JP2003313201A - Sulfated cellulose-containing, corneal or conjunctival- disease curative or preventive medicine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a low molecular-weight sulfated cellulose, which has a high curative effect on a corneal or conjunctival trouble caused by a physical, chemical or mechanical damage to the cornea or conjunctiva and on an endogenous corneal or conjunctival trouble. <P>SOLUTION: The sulfated cellulose having therein a hydroxyl group sulfated is a sulfated cellulose in which the numbers of SO<SB>3</SB>groups on a monosaccharide repeating unit is on the average 2.6 or more; the salt of the above sulfated cellulose is the one which is pharmacologically permissible. The curative or preventive medicine for a corneal or conjunctival disease and the curative or preventive medicine for a dry eye have each as an effective component the above sulfated cellulose or a slat thereof which is pharmacologically permissible. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to sulfated cellulose and a therapeutic or prophylactic drug for corneal or conjunctival diseases containing the same as an active ingredient.

[0002]

2. Description of the Related Art A specific reaction such as an enzyme reaction or an antigen-antibody reaction exists in a living body and forms a precise in-vivo system. Recent advances in sugar chain engineering in these fields are remarkable, and examples of biopolymers having sugar chains include proteoglycans of the cell wall of plant cells that contribute to cell stabilization, cell differentiation, proliferation, adhesion, and Examples include glycolipids that affect migration and the like, and glycoproteins that are involved in cell-cell interaction and cell recognition, etc., but the sugar chains of these macromolecules act for each other, assist, proliferate or inhibit their functions, It has come to be considered that it controls advanced and precise biological reactions.

Glucosaminoglycan (hereinafter referred to as GAG) is an acidic polysaccharide composed of repeating disaccharides, and the disaccharide having a basic structure is composed of uronic acid or galactose and hexosamine. In addition, GAG is classified into 6 types, hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin and heparan sulfate, according to the difference in constituent monosaccharides such as uronic acid and hexosamine, and the difference in the number of sulfate groups and the bonding position. . GAGs other than hyaluronic acid are known to be distributed in various tissues as proteoglycans covalently bound to proteins in nature. Proteoglycans not only function as a support that is a major component of the extracellular matrix, but also may be involved in cell growth processes leading to cell adhesion, migration, proliferation, differentiation and morphogenesis. It is believed that this is largely due to the structural specificity of GAG.

The use of these GAGs in medicine is as follows.
In addition to being used for blood coagulation of heparin, it is used to prevent infections against viruses such as AIDS and to use hyaluronic acid or chondroitin sulfate for corneal diseases. It is also described that persulfated chondroitin sulfate having an average of 3.5 or more O-sulfate groups as a sulfated polysaccharide has heparin-like anticoagulant activity (JP-A-11-16600).
1). US Pat. No. 6,063,773 describes that high molecular weight sulfated cellulos is effective against AIDS or herpes virus. However, there is no description about the relationship between sulfated cellulose and corneal disorders.

The cornea is located in the anterior pole of the eyeball and transparently covers the anterior lens of the eye. The epithelial cell layer, Bowman's membrane, parenchymal layer,
It is a tissue having an orderly layered structure composed of a Descemet's membrane and an endothelial cell layer.

The corneal stroma is mainly composed of collagen and proteoglycan. Proteoglycan is a mucopolysaccharide-protein complex, which is a compound in which GAG and a core protein are covalently bound.

[0007] As proteoglycans constituting the corneal stroma, mainly Lumican whose GAG is ketaran sulfate,
Decorin, whose GAG is chondroitin / dermatan sulfate, is mentioned, and the length of chondroitin / dermatan sulfate side chain corresponds to the center-to-center distance of collagen fibrils. It has been proposed that it is involved in regulation and constructs a three-dimensional lattice structure of the corneal stroma (Scoto, JE: Proteogl
ycan-fibrillier collagen interaction., JE: Morphom
etry of cupromeromeronic blue-stained proteoglycan
molecules inanimal corneas, versus that of purifie
d proteoglycans stained in vitro, implies that tert
iary structures contribute to corneal ultrastructu
re., J.Anat., 180,155-164,1992), Benedek, GB: Theory
of transparency of the eye., Appl.Opt.10,459-473,1
971).

Of the GAGs that compose the cornea, ketalan sulfate is particularly important, and 50% of the total corneal proteoglycans are used.
-70% is dominated by Lumican whose gag is ketalane sulfate. In the normal corneal stroma, lumican plays an important role in the arrangement of collagen fibers, adjustment of fiber gaps, and the like. However, when the cornea or the corneal stroma is injured for some reason, the luciferin of the parenchyma is depleted, and decorin, which is larger than the normal size, becomes predominant. Such changes in proteoglycan increase the distance between collagen fibers and the diameter of collagen fibrils, and the distance between collagen fibers and the diameter of collagen fibrils is 1/2 of the wavelength of light.
When disturbed over a distance of (200-250 nm), light is scattered in the cornea and causes corneal (parenchymal) opacity (Asail, KK, et al .: Wound healing inresponse to ke
ratorefeactive surgery., Surv.Ophalmol., 38,289-302,
1993).

