JPH09255576A - Antiinflammatory agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antiinflammatory agent having an excellent secretin- inhibiting action, an excellent antiinflammatory action and an excellent cancer metastasis-inhibiting action, low in cytotoxicity, and acting on inflammations such as pneumonia and ulcers, cancer metastasis, etc. SOLUTION: This antiinflammatory agent contains an N-acetylneuraminic acid of the formula [R is H, SO3 H;(n) is an integer of 1-1000; wherein the number of the SO3 H residues is 0.1-3 per N-acetylneuraminic acid residue], its homopolymer sulfate ester or its pharmacologically acceptable salt as an active ingredient. The compound of the formula is obtained e.g. by reacting 1 pt.wt. of Nacetylneuraminic acid homopolymer with 0.2-30 pts.wt. of a sulfating agent in the presence of 0.5-200 pts.wt. of a catalyst (e.g. pyridine) in a solvent (e.g. DMF) at 0-90 deg.C for approximately 0.2-100 hrs.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anti-inflammatory agent, a selectin inhibitor, a cancer metastasis inhibitor, and a therapeutic agent for ischemia-reperfusion injury.

[0002]

BACKGROUND OF THE INVENTION The selectin family (L-selectin, P-selectin, E-selectin) is a cell adhesion receptor molecule having a sugar chain as a ligand in the vascular system,
It is closely related to inflammation and cancer metastasis (because it reacts with sialyl Lewis ^x antigen).

As a ligand for the selectin family, sulfatide and its position-substituted isomer of a sulfate group are known (Lipid Biochemistry, Vol. 37, 1995).
Year; International Immunology, Vol.7, No.7, pp1107-
1113, 1995). However, there is a problem that sulfatide and its isomers are very difficult substances to obtain.

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel selectin inhibitor, anti-inflammatory agent, cancer metastasis inhibitor and ischemia-reperfusion injury therapeutic agent.

[0005]

As a result of repeated studies in view of the above problems, the present inventors have found that sulfuric acid esters of N-acetylneuraminic acid homopolymers such as N-acetylneuraminic acid and colominic acid and colominic acid are It has been found that all of them are useful as a selectin inhibitor, can be expected to be effective as a cancer metastasis inhibitor and a therapeutic agent for ischemia-reperfusion injury, and further that the sulfate ester is useful as an anti-inflammatory agent.

That is, the present invention provides the following selectin inhibitors and anti-inflammatory agents. The selectin inhibitor of the present invention has an activity of inhibiting P-selectin and L-selectin from binding to various selectin-binding cells such as leukocytes and renal distal tubular epithelial cells, and inhibits the action of selectin.

The selectin inhibitor and anti-inflammatory agent of the present invention are useful for humans and mammals such as dogs, cats, cows, horses, pigs and the like.

Item 1. The following formula (I)

[0009]

Embedded image

[In the formula, R is the same or different and represents a hydrogen atom or SO ₃ H, and n is an integer of 1 to 1000. However, the number of SO ₃ H residues per molecule of N-acetylneuraminic acid residue is 0.1 to 3. ] The anti-inflammatory agent which uses N-acetylneuraminic acid represented by these, or the sulfuric acid ester of its homopolymer, or its pharmaceutically acceptable salt as an active ingredient.

Item 2. The following formula (I)

[0012]

Embedded image

[Wherein R is the same as above. n is 3 to 100
Indicates an integer of 0. However, when n is 3 to 11, the number of SO ₃ H residues per molecule of N-acetylneuraminic acid residue is 0.1 to 3, and when n is 12 or more, N-
The number of SO ₃ H residues per molecule of acetylneuraminic acid residue is 0 to 3. ] The selectin inhibitor which uses the N-acetyl neuraminic acid homopolymer represented by these, its sulfuric acid ester, or its pharmaceutically acceptable salt as an active ingredient.

