as an active ingredient, and the aforementioned pharmaceutical composition contains an effective The dosage of 8-hydroxyisoflavone can inhibit or alleviate the diseases or symptoms associated with increased cyclooxygenase-2 activity, and is used for the preparation of a pharmaceutical composition that inhibits cyclooxygenase-2 activity.

In order to make the above and other objects, features, advantages and embodiments of the present invention more obvious and understandable, the detailed description of the attached drawings is as follows: [FIG. 1] illustrates an isoflavone derivative according to an embodiment of the present invention or The results of the production of prostaglandin E ₂ (PGE ₂ ) by polymyxin B (PMB) on LPS-stimulated mouse microglia cell line BV2.

[FIG. 2] is a graph showing the progress curves of different concentrations of isoflavone derivatives inhibiting human recombinant COX-2 enzyme activity in test tubes according to an embodiment of the present invention.

[FIG. 3] is a graph showing that the concentration of isoflavone derivatives according to an embodiment of the present invention inhibits the activity of human recombinant COX-2 enzyme.

[FIG. 4] is a Lineweaver-Burk double reciprocal diagram that inhibits the activity of human recombinant COX-2 enzyme in a test tube experiment of isoflavone derivatives according to an embodiment of the present invention.

As mentioned above, the present invention provides a cyclooxygenase-2 (COX-2) inhibitor and a pharmaceutical composition containing the same and its application, wherein the cyclooxygenase-2 inhibitor includes 8-hydroxyisoflavone Glycoside (8-hydroxydaidzein; 8-OHD) as an active ingredient can effectively inhibit cyclooxygenase-2 activity.

The "cyclooxygenase-2 (COX-2)" activity referred to herein refers to the use of a foreign inflammatory factor to induce cell inflammatory response and increase its COX-2 activity, not endogenous COX-1 activity. In one embodiment, the aforementioned COX-2 activity is evaluated by inducing an in vitro inflammatory response with lipopolysaccharides (LPS), thereby increasing COX-2 activity.

The in vitro inflammatory response referred to herein refers to the inflammatory response in "cells", and the cell type is not particularly limited. In one example, it may be, for example, microglia. Microglia are macrophages that reside mainly in the central nervous system and are responsible for brain development and repair of tissues. In the central nervous system of healthy adults, static dormant microglial cells monitor the tissue. When the central nervous system is facing actual or potential danger, it can quickly switch to the activated state and transfer to the lesion, where it proliferates and exerts phagocytic activity to remove tissue fragments. , Damaged cells or microorganisms. Increased COX-2 activity in micelles will also increase the biosynthesis of prostaglandins (such as PGE ₂ and PGG ₂ ). In addition, increased COX-2 activity is also associated with the progression of many cancers, such as colorectal cancer.

The "isoflavone derivative" referred to herein refers to 8-hydroxydaidzein (8-hydroxydaidzein; 8-OHD, also known as 4',7,8-trihydroxyisoflavone), and its structure is as shown in the formula (I) shows:

Isoflavones are dietary plant estrogens, almost produced by the Fabaceae plant. Soybean contains some of the more widely known free isoflavones such as genistein (genistein; 4', 5, 7-trihydroxyisoflavone, whose structure is shown in formula (II)), soybean isoflavone (daidzein; 4', 7' -dihydroxyisoflavone) and two other deglycosyl isoflavones (aglycone isoflavones) and glycerides (glycitein; 4', 7'-dihydroxy-6-methoxyisoflavone). Because isoflavones have estrogen activity, many clinical studies have revealed that genistein and soy isoflavones can help prevent breast cancer, prostate cancer, cardiovascular disease and osteoporosis, and relieve the symptoms of menopause.

Past studies have confirmed that genistein can pass through the blood-brain barrier (BBB), and has the potential to be used as a drug for the treatment of neurodegenerative disease. However, past studies have not confirmed whether 8-OHD has the function of inhibiting COX-2 activity.

The present invention has been confirmed by experiments that 8-OHD has the effect of inhibiting COX-2 activity in vitro or in a test tube, and the efficacy is better than genistein, and has the potential as a COX-2 inhibitor. The term "inhibition of COX-2 activity" referred to herein means that it can directly or indirectly inhibit the catalytic activity of COX-2 and regulate the biosynthesis of its products (such as PGE ₂ and PGG ₂ etc.) to achieve inhibition, slow down or prevention The effects of inflammation or cancer, such as colorectal cancer. It should be noted here that the 8-OHD of the present invention excludes downregulation of the biosynthesis of its products by directly or indirectly inhibiting the expression of COX-2 (for example, inhibiting the expression level of COX-2 mRNA or protein).

