JPH0892104A - Heparin-derived human ii-type phospholipase a2 inhibitor - Google Patents

Abstract

PURPOSE: To obtain the inhibitor effective on various inflammatory diseases, asthma, allergic diseases, etc., containing, as active ingredient, a sulfated sugar based on a heparin derivative having the link inhibitory activity for heparin and inflammation focus-derived phospholipase A2 . CONSTITUTION: This human II-type phospholipase A2 inhibitor contains, as active ingredient, a sulfated sugar obtained, for example, by enzymatic treatment of heparin with heparitinase I or heparitinase I, IV, etc., or treating heparin with periodic acid followed by, if needed, an alkali (esp. with a molecular weight of about 2000-15000). The sulfated sugar is esp. pref. a compound of formula I [R1 and R4 are each H or SO3 R (R is H or an alkali metal); R2 and R3 are each H or an alkali metal; R' and R" are each H or methyl] or formula II (R" is H or OR"). A medicinal composition can be obtained by combining this active ingredient with a pharmaceutically permissible vehicle, having effective on disseminated intravascular coagulation, ischemic angiopathy, restenosis, ulcer, septicemia, ARDS, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sulfated sugar having a binding inhibitory activity on phospholipase A ₂ and heparin. More specifically, the present invention relates to phospholipase A derived from local inflammation.
₂ relates to a sulfated sugar based on a heparin derivative having a binding inhibitory activity between heparin and ₂ .

[0002]

BACKGROUND OF THE INVENTION Proteoglycans are macromolecules in which one or more sugar chains of glycosaminoglycan are bound to a core protein, and they are present in the cell space or the cell surface. It is a substance with structural diversity involved in cell-cell or cell-matrix interactions. Heparin, which is a sulfated glycosaminoglycan consisting of a repeating disaccharide of glucosamine and glucuronic acid or iduronic acid modified with a sulfate group, was discovered in 1916 as a substance having anticoagulant activity. It has been used in medicine as an important therapeutic drug for thrombosis for over 50 years by utilizing the anticoagulant activity which is the origin of the discovery. To date, in addition to anticoagulant activity, heparin-like polysaccharides have high affinity for cell growth factors such as basic fibroblasts, contribute to the formation of higher-order structure of extracellular matrix, etc. Is being clarified, and attention is also paid to its anti-inflammatory action. These diverse biological activities are believed to be based on the structural diversity of heparin, such as the binding site for antithrombin III (Constant AA
van Boeckel, and Maurice Petitou; ANGEWANDTE CHEMI
E International Edition in English 32 , 1671-
1818 (1993)) and the binding site of basic fibroblast growth factor (Marco Maccarana, Benito Casu, and Ul)
f Lindahl; The Journal of Biological Chemistry 26
8 , 23898-23905 (1993)) are known to be different structures in heparin.

In recent years, type II PLA ₂ (membrane-bound PLA ₂ ) has been purified from human inflammatory diseases and inflammatory localities of animal models of inflammation, and its properties have been clarified (Ichiro Kudo, Keizo Inoue; Biochemistry 64 , 1330-1344 (1992)).
This enzyme is believed to promote the inflammatory response,
A drug that inhibits the activity of this enzyme is expected to exhibit an anti-inflammatory action, and is considered to be useful for treating inflammatory diseases and allergic diseases.

Moreover, this type II PLA ₂ has a strong affinity for heparin and is secreted extracellularly with inflammation. Further
In order for the physiological activity of type II PLA ₂ to work, like FGF,
It is necessary to be taken up by other target tissues or cells, and it has been clarified that heparin or heparan sulfate plays an important role in the phenomenon of migration to such tissues (Y. Suga, Murakami, et. Al.,
Eur J. Biochem. (1994)).

Accordingly, the binding site or a derivative of the type II PLA ₂ in heparin, a substance that inhibits the binding of type II PLA ₂ and heparin or the like, prevents the migration to the tissue such that the enzyme is targeted, Further, it is expected to have an anti-inflammatory action which also inhibits the exertion of its physiological activity.