Item 3. The following formula (I)

[0015]

[Chemical 6]

[Wherein R is the same as above. n is 3 to 100
Indicates an integer of 0. However, when n is 3 to 11, the number of SO ₃ H residues per molecule of N-acetylneuraminic acid residue is 0.1 to 3, and when n is 12 or more, N-
The number of SO ₃ H residues per molecule of acetylneuraminic acid residue is 0 to 3. ] The P-selectin inhibitor which uses N-acetyl neuraminic acid homopolymer represented by these, its sulfuric acid ester, or its pharmaceutically acceptable salt as an active ingredient.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION In an anti-inflammatory agent containing the compound of the general formula (I) of the present invention as an active ingredient, n is 1 to 1000,
It is preferably 1 to 500, more preferably 1 to 300, and particularly 4 to 200.

In the (P-) selectin inhibitor containing the compound of the general formula (I) of the present invention as an active ingredient, n is 3 to 1
000, preferably 3 to 500, more preferably 3 to 3
00, especially 4-200.

The (P-) selectin inhibitor of the present invention comprises n
When it is 12 or more, preferably 50 or more, it functions as an inhibitor even if it is not a sulfate ester, but a sulfate ester is preferable because the inhibitory activity is enhanced.

In the compound of general formula (I) of the present invention,
SO ₃ H per molecule of N-acetylneuraminic acid residue
The number of residues is 0.1-3, preferably 0.3-2, more preferably 0.3-1.5, especially 0.5-1.5.

The anti-inflammatory agent comprising the sulfuric acid ester of N-acetylneuraminic acid homopolymer of the present invention as an active ingredient is n
May be a single compound of only one kind, and 1 to 1
It may be a mixture of a plurality of N-acetylneuraminic acid homopolymers having different values of n within the range of 000.

The (P-) selectin inhibitor containing the sulfuric acid ester of the N-acetylneuraminic acid homopolymer of the present invention as an active ingredient may be a single compound in which n is only 1 type, and is 3 to 3. It may be a sulfate ester of a plurality of N-acetylneuraminic acid homopolymers having different n values within the range of 1000, or a mixture of N-acetylneuraminic acid homopolymers in which n is 12 or more.

The compound of the general formula (I) of the present invention is known and, for example, according to the method described in International Publication WO94 / 17123, 1 part by weight of N-acetylneuraminic acid homopolymer is added to a solvent (DMF, Catalyst (pyridine, triethylamine, etc.) in the presence or absence of DMSO, etc.
0.5 to 200 parts by weight, sulfating agent (sulfatrioxide / pyridine, sulfatrioxide / trimethylamine, chlorosulfonic acid, piperidine sulfuric acid, etc.) 0.2 to 3
The desired sulfuric acid ester of N-acetylneuraminic acid homopolymer is obtained by reacting with 0 part by weight at 0 to 90 ° C. for about 0.2 to 100 hours. After the sulfation reaction, it can be purified by a known purification method (concentration, gel filtration, various chromatography such as ion exchange, reprecipitation, dialysis and the like).

As the N-acetylneuraminic acid homopolymer, a homopolymer having a certain degree of polymerization (n) may be used, and a mixture of natural N-acetylneuraminic acid homopolymers such as colominic acid may be used. Is also good.

In the present invention, pharmaceutically acceptable salts include alkali metal salts such as sodium, potassium and lithium, and alkaline earth metal salts such as magnesium and calcium.

The pharmaceutical composition containing the sulfuric acid ester of N-acetylneuraminic acid homopolymer represented by the general formula (I) of the present invention or a salt thereof and a pharmaceutically acceptable additive is an anti-inflammatory agent, selectin. It is useful as an inhibitor, a cancer metastasis inhibitor, and a therapeutic agent for ischemia-reperfusion injury. The pharmaceutical composition is not particularly limited, but various forms such as injections, tablets, capsules, granules, oral preparations such as fine granules and emulsions, injections, suppositories, ointments and the like. Can be used in.

As the anti-inflammatory action, specifically, pneumonia,
It has effects on inflammation such as ulcer, phlebitis, arteritis, rheumatoid arthritis, adult respiratory distress syndrome (ARDS), ischemic brain / heart disease, atopic dermatitis, infiltration after organ transplantation.