In some embodiments, when applied to the preparation of a pharmaceutical composition that inhibits cyclooxygenase-2, the effective dose of 8-hydroxyisoflavone (8-OHD) is not particularly limited. In some embodiments, the effective dose of 8-hydroxyisoflavone glycoside (8-OHD) to neural macrophages in vitro may be, for example, 12.5 μM to 25 μM .

The aforementioned pharmaceutical composition may optionally contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier referred to herein refers to a conventional adjuvant and/or vehicle other than the carrier, diluent, nucleic acid adjuvant used to deliver the active ingredient to the individual itself. , Or added to the above composition to improve the handling or storage properties of the composition, or to allow or help the dosage unit of the composition to form an excipient or any substance suitable for pharmaceutical composition and convenient for administration. The aforementioned pharmaceutically acceptable carrier should not destroy the pharmacological activity of the active ingredient, and should be non-toxic when delivering a sufficient therapeutic dose of the active ingredient.

The aforementioned applicable pharmaceutically acceptable carriers may be well known to those who are generally familiar with the general knowledge of manufacturing pharmaceutical compositions, and include but are not limited to buffers, diluents, disintegrants, adhesives, adhesives, wetting agents, Polymers, lubricants, slip agents, substances added to mask or counteract bad tastes or odors, dyes, fragrances, and substances added to improve the appearance of the composition. Specific examples of the aforementioned pharmaceutically acceptable carriers may include, but are not limited to, citrate buffer, phosphate buffer, acetate buffer, bicarbonate buffer, stearic acid, magnesium stearate, magnesium oxide , Sodium and calcium salts of phosphoric acid and sulfuric acid, magnesium carbonate, talc, gelatin, gum arabic, sodium alginate, pectin, dextrin, mannitol, sorbitol, lactose, sucrose, starch, gelatin, cellulose (Cellulose esters and cellulose alkyl esters such as alkanoic acid), low melting waxes, cocoa butter, amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g. serum albumin), ethylenediaminetetraacetic acid ( EDTA), dimethyl sulfoxide (DMSO), sodium chloride or other salts, liposomes, mannitol, sorbitol, glycerol or powder, polymers (such as polyvinylpyrrolidone, polyvinyl alcohol and polyethylene glycol Alcohol) and other pharmaceutically acceptable substances.

In application, the above-mentioned pharmaceutical composition may be administered via intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection or oral route.

The following uses several embodiments to illustrate the application of the present invention, but it is not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various modifications and changes without departing from the spirit and scope of the present invention. Retouch.

Example 1. Preparation of isoflavone derivatives

8-hydroxyisoflavone (8-OHD) refers to the method disclosed by Esaki et al. in the paper published in the journal Biosci.Biotechnol.Biochem. 62, pages 740-746 in 1998, using Aspergillus oryzae ) 8-OHD is isolated from the fermented soybean fermented product, and is listed here as a reference.

In short, the soybean fermentation product (500 g) was refluxed with methanol (5 L) for 3 hours. The obtained methanol crude extract (102g) was suspended in water (0.1L), extracted with n-hexane and ethyl acetate respectively, and concentrated in vacuo to obtain n-hexane layer extract (54g) and ethyl acetate layer extract (5.43g) and water layer extract (37g).

The extract of the ethyl acetate layer was used in a silica gel column in order of n-hexane/ethyl acetate in a volume ratio of 3:1, n-hexane/ethyl acetate in a ratio of 1:1, ethyl acetate, and acetic acid in a volume ratio of 1:1 Ethyl acetate/methanol and methanol were eluted. Next, the ethyl acetate layer extract obtained was re-used in semi-preparative C-18 ODS2 OBD Prep Column (Spherisorb, 80Å, 5 μm, 10 mm×250 mm), according to the foregoing High-performance liquid chromatography (HPLC) was carried out repeatedly. The mobile phase is composed of Eluent A (deionized water containing 0.1% v/v acetic acid) and Eluent B [acetonitrile; Merck]. When performing extraction, the extraction solution B was isocratically extracted with 14% v/v for 10 minutes, and then the extraction solution B was linearly increased from 20% v/v to 40% at a fixed flow rate of 3 mL/min v/v, the process takes more than 50 minutes. According to the peak value of 8-OHD, each elution product was collected, which was white powder (14.7 mg) after lyophilization.