The present inventors have found a limited heparin-degraded fraction having a binding property to type II PLA ₂ , and found that this limited heparin-degraded fraction can inhibit the binding between type II PLA ₂ and heparin. The present invention has been confirmed.

[0007]

That is, the present invention provides a hepa
Human type II PLA containing phosphorus-derived sulfated sugar as an active ingredient ₂
Inhibitor and Effective Sulfated Sugar Derived from Heparin and Pharmacy
A pharmaceutical composition comprising a pharmaceutically acceptable excipient.

The heparin-derived sulfated sugar of the present invention means heparin (molecular weight of about 6 × 10 ³ to 2 × 1) derived from various organs such as liver, small intestine and lung of mammals such as human, pig and cow.
0 ⁴ ) or a heparin derivative in which a hydroxyl group and / or an amino group such as heparan sulfate is sulfated.

Such a sulfated sugar derived from heparin of the present invention is obtained by, for example, treating heparin with heparitinase I or heparitinase I, IV or the like, or treating heparin with periodic acid and then treating with alkali if necessary. The sulfated sugars obtained by the above method, and particularly the sulfated sugars having a molecular weight of about 2,000 to about 15,000 can be mentioned (Fransson, LA Carbohyd, Res. 80 , 131).
~ 145, 1980). Specifically, among such sulfated sugars, the molecular weight is about 3,000, about 7,000.
Or about 10,000, especially about 10,000
Those of 0 can be mentioned as preferable ones.

More specifically, among the sugar sequences of heparin that are limitedly decomposed by heparitinase I treatment, the following formula [I]:

[0011]

[Chemical 3]

[In the formula, R ₁ and R ₄ each independently represent a hydrogen atom or a —SO ₃ R group (R represents a hydrogen atom or an alkali metal), and R ₂ and R ₃ each independently represent a hydrogen atom. Or an alkali metal, and R ′ and R ″ each independently represent a hydrogen atom or a methyl group.], Or a sulphated sugar containing a sulphated disaccharide represented by: or by treatment with heparitinase I and IV, or The following formula [II] that can be obtained by chemical synthesis

[0013]

[Chemical 4]

[In the formula, the definitions of R _{1 to} R ₄ , R and R'are the same as those in the formula [I], and R '"is a hydrogen atom or OR" (the definition of R "is the formula [I]. When the broken line indicates a carbon-carbon single bond, R '"indicates a hydrogen atom, and when the broken line does not indicate a carbon-carbon single bond, R'" indicates OR ". Indicates. ] A sulfated sugar including a sulfated disaccharide represented by That is, as the sulfated sugar of the present invention, those in which the minimum unit has the above formula [I] or [II] are also preferable.

In the above formulas [I] and [II], examples of the alkali metal include sodium and potassium. In addition, the amino group of the sulfated disaccharide,
Sulfated sugars of the present invention also include those in which a carboxyl group or an aminosulfone group forms a salt with an inorganic acid such as hydrochloric acid or an organic acid, or those which are substituted with a known protective group for a sugar functional group. include.

As described above, the sulfated sugar derived from heparin of the present invention may be any of those obtained by purifying a limited degradation product derived from naturally occurring enzyme treatment and / or acid treatment, and those obtained by synthesis. Among such sulfated sugars, R ₁ , R ₄ in the above formula [I] may be used.
Is a —SO ₃ R group (the definition of R is the same as in formula [I]), R ₂ and R ₃ are alkali metals, and R ′ and R ″ are methyl groups. , Among them Table 1
MO-SS1 described above, or in the above formula [II], R ₁ and R ₄ are —SO ₃ R groups (the definition of R is the same as in formula [I]), or R ₁ and R ₄ One is a hydrogen atom and the other is a —SO ₃ R group, R ₂ and R ₃ are hydrogen atoms, R ′ and R ″ are hydrogen atoms, and the broken line represents a carbon-carbon single bond. Sugars, especially diS1, diS2 and tri-S described in Table 1, and particularly MO-SS1 and tri-S among these, can be mentioned as preferable ones.