Regarding cancer metastasis, it can be expected to be effective in suppressing metastasis of cancers related to sialyl Lewis ^x antigen, for example, small cell lung cancer and neuroblastoma cancer.

When a pharmaceutical composition of the present invention containing a sulfuric acid ester of N-acetylneuraminic acid homopolymer as an active ingredient is prepared as an injection, additives include pH adjusting agents, buffers, stabilizers, etc. Examples include a tonicity agent, a local anesthetic, and the like, and by appropriately mixing these, an injection preparation can be prepared. The formulation may be intravenous, intramuscular,
It is administered subcutaneously or intraperitoneally.

Additives for preparing the pharmaceutical composition of the present invention as an oral preparation include excipients, disintegrants, lubricants, binders, flavoring agents, flavoring agents and the like.

Additives when the pharmaceutical composition of the present invention is prepared as a suppository include bases and surfactants.

Additives for preparing the pharmaceutical composition of the present invention as an ointment include bases, surfactants and the like.

The amount of the sulfuric acid ester of the N-acetylneuraminic acid homopolymer of the present invention, which is the active ingredient contained in the above-mentioned various preparations, varies depending on the age, sex, weight and symptoms of the patient to be administered. However, in general, 1 to 100 mg per oral dosage unit for adult humans
About 0.1 to 20 mg for injections, 1 to about suppositories
It is preferably about 50 mg, and the daily dose for a human adult varies depending on the dosage form, but is 1 to 100.
It is about 0 mg, preferably about 10 to 200 mg.

[0034]

INDUSTRIAL APPLICABILITY The sulfuric acid ester of N-acetylneuraminic acid homopolymer of the present invention has excellent selectin inhibitor activity, anti-inflammatory activity and cancer metastasis inhibitory activity, and low cytotoxicity, so that it is a selectin inhibitor. , Useful as an anti-inflammatory agent. Further, the sulfate ester of the N-acetylneuraminic acid homopolymer of the present invention is also useful as a cancer metastasis inhibitor. Furthermore, it can be used for long-term administration.

[0035]

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

Reference Example 1 N-Acetylneuraminic Acid Homopolymer Sulfate Ester
Manufacturing of colominic acid (Purchased by: Nakarai Tesque, Inc.) 90m
g in 20 ml of dimethylformamide,
7 mg of sulfatrioxide / pyridine complex and 37
Add mg of dimethylaminopyridine and add 24 at 30 ° C.
Stirred for hours. Then, the mixture was cooled with ice and added dropwise to 50 ml of 1M sodium hydrogen carbonate. The solid matter is separated by filtration, and the dialysis tube (VISKASE SALES CO
RP. Dialysis was performed using the product (pore diameter: 24 Å). After dialysis, it was passed through a sodium ion type ion exchange resin (Amberlite IR-120B, manufactured by Organo Co.) column. Column effluent under reduced pressure 5m
Concentrated to l. Lyophilize the concentrate to 126 mg
Was obtained. The obtained colamine sulfate ester is n
(Average value) = 50 (determined from the average molecular weight measured by gel filtration using HPLC).

In addition, the rhodizonic acid method
hod) (Analytical Biochemistry (Anal. Bio
chem.), 41, 47 (1971)], the number of sulfuric acid ester groups per molecule of N-acetylneuraminic acid in the sulfuric acid ester of colominic acid was 1.1.

Reference Example 2 Colominic acid (purchased by: Nacalai Tesque, Inc.) 90 m
715 mg of sulfatrioxide / pyridine complex and 56 mg of dimethylaminopyridine were added to g, and 3
N (average value) = 50 (determined from average molecular weight measured by gel filtration using HPLC) and N-acetylneuraminic acid in the same manner as in Reference Example 1 except that the mixture was stirred at 0 ° C. for 48 hours. Number of sulfate ester groups per molecule = 1.
6 colominic acid sulfate ester was obtained.

