JPH07304667A - Venous cell adhesive molecule-1 production inhibitor - Google Patents

Abstract

PURPOSE:To obtain a new medicinal agent presenting no cytotoxicity, highly inhibiting the production of venous cell adhesive molecule-1 (VCAM-1) by human venous endothelial cells, thus useful as a metastasis inhibitor, anti-inflammatory agent, medicine for e.g. arteriosclerosis. CONSTITUTION:This VCAM-1 production inhibitor contains, as active ingredient, jolkinolide B of formula I or an alaninol derivative of formula II. The jolkinolide B of formula I is obtained by isolation from Euphorbia jolkini Boiss. followed by purification, the alaninol derivative of formula II from Euphorbia fischeriana Steud. The dose of the above compounds each is normally about 0.01-500mg/kg a day, and it is preferable that these compounds each be contained, as the active ingredient, at ca.0.01-99.99wt.% in the corresponding preparation.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a venous cell adhesion molecule-1.
The present invention relates to a drug having an action of inhibiting the production of (VCAM-1), which includes a cancer metastasis inhibitor, an anti-inflammatory agent, arteriosclerosis,
It is useful as a therapeutic agent for transplant rejection, rheumatoid arthritis, sarcoidosis and the like.

[0002]

2. Description of the Related Art VCAM-1 is a venous cell adhesion molecule (V
CAM) is a glycoprotein with a molecular weight of 90,000 newly discovered in 1989 (Osborn, L., et al, Cell 59, 12).
03, 1989). Expression of VCAM-1 is induced by stimulating vascular endothelial cells with inflammatory cytokines such as TNF (tumor necrosis factor) and IL-1 (interleukin-1), and is a ligand for vascular endothelial cells and VCAM-1. V
Cells expressing LA-4 (lymphocytes, macrophages,
Eosinophils, basophils, melanoma, etc.).

In addition to VCAM-1 in the inflammatory response, P
-Adhesion molecules such as selectin, ELAM-1, and ICAM-1 are involved. Among them, the adhesion via VCAM-1 and ICAM-1 is particularly strong, and these functions play an important role in actual diseases. It is said that It has been reported that VCAM-1 expression is increased in vascular endothelial cells in a wide range of local lesions such as arteriosclerosis, allograft rejection, cancer metastasis such as melanoma, and various acute and chronic inflammatory diseases. Therefore, a substance having an action of suppressing VCAM-1 production of this vein endothelial cell is considered to be effective as a therapeutic drug for these diseases, but such a substance has not been reported until now.

On the other hand, in the medical field, although inflammatory diseases are not directly life-threatening, there are many patients suffering from symptoms due to various causes, and more effective therapeutic agents with fewer side effects are always required. Moreover, although the metastasis of cancer is the main cause of death due to cancer, the preventive method and the therapeutic method thereof have not yet been established.

[0005]

SUMMARY OF THE INVENTION Therefore, according to the present invention, V
It is an object to provide a VCAM-1 production inhibitor containing a substance having an action of inhibiting CAM-1 production as an active ingredient.

[0006]

Means for Solving the Problems As a result of searching various compounds, the present inventors have found that VCA by venous endothelial cells.
The present invention has been completed by finding a substance showing an action of inhibiting the production of M-1.

That is, the present invention provides the following formula (1)

[0008]

[Chemical 3]

A venous cell adhesion molecule-1 (VCAM-1) production inhibitor containing jorquinolide B as an active ingredient, and the following formula (2):

[0010]

[Chemical 4]

The present invention provides a venous cell adhesion molecule-1 (VCAM-1) production inhibitor containing an alaninol derivative represented by the following as an active ingredient.

The jorquinolide B represented by the above formula (1) according to the present invention is a compound obtained by separating and purifying it from Euphorbia jolkini Boiss. By the method described in the literature (D. Uemura and Y. Hirat
a, Tetrahedron Letters No. 15, 1387, 1972.).

The alaninol derivative represented by the above formula (2) according to the present invention can be obtained by separating and purifying from the wolf venom (Euphorbia fischeriana Steud.) By the method described in the literature (D. Uemura et al., Che
m. Lett., 537, 1975.).

The VCAM-1 production inhibitor containing the above compound of the present invention as an active ingredient can be provided by oral administration, parenteral administration (subcutaneous, intravenous, intramuscular, sternum injection, etc.) or rectal administration. The dose of the above compound varies depending on the patient's symptoms, age, administration method, etc., but is usually 0.
It is about 01 to 500 mg / kg / day. For administration, the compound can be used as it is,
Usually, it is used as a formulation. For formulation, pharmacologically and pharmaceutically acceptable components of the formulation such as conventional diluents, carriers, fillers, additives, etc. may be used, and the methods usually used in this field may be applied. it can. Further, it is also possible to prepare a sustained-release preparation by a known technique. As the form of the preparation, tablets, granules, fine granules, powders, capsules, syrups, elixirs, injections, eye drops, eye ointments or suppositories are used. The amount of active ingredient contained in the preparation is 0.
It is about 01 to 99.99%.