[0017]

[Table 1]

Further, the above-mentioned formulas [I] and [II], in particular, the dimer in which the sulfated disaccharides shown in Table 1 are linked via a crosslinking agent are also included in the sulfated sugar of the present invention. Be done.

Hereinafter, a method for producing a sulfated sugar derived from heparin, which is an active ingredient of the human type II PLA ₂ inhibitor of the present invention, will be described, for example, in the case of treating heparin with periodic acid.

First, heparin derived from porcine small intestine was prepared according to a conventional method.
Is treated with periodate to reduce its molecular weight.
Purify with a C (Asahi Kasei) column. Such periodic
In acid treatment, following the periodate treatment,
It is preferable to carry out an alkali treatment. In this way
Regarding the obtained low molecular weight sulfated sugar, human type II PLA ₂
ELISA (Enzyme Linked Immu) using specific antibody
nosolvent Assay) system, human type II PLA₂And hepa
The binding inhibitory activity of phosphorus is measured. This measurement method is
Human type II PLA in vitro₂Reproduce the binding of heparin
In order to do so, we conjured with Bovine Serum Albumin (BSA)
Heparin derived from porcine small intestine (Analytical Bio
chemistry126, 414-421 (1982))
The coated 96-well plate is used. This
Human type II PLA on the plate ₂Binds well.
Therefore, human type II PLA in the presence of the sulfated sugar of the present invention₂
Human type II PLA₂Specific monoclonal antibody
LAM1-1 (described in Japanese Patent Application No. 5-255646)
Can be obtained by
Human type II PLA that binds to heparin derived from small intestine₂Detect
And anti mouse IgG Alkali-phosphatase conjugate
More substrate (disodium para-nitrophenyl phosphate)
OD to develop color₄₀₅To measure. As a result, human type II PL
A₂On the plate due to the presence of the sulfated sugar of the present invention.
Binding to coated porcine small intestine-derived heparin
If an inhibitory phenomenon is confirmed, human type II PLA₂of
Human type II PLA capable of inhibiting physiological activity₂Have connectivity
A sulfated sugar derived from heparin is obtained.

The sulfated sugar derived from heparin of the present invention is usually administered systemically or locally in oral or parenteral form. The dose varies depending on the age, symptoms, therapeutic effect, administration method, administration time, etc., but is usually administered within a range of 1 mg to 1000 mg per adult, for example, once to several times a day orally. Alternatively, it is parenterally administered, or preferably is continuously administered intravenously in the range of 1 hour to 24 hours per day. Of course, as mentioned above,
Since it varies depending on various conditions, a dose smaller than the above dose may be sufficient in some cases, or a dose exceeding the range may be necessary in some cases.

The human type II PLA ₂ inhibitor containing a heparin-derived sulfated sugar of the present invention as an active ingredient comprises the above-described effective amount of the sulfated sugar and a pharmaceutically acceptable excipient. By forming a pharmaceutical composition, a solid or liquid pharmaceutical composition may be directly or, if necessary, a known lubricant such as magnesium stearate or talc; binding of gelatin, ceramic, polyvinylpyrrolidone, methylcellulose or the like. Agents; disintegrators such as sodium alginate and starch; moisturizers; antioxidants; absorption promoters; surfactants; isotonic agents and the like, together with known methods such as soft capsules, hard capsules, tablets, pills, and granules. It is provided as an agent, powder, suspension, liquid, syrup, emulsion, elixir and other oral agents, injections, suppositories, pessaries or external preparations (liquids, ointments, coatings).

The pharmaceutically acceptable excipients of the present invention include vegetable oils (eg corn oil, cottonseed oil, coconut oil, almond oil, peanut oil, olive oil, etc.), oily esters of medium-chain fatty acid glyceride oil, etc. Mineral oil, glycerin esters such as tricaprylin and triacetin, gums such as tragacanth and arabic gum, alcohols such as ethanol, physiological saline, propylene glycol, polyethylene glycol, petroleum jelly oil, cellulose derivatives (crystalline cellulose, hydroxypropyl cellulose) , Hydroxypropylmethylcellulose, methylcellulose), polyvinylpyrrolidone, dextrin, lactose, annitol, sorbitol, starch and the like.

