WO2008020495A1

WO2008020495A1 - Novel inhibitor

Info

Publication number: WO2008020495A1
Application number: PCT/JP2007/000865
Authority: WO
Inventors: Kazuaki Kakehi; Yuki Matsuno; Yuji Matsuzaki; Junichi Kumada; Masato Hanyu
Original assignee: Tokyo Cemical Industry Co., Ltd.
Priority date: 2006-08-15
Filing date: 2007-08-10
Publication date: 2008-02-21
Also published as: JPWO2008020495A1

Abstract

A novel hyaluronidase inhibitor can be provided by using dermatan sulfate or a pharmacologically acceptable salt thereof, preferably dermatan sulfate or a pharmacologically acceptable salt thereof having a weight average molecular weight of 10,000 to 50,000 as an active ingredient of a mammalian hyaluronidase inhibitor. The hyaluronidase inhibitor can overcome a problem that hyaluronic acid (which is conventionally used in a pharmaceutical or cosmetic product) is degraded by hyaluronidase to lose its desirable effect.

Description

Specification

New inhibitor

Technical field

[0001] The present invention relates to an inhibitor for a specific hyaluronidase having an action of degrading hyaluronic acid, which is applied to pharmaceuticals and cosmetics.

Background art

[0002] Hyaluronic acid is extremely useful as a pharmaceutical or a cosmetic, but a hyaluronidase inhibitor has been demanded because it is easily decomposed. As a hyaluronidase inhibitor used with such hyaluronic acid, it is considered preferable to use the same saccharide as hyaluronic acid as an inhibitor for reasons such as stability.

[0003] Patent Document 1 describes that acidic polysaccharides extracted from shellfish meat are useful as a hyaluronidase inhibitor but does not contain dermatan sulfate.

[0004] Patent Document 2 discloses the use of chondroitin sulfate as a hyaluronidase inhibitor, but there is a description suggesting that chondroitin sulfate A and C have such an action. On the other hand, it has not been disclosed or suggested that dermatan sulfate exhibits a hyaluronidase inhibitory effect.

[0005] Patent Document 3 describes that various sulfated polysaccharides can be used as microorganism-derived hyaluronidase inhibitors, but does not disclose that dermatan sulfate alone can be used as a hyaluronidase inhibitor. Furthermore, there is no disclosure or suggestion of the use as an animal-derived hyaluronidase inhibitor.

Patent Document 1: Japanese Patent Application Laid-Open No. 6_2 1 9 95 4

Patent Document 2: Japanese Patent Laid-Open No. 7-8 2 1 3 1

Patent Document 3: Japanese Patent Laid-Open No. 2 00 0 _ 1 7 8 1 9 6

Disclosure of the invention

Problems to be solved by the invention [0007] Although various such hyaluronidase inhibitors have been studied, there are no polysaccharides currently in practical use as hyaluronidase inhibitors. In particular, there is no inhibitor exhibiting an inhibitory action on mammalian-derived hyaluronidase, and such a novel inhibitor has been demanded.

Means for solving the problem

[0008] The inventors of the present invention, as a result of diligent studies to solve the above-mentioned problems, have surprisingly been able to inhibit hyaluronidase superior to dermatan sulfate, which is one of the same glycosaminoglycans as hyaluronic acid. It was found that there was an effect, and the present invention was conceived. The gist of the present invention is as follows.

[0009] 1. A hyaluronidase inhibitor containing dermatan sulfate as an active ingredient.

2. The hyaluronidase inhibitor according to 1, wherein the weight average molecular weight of dermatan sulfate is 10,000 to 50,000.

3. The hyaluronidase inhibitor according to 1 or 2, which exhibits an inhibitory action against a mammalian type hyaluronidase (EG3.2.1.35).

4. Use of dermatan sulfate as a hyaluronidase inhibitor.

The invention's effect

[0010] The present invention provides a novel hyaluronidase inhibitor.