As the above-mentioned components for the preparation, preparations for oral administration (oral preparations), preparations for injection (injection preparations), mucosal administration agents (buccal, troches, suppositories etc.), external preparations (ointments, patches etc.) Appropriate components are used depending on the administration route such as. For example, in the case of oral agents and agents for mucosal administration, excipients (eg:
Starch, lactose, crystalline cellulose, lactose calcium, magnesium aluminometasilicate, anhydrous silicic acid), disintegrant (eg carboxymethylcellulose, carboxymethylcellulose calcium), lubricant (eg magnesium stearate, talc), In the case of injections, pharmaceutical ingredients such as coating agents (eg, hydroxyethyl cellulose) and corrigents, and in the case of injections, solubilizers or solubilizers (eg, distillation for injections) that can constitute aqueous injections. For formulations such as water, physiological saline, propylene glycol), suspending agents (eg surfactants such as polysorbate 80), pH adjusters (eg organic acid or its metal salts), stabilizers, etc. When the component is an external preparation, it is an aqueous or oily solubilizer or solubilizing agent (eg alcohol, fatty acid ester), adhesive (eg:
Pharmaceutical ingredients such as carboxyvinyl polymer, polysaccharides) and emulsifiers (eg surfactants) are used.

The above-mentioned preparation can be prepared by a known production method, for example, the method described in the Japanese Pharmacopoeia, 10th Edition General Rules for Preparation or a method with appropriate modification.

[0017]

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

Test Example 1. Human VCAM-1 Production Inhibition Test Normal human umbilical vein endothelial cells (CS-VE) were supplemented with 10 ng / mL of human recombinant basic FGF.
% CHF-MCBD131 medium containing fetal bovine serum,
Prepared to 1 × 10 ⁴ cells / mL and treated with collagen I 2
0.5 mL each was dispensed to a 4-well plate and cultured in a CO ₂ incubator (CO ₂ 5%, humidity 100%, 37 ° C.) until confluence was reached. CHL containing 10% fetal bovine serum
-Replace with MCBD131 medium and replace with human recombinant T
NF-α was adjusted to 500 U / mL and 35 μL thereof was added. Then, the compounds (1) and (2) were added to a predetermined concentration and cultured in a CO ₂ incubator (CO ₂ 5%, humidity 100%, 37 ° C.) for 18 hours. VCAM-1 in this supernatant was quantified using a human VCAM-1 ELISA kit. The 50% production inhibitory concentration (IC ₅₀ ) was determined from the percentage of the measured VCAM-1 amount relative to that of the control group. The results are shown in Table 1.

[0019]

[Table 1]

Test Example 2. Cytotoxicity test The above compound was subjected to a cytotoxicity test by the following method, and it was confirmed that no toxicity was observed at the above concentration. 24 after collecting the supernatant used in the test in Test Example 1
Add 1.1 mg / mL of MTT aqueous solution to the well plate at 0.
15 mL was dispensed and viable cells were counted by the MTT method. 50% growth inhibitory concentration (IC
₅₀ ) asked. The results are shown in Table 2.

[0021]

[Table 2]

Test Example 3. TNF-α receptor binding test The above-mentioned compound was subjected to a TNF-α receptor binding test by the following method, and it was confirmed that the above-mentioned concentration did not inhibit the binding of TNF to the receptor. 1 x 10 ⁷
3 cultured human promyelocytic leukemia cells (HL-60) were suspended in 1 mL of RPMI1640 medium solution containing the compound (1) or (2) at a predetermined concentration and containing 10% fetal bovine serum.
Sensitization was performed at 7 ° C for 30 minutes. After washing with PBS containing 0.5% bovine serum albumin (binding solution), 4 × 10 ⁷ cells / mL cell suspension was prepared with the same solution. 25 μ of the prepared cell suspension was added per well of a 96-well plate.
25 μL of ¹²⁵ I-TNF adjusted to L and 2 nM and 50 μL of binding solution were added, and the mixture was reacted in ice for 90 minutes. 70 μL of the reaction mixture was added to 1 M sucrose-containing PB
Layer on an Eppendorf tube containing 1 mL of S, 12
The reaction was stopped by centrifugation at 000 rpm for 4 minutes. After removing the supernatant, the cells were washed with the binding solution, and the radioactivity of the cells was measured with a gamma counter.
The nonspecific binding number (radioactivity measured by adding 400 nM unlabeled TNF to the above reaction mixture by mixing) was subtracted from this value (total binding number) to determine the specific binding number, and the control group (10 The ratio of the specific binding (sensitization with RPMI1640 medium supplemented with% fetal bovine serum) was shown in percentage. The results are shown in Table 3.

[0023]

[Table 3]

[0024]

INDUSTRIAL APPLICABILITY The compound of the present invention has a strong inhibitory effect on the production of VCAM-1 by human venous endothelial cells within the range of not showing any cytotoxicity, and is a cancer metastasis inhibitor, an anti-inflammatory agent and atherosclerosis. It is expected to be used as a therapeutic drug for transplant rejection, rheumatoid arthritis, sarcoidosis, etc.
The mechanism of action is not merely inhibition of TNF stimulation.

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[Procedure amendment]

[Submission date] April 5, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0020

[Correction method] Change

[Correction content]

Test Example 2. Cytotoxicity test The above compound was subjected to a cytotoxicity test by the following method, and it was confirmed that no toxicity was observed at the above concentration. 24 after collecting the supernatant used in the test in Test Example 1
Add 1.1 mg / mL of MTT aqueous solution to the well plate at 0.
15 mL was dispensed and viable cells were counted by the MTT method. 50% inhibition concentration of percentage relative to the control group measurements (IC ₅₀₎
I asked. The results are shown in Table 2.

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Claims (2)

[Claims] 1. The following formula (1): A venous cell adhesion molecule-1 production inhibitor containing jolquinolide B as an active ingredient. 2. The following formula (2): A vein cell adhesion molecule-1 production inhibitor containing the alaninol derivative represented by