The heparin limited degradation fraction of the present invention is human II
It has an action of inhibiting the binding between type PLA ₂ and heparin. Therefore, in these heparin-limited degradation fractions, type II PLA ₂ secreted in the body is transferred to other tissues or cells and its activity is exerted. By suppressing the phenomenon, various inflammatory diseases (eg, rheumatoid arthritis, organ inflammation), asthma,
Allergic disease, generalized intravascular coagulation (DIC),
It is effective for the prevention and / or treatment of ischemic vascular disorder (myocardial infarction, cerebral infarction), restenosis (restenosis), ulcer, sepsis, ARDS, nephritis, pancreatitis, its serious injury and multi-organ disorder. In particular, if a composition in which the structure of heparin involved in the binding to type II PLA ₂ is specified and only that site is extracted or synthesized by limited decomposition by treatment with an enzyme or periodate can be used, side effects of heparin administration The risk of (systemic hemorrhage, thrombocytopenia, etc.) is reduced, and it becomes possible to enhance the anti-inflammatory action or to administer an effective dose capable of exerting the anti-inflammatory action.

[0025]

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

Reference Example 1 (1) Preparation of Human Type II PLA ₂ The type II PLA ₂ cDNA was reported by JJ Seilhamer et al.
Biol. Chem., 264, 5335 (1989)). That is, RN derived from human placenta
Reverse transcriptase RA from A (purchased from Clontech)
A cDNA library was prepared by using V-2 (Takara Shuzo) according to the attached protocol.

Next, (5 ') AACTCTAGACCA
TGAAGACCCTCC (3 ') and (5') AG
AAGATCTCCTGCCTGGGCCTCTA
The type II PLA ₂ cDNA was amplified by the PCR method using the single-stranded synthetic DNA primer of the sequence (3 ′). The reaction was performed by using Taq polymerase manufactured by Perkin Elmer Co., Ltd. according to the attached protocol. About 560
The bp type II PLA ₂ cDNA fragment was purified by agarose gel electrophoresis.

Subsequently, type II PLA ₂ cDNA was synthesized with synthetic D
Baculovirus recombinant vector pAC YM1 (Matsuura, Y. et al, T. Gen. Viro
L., 68, 1233-1250 (1987); National Institute of Health, Granted by Dr. Zenji Matsuura) BamH
Incorporated into I site.

[0029] The baculovirus (Ac
Recombinant virus was obtained by transfecting insect cells Sf-9 with NPV) DNA. Sf
-9 cells were purchased from Invitrogen and the attached protocol was followed for the procedure of transfection and purification of recombinant virus.

Next, Sf-9 cells were statically cultured on a scale of 20 ml / flask (manufactured by Falcon), and MOI =
1 was infected with the above recombinant virus and cultured at 25 ° C. for 4 days, and the culture supernatant was recovered.

From this culture supernatant, a heparin sepharose column (Pharmacia CL-6B) was used.
Type II PLA ₂ was purified. That is, φ1.5 × 5 cm
After passing 1.1 liters of culture supernatant using the column of 10 ml, 50 ml of 50 mM Tris.HCl buffer (p
H7.4), 50 ml of 0.3 M NaCl / 50 mM
Tris.HCl (pH 7.4) buffer, 50 ml of 0.6 M NaCl / 50 mM Tris.HCl (p
H7.4) buffer solution was sequentially passed through to obtain 0.6 M Na.
Type II PLA ₂ was eluted in the Cl fraction.

Next, this eluate was added to Waters 600E.
Reversed phase column (Vyd combined with HPLC system
It was further purified by applying it to an ac ^™ C-4 column, φ2.2 × 25 cm). The buffer used was 0.1% TFA / H ₂ O.
A linear gradient (60 minutes) of 0% to 50% of acetonitrile was applied at. The purification method described here and the antibody production method described below are described in Methods in Enzymology, 197,
223-233 was used as a reference. Thus, type II PLA ₂
Was purified and used for immunization as described below. The purified type II PLA ₂ has a single peak in HPLC, and Laemli has a single peak.
There was a single band in PAGE of the system.