Brief Description of Drawings

[001 1] FIG. 1 is a diagram showing a capillary electrophoresis electrophoresis chart for confirming the inhibitory action on hyaluronidase inhibitory action by the concentration of dermatan sulfate. The horizontal axis shows the detection time.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail by the best mode for carrying out the invention.

[0013] 1. Inhibitor of the present invention

The inhibitor of the present invention comprises dermatan sulfate as an active ingredient

[0014] The origin of the dermatan sulfate used in the inhibitor of the present invention is not particularly limited. Dermatan sulfate that has been separated and purified from biological materials such as milk crowns (eg, chicken crowns), skin, umbilical cords, and internal organs can be used, but those derived from pig skin or chicken crowns are preferred.

[0015] Separation of dermatan sulfate ■ Purification includes crushing, extraction, enzymatic degradation (for example, degradation with protease), precipitation with organic solvent (for example, alcohol), salting out, salt solution, various chromatographies, etc., or combinations thereof It can be carried out by a method usually used for separation and purification of glycosaminoglycans.

[0016] The dermatan sulfate having a weight average molecular weight used in the inhibitor of the present invention may be a pharmaceutically acceptable salt thereof. Examples of pharmaceutically acceptable salts of dermatan sulfate include salts with inorganic bases such as alkali metal salts (sodium salt, lithium salt, potassium salt, etc.), alkaline earth metal salts or ammonium salts, or organic amines. A salt with an organic base such as a salt (diethanolamine salt, cyclohexylamine salt, triethylamine salt, triethanolamine salt, etc.) or an amino acid salt (arginine salt, lysine salt, etc.) can be used. Of these, sodium salts are preferred.

[0017] The weight average molecular weight of the dermatan sulfate used in the inhibitor of the present invention is not particularly limited, but it is preferably included in the range of 10,000 to 50,000, particularly preferably about 30,000 to 40,000.

[0018] The dermatan sulfate that can be used in the inhibitor of the present invention preferably has the following characteristics. Note that dermatan sulfate having such preferable characteristics is hereinafter abbreviated as D S 1.

[0019] (A) High-performance liquid chromatography when digested with chondroitinase A B C

—2-acetoamide in unsaturated disaccharides analyzed in —— 2-deoxy— 3 _

0 _ (4—Deoxy 1 _ L—Threoxy 1 4 _Enopyranosyluronic acid

) _6_sulfo_D—galax I (A D i — 6 S) is about 4 to 25%, preferably about 7 to 16%.

[0020] (B) When chondroitinase B is allowed to act, about 3 wt% to about 17 wt%, preferably about 5 wt% to 15 wt% is not digested. [0021] (C) The molecular weight range measured by the multi-angle light scattering method is 10,000 Dalton to 200,000 Dalton.

The dermatan sulfate preferably further has the following characteristics.

[0022] (D) The molecular weight distribution measured by the multi-angle light scattering method is 79 to 99% for fractions with a molecular weight of 40,000 daltons or less, 1 to 20% for fractions with 40,000 to 100,000 daltons, 1 A fraction with a fraction of 0,000 Dalton or more is detected as a fraction composition of 1% or less.

[E] (E) The weight average molecular weight measured by the multi-angle light scattering method is about 28,000 to about 4

30,000 Daltons, preferably about 320,000 to about 38,000 Daltons.

[0024] (F) The weight average molecular weight measured by gel filtration high performance liquid chromatography (GPC-HPLCC) method is about 3 million to 46,000 daltons, preferably about 3.

50,000 to about 41 thousand daltons.

[0025] (G) Molecular weight distribution measured by GP CH PLC method is 5-30% for fractions with a molecular weight of 20,000 daltons or less, 70-95% for fractions with 20,000-80,000 daltons, 80,000 daltons The above fraction is detected as a fraction composition with 0%.

[0026] (H) Chondroitinase The composition of the unsaturated disaccharide when digested with ABC is as follows.

■ 2-acetoamide 1-deoxy _3_0_ (4-deoxy _1 _ L-sl-hexoxy 4 _enopyranosyluronic acid) 1 6 _sulfo _D_galax I

The content of (Δ D i −6 S) is about 4 <½ to about 25%, preferably about 7 <½ to about 16%.