The white powder was analyzed by mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR spectrometry) and confirmed to be 8-OHD. In addition, the ultra-high performance liquid chromatography (UPLC) is used to detect the white powder, and its purity is as high as 99%.

98%；Sigma-Aldrich,St.Louis,MO,USA)。 Genistein is a commercially available product (Genistein; purity

98%; Sigma-Aldrich, St. Louis, MO, USA).

Example 2. Evaluation of the inhibitory effect of isoflavone derivatives on COX-2 activity

1. Cell culture

The cell pattern used in this example is the mouse micellar cell line BV2 (hereinafter referred to as BV2 cell line). The BV2 cell line was cultured in Dulbecco's Modified Eagle Medium (DMEM) and nutrient medium (nutrient mixture) F-12 mixed DMEM/F12 medium (Invitrogen Life technologies, Calsbad, CA, USA) In the DMEM/F12 culture solution, 10% fetal bovine serum (HyClone, Logan, UT, USA), 100 U/mL penicillin and 100 μg/mL streptomycin were added. The BV2 cell line is cultured in a constant temperature incubator with 5% CO ₂ , 37°C, and 95% ambient humidity.

2. Evaluate isoflavone derivatives to inhibit microglial prostaglandins (PGE ₂ ) Biosynthesis

This example uses polymyxin B (10 μg/mL polymyxin B, PMB; Sigma-Aldrich, St. Louis, MO, USA), the isoflavone derivative to be tested (ie, 8-OHD or Gen) or a solvent [ 0.1% dimethyl sulfoxide (DMSO), veh] treated BV2 cells (cell concentration 1×10 ⁶ /mL) for 30 minutes, and then treated with LPS (10 ng/mL; Escherichia coli O111: B4, Sigma- Aldrich, St. Louis, MO, USA) stimulate cells for 20 hours, collect the cell supernatant of each group, and use a commercially available ELISA kit (eg PGE ₂ ELISA Kit; Abcam, Cambridge, UK) according to the user manual provided by the manufacturer , The amount of PGE ₂ produced by the cells was detected, and the results are shown in FIG. 1.

Please refer to FIG. 1, which shows that isoflavone derivatives or polymyxin B (PMB) produce prostaglandin E in LPS-stimulated mouse microglia cell line BV2 (hereinafter referred to as BV2 cells) according to an embodiment of the present invention. ₂ (PGE ₂ ) results. In Fig. 1, the value is represented by the mean value ± standard deviation (SD) (n=3). Figure number ** represents that this group is statistically significantly different from the solvent group (veh) without LPS treatment ( p <0.01). Figure number ## represents a statistically significant difference ( p <0.01) compared to the LPS-treated solvent group (v).

The results in Figure 1 show that the LPS-treated solvent group (v, 10 ng/mL) was based on the amount of PGE ₂ production (153 ± 77 pg/mL) of the non-LPS-treated solvent group (veh, 0.1% DMSO). LPS and 0.1% DMSO) increase the production of PGE ₂ (1633±88pg/mL), use PMB (LPS inhibitor) or isoflavone derivatives to be tested (ie 8-OHD) to treat LPS-stimulated BV2 cells, can reduce The amount of PGE ₂ produced is dose-dependent.

In other experiments, the treatment of LPS-stimulated BV2 cells with 8-OHD could not inhibit the expression of COX-2 at the level of transcription or translation (not shown), which means that 8-OHD reduces PGE ₂ production by other means. the amount.

3. Assess the effect of 8-OHD on COX-2 enzyme kinetics

Most nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the production of their products PGE ₂ and PGG ₂ by inhibiting the catalytic activity of COX-2. This example uses a commercially available COX-2 fluorescent detection kit (eg Fluorometric COX-2 inhibitor screening kit; Biovision, Milpitas, CA, USA). According to the manual provided by the manufacturer, different concentrations (0-100 μM, After the isoflavone derivative (ie 8-OHD) with 0 μM as the control group reacted with human recombinant COX-2 for an unequal time (0 to 400 seconds), the reactants were detected at the excitation (Ex) wavelength (535 nm) and Emission (Em) wavelength (587nm) absorbance value, and the relative fluorescence unit (relative fluorescence unit per second; RFU/sec) to calculate the initial catalytic rate of COX-2, the results are shown in Figure 2.