(2) Monochrome that reacts with type II PLA ₂
Preparation of null antibody (LAM1-1) 10 μg of type II PLA2 prepared in Example 1 was treated with Freund's complete adjuvant (manufactured by BACTO, 1:
In addition to 1), Balb / c mice (6 weeks old, male) were administered i. p. Was administered. After this was done 4 times, the 5th time, i. v. Was administered.

Immediately before cell fusion, the mouse was killed, splenocytes were homogenized in PBS, the residue was filtered through a nylon mesh, and then washed once with PBS. 2 × 10 ^{7 of} these splenocytes were converted into mouse myeloma (P3-X63-Ag
8-U1) 4 × 10 ⁷ and common method (Kohler, Milstein; Natu
re, 256, 495-497 (1975)).

That is, both cells were mixed, spun down, and then Cel
After 1 pack, 1 ml of 50% polyethylene glycol (Wako Pure Chemical Industries, 2000) HAT medium (GIT medium, Wako Pure Chemical Industries) was added dropwise over 2 minutes, and polyethylene glycol was diluted with 10 ml of HAT medium over 5 minutes. After making up to 100 ml with the same medium,
It was dispensed into 5 96-well plates at 00 μl / well. After culturing at 37 ° C. in 5% CO ₂ for 1 week, half the amount (100
μl / well) was removed, and HT medium (GIT medium, Wako Pure Chemical Industries, Ltd.) was added (100 μl / well). Two weeks later, screening with a plate coated with type II PLA ₂ described below was performed.

That is, 50 mM Na of type II PLA ₂
CO ₃ buffer solution (pH 9.5) solution (1 μg / ml) was added to 9
50 μl of each well was dispensed into a 6-well plate (Falcon, PVC) and left at 37 ° C. for 1 hour. After washing, add 3% BSA / PBS to each well to 200
μl was added and blocking was performed at 37 ° C. for 1 hour. After washing again, add 50 μl of culture supernatant to each well and
It was left to stand for 3 hours and washed 3 times with 0.05% Tween / PBS.

Next, a goat anti-mouse IgG-alkaline phosphatase conjugate (Ta) diluted 2000-fold with PBS containing 3% BSA and 0.2% skim milk was prepared.
50 μl was added to each well, and left at room temperature for 1 hour. It was washed again, and a 1 mg / ml solution of p-nitrophenyl phosphate disodium salt (Wako Pure Chemical Industries, Ltd.) dissolved in 1 M diethanolamine buffer (pH 9.8) containing 0.25 mM magnesium chloride was added to each well at 100 μm / well.
1 was added and reacted at room temperature for 30 minutes. The absorbance at 405 nm was examined using an ELISA reader measuring instrument for 96-well plates (Vmax, Molecular Davice), and hybridomas secreting a monoclonal antibody that binds to type II PLA2 were selected.

Regarding the hybridoma selected in this way,
And cloned twice by limiting dilution method
did. Specifically, 10 cells of mouse peritoneal exudate were placed in HT medium.
⁶Dispense into each well what was prepared at a rate of
The cells were suspended in HT medium at a rate of 0.5 per well.
Hybridoma cells were seeded. 5% CO₂CO ₂
Incubate at 37 ℃ for 2 weeks in an incubator and culture
The nutrient supernatant was screened by the above-mentioned ELISA method,
Establish a strain by picking up a single colony
It was

[Reference Example 2] Preparation of heparin-binding plate 10 μg / m 2 in each well of a 96-well plate for ELISA
50 μl each of 10 mM phosphate buffered saline (hereinafter PBS) containing 1 L of BSA conjugated Heparin was added, and 37
After incubating at ℃ for 1 hour or more, wash twice with distilled water, then 3mg / ml BSA-10mM PBS
200 μl of the solution was added to each well, incubated at 37 ° C. for 1 hour, and then stored at 4 ° C.