■ 2-acetamide 1-deoxy 1 3 _0_ (4-deoxy 1 _ L-threo-hexyl 4 _enopyranosyluronic acid) 1D-galax! (A D i -O S) content of about 4% to about 10%, preferably about 4.5% to about 10%

■ 2-acetoamide 1-deoxy 1 3 _0_ (4-deoxy 1 _ L—threo hex 1 _enopyranosyluronic acid) 1 4 _0_sulfo _ D—galax I The content of D i -4 S) is about 63% to about 85%, preferably about 68% to about 84%. ■ 2 _acetoamide— 2—deoxy— 3 _0_ (4—deoxy _2_0—sulfoic acid _ L—threo monohexyl 4 _enopyranosyluronic acid) _6_0_sulfo _D_galax I (AD i _d i SD) content of about 0.5% to about

2.2 <½, preferably about 0.8% to about 1.7 <½.

■ 2—acetamido— 2—deoxy— 3 _0_ (4—deoxy _2_0—sulfoic acid _ L—threo monohexyl 4 _enopyranosyluronic acid) _4_0_sulfo _D_galax I (AD i _d The content of i SB) is from about 2% to about 8%, preferably from about 3% to about 7%.

■ 2-acetoamide 1-deoxy 1 3 _0_ (4-deoxy 1 _ L—threo 1 hex 4 _enopyranosyluronic acid) 1,4,6-bis 1 0—sulfo 1 D—galac The content of IS (AD i _d i SE) is about 0% to about 2%.

[0027] The names and abbreviations of the various unsaturated disaccharides were as described in Yoshida, K. et al., Analytical Biochemistry, 177, 327-332 (1989).

[0028] The most preferable DS 1 characteristics among these DS 1 are shown in Table 1 below.

[0029] [Table 1]

[0030] This DS 1 can be produced, for example, by the following method.

[0031] About 2 to 3 times the amount of water is added to one weight of chicken crown, minced and then boiled. After cooling, protease (Pronaise, Kaken Pharmaceutical Co., Ltd.) is added for hydrolysis. . After adding the benzalkonium chloride solution to the hydrolyzate, filter through diatomaceous earth, Discard the filtered supernatant to obtain the remaining diatomaceous earth. This diatomaceous earth is suspended in a sodium chloride solution, ethanol is added, the resulting precipitate is allowed to stand, and the resulting precipitate is dried to obtain a powder.

[0032] This powder is dissolved in water to prepare a 10% solution, and heparin / heparan sulfate is removed by nitrous acid treatment by Shively and Gonrad method. That is, a solution in which the above powder is dissolved is mixed with a 0.1% nitrous acid aqueous solution and left at room temperature, and then the precipitate is removed by filtration. Adjust the pH of the filtrate to 10.5, add sodium chloride to a final concentration of 1%, and add ethanol with stirring to a final concentration of 48%. Activated carbon is added to the resulting precipitate and suction filtered. The filtrate is passed through an ion exchange resin Diaion SA-12A (Mitsubishi Chemical Corporation), desalted, and ethanol is added to the filtrate to obtain DS 1. .

[0033] The obtained DS 1 can be subjected to molecular ultrafiltration with a membrane (Amicon) having a molecular exclusion limit of 1 million daltons and washed with a sodium chloride solution of about 0.2 M. preferable.

[0034] The characteristics of the DS 1 can be confirmed by the following method.

[0035] (a) Measurement of average molecular weight by G P C-H P L C method

The average molecular weight of DS 1 can be measured according to the method of Arai et al. (Biochem. Biophys. Acta, 1117, 60-70, 1992), and chondroitin sulfate with a known molecular weight (molecular weight 39 1 00, 1 8000, 8050 ) And sodium hyaluronate (molecular weight: 104000) as standard, and can be determined by elution time in gel filtration (GPC-HPLC) using high performance liquid chromatography. As the column, TSK gel G4000PWXL, G3000PWXL and G2500PWXL (each 07.8 × 300 mm, Tosohichi Co., Ltd.) can be used. The solvent is a 0.2 mol / L sodium chloride solution, the flow rate is 0.6 ml / min, and a differential refractive index detector (R （800, Tosoichi Co., Ltd.) can be used as the detector.