Please refer to FIG. 2, which is a graph showing the progression curves of different concentrations of isoflavone derivatives inhibiting human recombinant COX-2 enzyme activity in a test tube according to an embodiment of the present invention.

The results from Figure 2 show that compared with the control group (0 μM 8-OHD), as the concentration of 8-OHD increases, the initial catalytic rate of COX-2 activity has a decreasing trend, and has a dose-dependent relationship, representing 8- OHD does have the effect of inhibiting COX-2 activity.

Please refer to FIG. 3, which is a graph illustrating the inhibition of COX-2 enzyme activity by an isoflavone derivative according to an embodiment of the present invention. The results from Figure 3 show that the IC ₅₀ obtained by linear regression is 8.9±1.2 μM. As the concentration of 8-OHD increases, the relative enzyme activity of COX-2 decreases, and the degree of inhibition of COX-2 activity is greater than that of Gen. The effect is more significant.

Please refer to FIG. 4, which illustrates a Lineweaver-Burk double reciprocal plot (Lineweaver-Burk double reciprocal plot) in which isoflavone derivatives inhibit COX-2 catalytic activity in vitro according to an embodiment of the present invention, in which the horizontal axis ( The x-axis represents the reciprocal (μM ^-1 ) of the concentration of the substrate (i.e. arachidonic acid) catalyzed by COX-2, and the vertical axis (i.e. the y-axis) represents the reciprocal of the initial catalytic rate of COX-2 producing PGG ₂ (1 /V; sec/RFU).

The results in Figure 4 show that the relationship between the reciprocal of the mass concentration and the reciprocal of the initial catalytic rate of the enzyme intersects below the x-axis, rather than on the x-axis (that is, non-competitive inhibition) or the y-axis ( (Competitive inhibition), which means that 8-OHD inhibits COX-2 activity is a mixed type of inhibition mechanism, that is, it contains both competitive and non-competitive inhibition mechanisms.

To sum up, although the present invention takes isoflavone derivatives of a specific structure, specific analysis modes or specific evaluation methods as examples, it illustrates the cyclooxygenase-2 inhibitors of the present invention and the pharmaceutical compositions containing the same and their applications, However, anyone with ordinary knowledge in the technical field to which the present invention belongs can know that the present invention is not limited to this, and the cyclooxygenase-2 inhibitor of the present invention and the pharmaceutical composition containing the same are within the spirit and scope of the present invention Its application can also use other analysis modes or other evaluation methods. For example, the pharmaceutical composition for inhibiting COX-2 activity of the present invention can also be used to inhibit or alleviate diseases or symptoms associated with increased cyclooxygenase-2 activity, and further be used to prepare a pharmaceutical for inhibiting cyclooxygenase-2 activity Use of the composition.

It can be seen from the above examples that the cyclooxygenase-2 (COX-2) inhibitor of the present invention and the pharmaceutical composition containing the same and its application have the advantage of using 8-hydroxyisoflavone as epoxidation The active ingredient of the enzyme-2 inhibitor, and the aforementioned pharmaceutical composition also contains an effective dose of 8-hydroxyisoflavone, so it can effectively inhibit or alleviate the diseases or symptoms related to the increased activity of cyclooxygenase-2, and is used in the preparation Use of a pharmaceutical composition that inhibits cyclooxygenase-2 activity.

Although the present invention has been disclosed in several embodiments as above, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs can be regarded as various without departing from the spirit and scope of the present invention. Changes and retouching, therefore, the scope of protection of the present invention shall be subject to the scope defined in the appended patent application.

Claims (5)

A cyclooxygenase-2 (COX-2) inhibitor contains 8-hydroxydaidzein (8-OHD) as an active ingredient. A pharmaceutical composition comprising an effective dose of 8-hydroxyisoflavone glycoside (8-OHD). The pharmaceutical composition according to item 2 of the patent application scope, wherein the effective dose of the 8-hydroxyisoflavone glycoside to inhibit COX-2 enzyme activity in vitro is 12.5 μM to 25 μM . An isoflavone derivative is used for preparing a pharmaceutical composition for inhibiting cyclooxygenase-2 activity, wherein the pharmaceutical composition contains an effective dose of 8-hydroxyisoflavone glycoside. The use of the isoflavone derivative according to item 4 of the patent application scope for preparing a pharmaceutical composition that inhibits cyclooxygenase-2 activity, wherein the 8-hydroxyisoflavone glycoside inhibits COX-2 enzyme activity in vitro The effective dose is 12.5 μM to 25 μM .

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