Reference Examples 3 to 8 In the same manner as in Reference Example 1, except that 1-hexamer of N-acetylneuraminic acid was used in place of colominic acid as a raw material, the results shown in Table 1 and Table 2 were obtained. Was obtained.

Example 1 (1) Therapeutic effect on pneumonia model in rats administered with cobra toxin (CVF) Long-Evans not infected with a specific pathogen
Evans) rats (male, 250-300 g) were used. Cobra venom (CVF) is a crude venom of Cobra ( Naja naja ) from Till et al., J.
Prepared as described by Clin. Invest. , 69 , 1126-1135 (1982). ¹²⁵ IB of 20 U CVF per kg of body weight
Rats were injected intravenously with a fixed amount of SA (0.5 μCi).

Thirty minutes after CVF injection, the rat was anesthetized with ketamine hydrochloride (100 mg / kg) (Paeke Davis and Co.),
Blood was collected from the dorsal aorta. As a negative control, a phosphate buffer solution (PBS, p
H7.4) was used and processed in the same manner as above. The lung vasculature is used every 30 minutes with 10 ml of PBS,
Perfusion was performed via the right ventricle. Then, the lungs were removed, the vascular system was perfused with 10 ml of sterile physiological saline, and then the radiation dose remaining in the tissues was measured by a gamma scintillation counter. The positive control is a measured value when the test substance is not administered.

Lung damage was identified by determining the increase in pulmonary vascular permeability (the ratio of ¹²⁵ I-BSA radioactivity present in lung tissue to the radioactivity present in 1 ml of venous blood obtained at death). did. The protection against lung injury was calculated by the following formula.

Protection rate (%) = 100 × [test substance value-negative control (PBS) value] / [positive control-negative control (PBS) value] (2) Tissue myeloperoxidase (MPO) activity The MPO activity of the tissue was measured as an index of sphere infiltration. A standard curve was constructed after a constant number of intraperitoneal neutrophils in glycogen-stimulated rats were added to normal rat lungs and the tissues were homogenized and then extracted (Warren et al., J. Clin I.
nvest. , 84 , 1873-1882 (1989)).

A lung sample was prepared by using 6 ml of a buffer solution (50 mM phosphate, pH 6.0) and a homogenizer (Polymer).
tron: Tekmer Co.) was set to 4 and homogenized for 4 × 10 seconds, then centrifuged at 4 ° C. (3000 g, 30 minutes). The MPO activity in the supernatant, the absorbance resulting from the consumption of H ₂ O ₂ in the presence of o- dianisidine (460N
It was evaluated by measuring the change in m). Immediately before administration of the CVF of (1) above, each test substance was measured for 1 m
Table 1 shows the results of intravenous administration of g / kg.

[0045]

[Table 1] Reduction rate of lung injury (%) Test substance n Sulfation degree Vascular permeability Neutrophil infiltrating NANA monomer 1- <2 <2 NANA dimer 2 <2 <2 Colominic acid 50 <2 <2 Reference example 3 1 1.6 37 37 32 Reference Example 4 2 1.3 14 14 Reference Example 5 3 1.2 6 11 Reference Example 6 4 1.1 58 49 Reference Example 7 5 1.1 1.1 37 35 Reference Example 8 6 1.0 22 26 Reference Example 1 50 1.1 52 46 Reference Example 2 50 1.6 42 35 In Tables 1 and 2: "NANA" represents N-acetylneuraminic acid.

"Sulfation degree" indicates the number of sulfate groups per molecule of N-acetylneuraminic acid.

(2) Inhibition of P-selectin-Ig binding (IC
₅₀ ) Action P-selectin (derived from human) is produced by Watanabe T., Song.
Y., Hirayama, Y., Tamatani T., Kuida K., Miyasaka
M., Biochem. Biophys. Acta, 1131, 321-324 (1992)
A chimeric protein (hereinafter, abbreviated as "P-selectin-IgG") of the selectin extracellular domain and human IgG prepared by genetic engineering, which was prepared according to the method described in 1., was used.