[0036] (b) Measurement of molecular weight by multi-angle light scattering method

Using a differential refractive index detector (Shodex RI-7U Showa Denko KK) and a multi-angle light scattering detector (DAWN, Wyatt Technology Corporation) as detectors, ASTRA software Square analysis is performed using the Jim Plot module of Ware (WyattTechnology CORPORATION).

[0037] The column is S B-806 HQ (08 x 3 O Omm, Showa Denko KK), the solvent is 0.1 mol / L sodium nitrate solution, and the flow rate is 1.0 mL / min.

[0038] (c) Analysis of chondroitinase B digestion pattern

Buffer A (0.001mol / L Calcium Acetate, 0.02mol / L Tris-HCU pH7.5) 0.03U Chondroitinase B (Seikagaku Corporation) dissolved in 10 L in DS 1 solution 100 1_ And digest at 37 ° C for 2 hours. Stop the reaction by heating in a boiling water bath for 1 minute, and analyze 10 L of the digested product equivalent to 100 g using G P C_H P L C at 40 ° C. The column used is a concatenation of TSK gel G4000PWXL, G3000PWXL and G2500PWXL (each ø7.8 X 30 Omm Tosohichi Co., Ltd.). The solvent is a 0.2 mol / L sodium chloride solution, the flow rate is 0.6 mL / min, and the detector is a differential refractive index detector (R 卜 8020, Tosoh Corporation) and an ultraviolet-visible detector (UV_8020, A230nm, east So-Co., Ltd.) can be used.

[0039] (d) Digestion with chondroitinase ABC

1% DS 1 solution 50 1_, buffer B (0.01 mol / L sodium acetate, 0.05 mol / LTris-HCU pH7.5) 0.5 L of chondroitinase ABC dissolved in 10 L, Digest at 37 ° C for 2 hours. Stop the reaction by heating in a boiling water bath for 1 minute, and centrifuge to remove insolubles.

[0040] To this degradation product, 0.5 U of chondroitinase ABC dissolved in buffer B 101_ is added and digested at 37 ° C for 6 hours. Stop the reaction by heating in a boiling water bath for 1 minute, and centrifuge to remove insolubles.

[0041] (e) Digestion with chondro 6-sulfatase

Add 0.25 U chondro-6-sulfatase dissolved in buffer C (0.02 mol / L Tris_Ac0H, pH 7.0) 50 1_ to the equivalent of 100 g of the digest (c) above. Digest at 0 ° for 24 hours to remove insolubles.

[0042] (f) Disaccharide composition analysis

Analyze each digest (d) or (e) using HPLC. . Column

Lichorosorb NH ₂ 40 x 250 mm (Merck) can be used. The flow rate is 1.0 mL / min, and 0.8 mol / L sodium hydrogenphosphate is flowed in a linear concentration gradient from 2% to 100% in 60 minutes. Based on the elution position of the unsaturated chondro-disaccharide kit (Seikagaku Corporation), various unsaturated disaccharides eluted during this period were identified at 232 nm, and the sum of the peak areas identified as unsaturated disaccharides The disaccharide composition ratio can be obtained by calculating as 100%.

[0043] (g) Measurement of intrinsic viscosity

The intrinsic viscosity of DS 1 can be measured according to the 13th revised Japanese Pharmacopoeia. As the measuring device, an automatic viscosity measuring device (VMC_052, Koiso Co., Ltd.) can be used. As the solvent, 0.2 mol / L sodium chloride solution can be used, and the same solution can be used to measure the flow time of the Ubeguchi-type viscometer. Viscosity is measured at 30 ± 0.1 ° C. The flow time is rounded off to 1/100 second, and the measured value within 3 seconds is used for the calculation of intrinsic viscosity.