BSA conjugated Heparin is Analytical
According to Biochemistry 126 , 414-421 (1982), porcine small intestine-derived heparin 20 mg, NaBH ₃ CN
20 mg and 10 mg of BSA were dissolved in 2 ml of a 0.2 M potassium hydrogen phosphate solution (pH 9.0) and reacted at 60 ° C. for 7 to 10 days to prepare.

Reference Example 3 Preparation of Heparin Limited Degradation Fraction (Periodic Acid Decomposition / Alkali)
Reprocessing) Fransson, LA, et. Al. Carbohyd Res 80 , 131-
145, 1980, heparin derived from pig small intestine (SI
GMA MW. 15,100) was subjected to periodic acid decomposition and alkali treatment to obtain three types of limited decomposition fractions, which were named HFO, HFP, and HFQ, respectively. When these limited decomposition heparins HFO, HFP, and HFQ are decomposed into molecular weight fractions, molecular weights of about 10,000, 7,000, and
It was estimated to be 3000. HFO, HFP, and HFQ were evaluated by the following human type II PLA ₂ binding inhibitory activity measurement.

The molecular weight of the limited decomposition heparin fragment was determined by using a heparin-derived oligosaccharide having a known molecular weight as a standard substance (GPC column (Asahipak GFA-3000 (Asahi Chem. Ind. Co, Osak)).
a, Japan), SynChropak 50 (CynChrom. Inc., Lafayet
te, IN, USA)) and 0.2 M PBS (pH 7.
0) was performed by HPLC (Shimadzu SPD-6A, C-R4A) (Y. Suda, D. Marques, et. A.
l., Thrombosis Res. 69, 501-508, 199
3).

[Measurement Method] Measurement of Human Type II PLA ₂ -Heparin Binding Inhibitory Activity A plate (96 wells) coated with BSA conjugated Heparin was preincubated at 37 ° C. for 1 hour and then washed twice with distilled water. .

The low molecular weight heparin limited degradation fraction was treated with Tris buffered saline (hereinafter TBS, pH 7.0) containing 1% chicken ovalbumin (hereinafter ovalbumin) at 100 μm.
A sample prepared by diluting 2-fold from g / ml was added at each concentration of 50 μl / well, and the mixture was mixed at 37 ° C. for 3 times.
Incubate for 0 minutes. Furthermore, 0.5 μg / ml
(TBS-1% ovalbumin solution) 5 human type II PLA ₂
0 μl / well was added to each well and the mixture was reacted at 37 ° C. for 30 minutes. After the reaction is completed, 10 mM PBS-0.5% Twe
Wash the plate with en20 (use the washing device, 3
After washing twice, change the direction of the plate and wash three more times). Next, 10 μM PBS-3% containing 1 μg / ml mouse anti-human type II PLA ₂ monoclonal antibody LAM1-1
Add 50 μl of BSA solution to each well 37
After reacting at ℃ for 1 hour, 10mM PBS-0.5
Wash the plate with% Tween 20 (using a washer, washing 3 times, then changing plate orientation and washing 3 more times). Finally, 200 with 10 mM PBS-3% BSA
50 μl of 0-fold diluted goat anti-mouse IgG alkaline phosphatase (TAGO) was added to each well and reacted at 37 ° C. for 1 hour, 10 mM PBS-0.
Wash the plate with 5% Tween 20 (using the washer, after washing 4 times, then changing the orientation of the plate and washing 4 more times). A substrate (1 mg / ml disodium paranitrophenyl phosphate, Wako Pure Chemical Industries, Ltd.) is added at 100 μl / well, color development is detected by OD _405, and an inhibitory activity (% inhibition) is determined.

[Example 1] According to the above measurement method, HFO, HFP, and HFQ, which are the periodate-limited heparin limited decomposition fractions obtained in Reference Example 3, were used in Reference Example 1
The binding inhibitory activity of the human type II PLA ₂ obtained in the above and heparin was evaluated. The results are shown in Fig. 1. All of HFO, HFP, and HFQ have binding inhibitory activity (inhibition rate%), and the heparin-limited fragment having a larger molecular weight has higher human type II P
It is found that the LA ₂ -heparin binding inhibitory activity is enhanced.