In the presence of a certain amount of P-selectin-IgG, the concentration of sulfate ester of N-acetylneuraminic acid or its homopolymer is increased so that the sulfate ester causes P-selectin-.
The effect of inhibiting the binding between IgG and leukocytes was observed. The binding of P-selectin-IgG to leukocytes binds phycoerythrin to human IgG.
It was measured by flow cytometric analysis using an antibody complexed to the Fc region of E. coli. As the sulfuric acid ester, the sulfuric acid ester of the N-acetylneuraminic acid tetramer obtained in Reference Example 6 (sulfation degree =
The results using 1.1) are shown in FIG. In addition, reference examples 1 to 1
The results for the 5, 7, and 8 sulfates are shown in Table 2. The IC ₅₀ is the concentration of P-selectin-IgG that inhibits the binding to leukocytes by 50%.

[0049]

[Table 2] Test substance for P-selectin-IgG binding n Sulfation degree inhibition (IC ₅₀ ) (μM) NANA monomer 1-> 900 NANA dimer 2> 5000 colominic acid 50 36.4 Reference example 3 1 1.6 > 900 Reference example 4 2 1.3> 5000 Reference example 5 3 1.2 83.9 Reference example 6 4 1.1 4.45 Reference example 7 5 5 1.1 33.7 Reference example 8 6 1.0 16. 7 Reference Example 1 50 1.1 0.58 Reference Example 2 50 1.6 0.37 In Table 2: "NANA" indicates N-acetylneuraminic acid.

From the results shown in Table 1, the sulfates of N-acetylneuraminic acid having various degrees of polymerization and degree of sulfation are excellent in anti-inflammatory action (suppression of inflammation in CVD pneumonia model) and action as P-selectin inhibitor. , Selectin inhibitors,
It was revealed that they are useful as P-selectin inhibitors and anti-inflammatory agents. In addition, since it has been revealed that cancer metastasis and ischemia-reperfusion injury are closely related to selectin, the selectin inhibitor of the present invention that selectively binds to selectin is a cancer metastasis inhibitor and an imaginary metastasis. It was revealed that it is also useful as a therapeutic agent for blood-reperfusion injury.

[Brief description of drawings]

FIG. 1 shows the inhibition rate of sulfate ester of N-acetylneuraminic acid tetramer with respect to P-selectin.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location C08B 37/00 C08B 37/00 H // C07H 13/06 C07H 13/06

Claims (3)

[Claims] (1) The following formula (I): [In the formula, R is the same or different and is a hydrogen atom or SO ₃
H is shown and n shows the integer of 1-1000. However, N-
The number of SO ₃ H residues per molecule of acetylneuraminic acid residue is 0.1 to 3. ] The anti-inflammatory agent which uses N-acetylneuraminic acid represented by these, or the sulfuric acid ester of its homopolymer, or its pharmaceutically acceptable salt as an active ingredient. 2. The following formula (I): [Wherein R is the same as above. n represents an integer of 3 to 1000. However, when n is 3 to 11, the number of SO ₃ H residues per molecule of N-acetylneuraminic acid residue is 0.1 to 10.
When n is 12 or more, the number of SO ₃ H residues per molecule of N-acetylneuraminic acid residue is 0 to 3
It is. ] The selectin inhibitor which uses the N-acetyl neuraminic acid homopolymer represented by these, its sulfuric acid ester, or its pharmaceutically acceptable salt as an active ingredient. 3. The following formula (I): [Wherein R is the same as above. n represents an integer of 3 to 1000. However, when n is 3 to 11, the number of SO ₃ H residues per molecule of N-acetylneuraminic acid residue is 0.1 to 10.
When n is 12 or more, the number of SO ₃ H residues per molecule of N-acetylneuraminic acid residue is 0 to 3
It is. ] The P-selectin inhibitor which uses N-acetyl neuraminic acid homopolymer represented by these, its sulfuric acid ester, or its pharmaceutically acceptable salt as an active ingredient.