[0044] Intrinsic viscosity is reduced viscosity (= (tS / tO_1) / C, tS: solution flow time, tO: solvent flow time, C: sample concentration (wt%)). The straight line obtained by plotting on the axis can be obtained from the intercept when the concentration is extrapolated to zero.

[0045] The hyaluronidase to which the inhibitor of the present invention exhibits an inhibitory effect is not particularly limited as long as it is an enzyme having an action of degrading hyaluronic acid, but it is particularly an endo-S_N-acetyl hexosaminida called a mammal type. Ize is preferred. Such a preferred enzyme is an enzyme belonging to EG3.2.1.35 in the enzyme classification.

[0046] The concentration at which the inhibitor of the present invention is used is preferably 0.3% or more, more preferably 0.5% or more, still more preferably 1% or more, and most preferably 2.5% or more compared to the concentration of hyaluronic acid. preferable. By mixing dermatan sulfate with hyaluronic acid at such a concentration, it is also possible to obtain a hyaluronic acid composition that is resistant to degradation by mammalian hyaluronidase.

Example 1 [0047] Dermatan sulfate was added to microorganism-derived hyaluronic acid, and the digestive action of Hedge testis-derived hyaluronanidase was examined.

[0048] Hyaluronic acid derived from actinomycetes (manufactured by Kibun Food Chemifa Co., Ltd .: weight average molecular weight 1,000,000) and dermatan sulfate derived from pig skin (Seikagaku Corporation): weight average molecular weight 2.0 After dispensing the aqueous solution containing the sample into a sample tube and adding 101 1_100 _________________ 's acetate buffer (pH 5.2), add Higgir testis-derived hyaluronidase (Roche). The reaction was allowed to proceed for 8 hours at 37 ° C.

[0049] [Table 2]

[0050] The reaction mixture was incubated for 5 minutes in a boiling water bath to stop the enzyme reaction, followed by centrifugation (15,000 xg) for 10 minutes, and the supernatant was analyzed by capillary electrophoresis by a conventional method. Table 3 shows the conditions for capillary electrophoresis.

[0051] [Table 3] f "^ jj-leoknian P ACE 誦

1

\ l T. Noso-ί u ¾ 6 o, sili ca o¾pill¾ y v 4 Ocwi x 50 ^ w i, d.

^) ^ η f Night SO miKiol L Φ ^ ll II ifi (pH 8.S) 100 o

Gisyl sulfate sodium

2 5 * C

Applied voltage 20 kV

ii charge ¾ person method liquid bath time garden Kasho method / 1 0 seconds 圜

Detection method 2 00 [0052] The results are shown in FIG. Some degradation of hyaluronic acid was observed in Sample 6 containing 1.25% dermatan sulfate, and low molecular weight hyaluronic acid was observed as a shoulder peak. Sample 4 containing 0.33% of dermatan sulfate began to see oligosaccharide peaks, and sample 2 containing 0.078% dermatan sulfate and sample 1 containing 0.039% showed almost no inhibitory effect. . Sample 7 with a dermatan sulfate concentration of 2.5% was found to completely inhibit the enzymatic reaction of mammalian hyaluronidase.

[0053] On the other hand, when a similar experiment was performed using chondroitin sulfate (derived from shark cartilage: manufactured by Seikagaku Corporation), which is known to be structurally similar to dermatan sulfate, the hyaluronidase inhibitory action was Not observed.

Industrial applicability

[0054] The present invention can be used as a new inhibitor for hyaluronidase used in cosmetics and pharmaceuticals.

Claims

The scope of the claims

[1] A hyaluronidase inhibitor comprising dermatan sulfate or a pharmacologically acceptable salt thereof as an active ingredient.

[2] The hyaluronidase inhibitor according to claim 1, wherein the weight average molecular weight of dermatan sulfate is 10,000 to 50,000.

[3] The hyaluronidase inhibitor according to claim 1 or 2, wherein the hyaluronidase inhibitor exhibits inhibitory action against a mammalian type hyaluronidase (EG3.2.1.35).

[4] Use of dermatan sulfate as a hyaluronidase inhibitor.