[Example 2] Heparin-complete enzyme-digested disaccharides listed in Table 1 according to the above-mentioned measurement method; diS1 and diS1
S2 (manufactured by Seikagaku Corporation) and the disaccharide (synthetic product) (MO-SS1), which is a component of the periodate / alkali treated fraction, were tested for the inhibitory activity (inhibition rate%) of human type II PLA ₂ and heparin It was measured.

The results are shown in FIG. From FIG. 2, diS1,
It is found that all of diS2, Tri-S and MO-SS1 have a concentration-dependent binding inhibitory activity between human type II PLA ₂ and heparin.

[Brief description of drawings]

FIG. 1 is a human type II P produced by the heparin-derived sulfated sugar (periodic acid limited degradation product) of Example 1 of the present invention.
The result of having evaluated the binding inhibitory activity of LA ₂ and heparin is shown. In the figure, ○, ×, and ■ are HFO (MW approximately 10,000), which are limited decomposition products of heparin with periodic acid,
HFP (MW about 7,000), HFQ (MW about 3,000)
0).

FIG. 2 shows the results of evaluation of the binding inhibitory activity between human type II PLA ₂ and heparin by the sulfated sugar (disaccharide) derived from heparin of the present invention in Example 2. In the figure, △, ●,
+ And ▲ are diS1 and diS, which are sulfated disaccharides, respectively
2, Tri-S, and MO-SS1.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Shoichi Kusumoto 1-1, Machikaneyamacho, Toyonaka City, Osaka Prefecture, Faculty of Science, Osaka University (72) Inventor, Yasuo Sumida 1-1, Machikaneyamacho, Toyonaka City, Osaka Prefecture Faculty of Science

Claims (8)

[Claims] 1. An inhibitor of human type II PLA ₂ containing a sulfated sugar derived from heparin as an active ingredient. 2. The inhibitor according to claim 1, wherein the sulfated sugar is obtained by enzymatically decomposing heparin. 3. The inhibitor according to claim 1, wherein the sulfated sugar is obtained by treating heparin with periodate. 4. A sulfated sugar is represented by the following formula [I]: [Wherein R ₁ and R ₄ are each independently a hydrogen atom or —S
O ₃ R group (R represents a hydrogen atom or an alkali metal), R ₂ and R ₃ each independently represent a hydrogen atom or an alkali metal, and R ′ and R ″ each independently represent a hydrogen atom or methyl. Group is included.] The inhibitor according to claim 1, which contains a sulfated disaccharide. 5. R ₁ and R ₄ are —SO ₃ R groups (the definition of R is the same as in formula [I]), R ₂ and R ₃ are alkali metals, and R ′ and R are R. 5. The inhibitor according to claim 4, wherein "" is a methyl group. 6. A sulfated sugar is represented by the following formula [II]: [In the formula, the definitions of R _{1 to} R ₄ , R and R ′ are the same as those in formula [I], and R ′ ″ is a hydrogen atom or OR ″.
(The definition of R ″ is the same as that in formula [I]), provided that when the broken line represents a carbon-carbon single bond, R ′ ″ represents a hydrogen atom and the broken line represents a carbon-carbon single bond. If not present, R '"indicates OR". ] The inhibitor according to claim 1, which comprises a sulfated disaccharide represented by the formula: 7. R ₁ , R ₄ are —SO ₃ R groups (the definition of R is the same as in formula [I]) or R ₁ , R 4
₄ is a hydrogen atom and the other is a —SO ₃ R group,
The inhibitor according to claim 6, wherein R ₂ and R ₃ are hydrogen atoms, R ′ and R ″ are hydrogen atoms, and the broken line represents a carbon-carbon single bond. 8. A pharmaceutical composition comprising an effective amount of a heparin-derived sulfated sugar and a pharmaceutically acceptable excipient.