In addition, when the cornea or the corneal stroma is injured, the molecular weight of the proteoglycan in the stroma also changes, which increases the water content due to the position of GAG sulfation and the ratio of GAG species. Is said to have affected (Hass
ell, JR, et al .: Proteoglycan changes during restora
tion of transparency in corneal scars., Arch.Bioche
m. Biophys., 222, 362-369, 1983).

Further, in patients with ecchymosis corneal dystrophy,
A large number of proteoglycans containing ketalan in the cornea lacking a sulfate group have been recognized (Miura, RJ, eta.
l.:Proteoglycan biosynthesis by human corneas from
patient with typel and 2 macular corneal dystroph
y., Jbiol, Chem., 265 (26), 15947-15955, 1990). The cause is a defect due to a gene abnormality of sulfotransferase to ketaran.

In order to maintain the normal translucency, it is necessary that a high proportion of lumican, which uses ketalane sulfate as GAG, is used. However, deficiency of sulfotransferase produces lumican and other proteoglycans, which have a sulfate-free ketalan as a constituent mucopolysaccharide. Their solubility is extremely low, and they are deposited in the cornea and cause visual impairment (Edward, DP, et al .: Macular dystrophy of
the cornea.A systemic disorder of keratan sulfate
metabolism., Ophthalmology, 97 (9), 1194-1200,1990)

[0012] As described above, various proteoglycans and various GAGs constructing the proteoglycans play an extremely important role in the formation of a regular and ordered lattice-like structure of the corneal stroma, and the cornea was damaged for some reason. In this case, it is presumed that the substance acting as a normal cornea plays an important role in healing corneal damage. In particular, the constitution of the sulfate group constituting GAG has a great influence. From this, it was considered that the polysaccharide having a sulfate group may have an important pharmacological effect on the cornea. Furthermore, there are reports of proteoglycan abnormalities and GAGs as the etiological factors of keratopathy and dry eye, in which the number of seriously ill patients continues to increase. Dry eye develops as a complication of retinal surgery, vitrectomy, radiation therapy for corneal cancer, etc. The main causes are dry cell mucin-producing goblet cell in conjunctiva and proteoglycan in conjunctiva, syndecan having keratan sulfate chain. It has been reported that the distribution of expression of -1, -1 and tenascin-C is different from normal (Hei
mann H, et al: Alterations in expression of mucin,
tenascin-C and syndecan-1 in the conjunctiva foll
owing retinal surgery and plaque radiotherapy., Gr
aefes Arch Clin Exp Ophthalmol, 239 (7): 488-495, 2
001). This suggests that dry eye and keratoconjunctival proteoglycan have a close relationship. In addition, there are many reports that sodium hyaluronate is effective as a symptomatic treatment for dry eye (Condon PI, et al: Dou
ble blind, randomized, placebocontrolled, crossove
r, multicentre study to determine the efficacy of
a0.1% (w / v) sodium hyaluronate solution (Fermavisc)
in the treatment of dry eye syndrome., Br J Ophtha
lmol, 83 (10), 1121-1124, 1999. Yokoi N, et al; Eff
ectiveness of hyaluronan on corneal barrier functi
on in dry eye., Br J Ophthalmol, 81 (7): 533-536, 19
97). However, in reality, many ophthalmologists evaluate that sodium hyaluronate preparations are clinically ineffective. In addition, as a result of diligent examination in our animal experiments, we could not find any corneal wound healing effect or dry eye preventive / therapeutic effect to sodium hyaluronate. Furthermore, it has been reported that no effect is observed even when instilled at the time of corneal wound, although GAGs other than sodium hyaluronate, such as chondroitin sulfate, keratan sulfate, and heparan sulfate, are important constituent GAGs of the cornea. Nakamura
M, et al; Concentration and molecular weight depen
dency of rabbit corneal epithelial wound healing o
n hyaluronan., Curr Eye Res, 11 (10): 981-986, 199
2) On the other hand, a hydroxypropylmethylcellulose preparation is used as a cellulose derivative, but toxicity such as DNA aggregation and radical production to conjunctival cells was observed (Debbasch C, et al; Cytotoxicity evaluation of t
hree tear substitutes used in the treatment of dry
eye syndromes., J Fr Ophtalmol, 23 (9): 863-869, 2
000), but cannot be said to be a satisfactory preventive / therapeutic agent. Under such circumstances, development of a novel corneal or conjunctival damage and dry eye preventive / therapeutic agent is desired.

[0013]

Therefore, from the above circumstances, a drug for preventing and treating corneal or conjunctival disorders and endogenous corneal or conjunctival disorders caused by physical, chemical or mechanical damage to the cornea or conjunctiva. Is to provide. As a method of safely and surely promoting healing of corneal or conjunctival disorders to achieve such an object, it is to provide a polysaccharide containing a sulfate group, particularly sulfated cellulose.

[0014]

The present invention provides a therapeutic agent or preventive agent for cornea or conjunctival wounds (hereinafter abbreviated as “repair agent for corneal wounds”) for treating or preventing corneal or conjunctival disorders caused by various causes. The present invention relates to cellulose having a sulfate group. When the effect of such sulfated cellulose on corneal injury was examined, a remarkable effect was observed when a sulfate group was introduced into low molecular weight cellulose. In particular, it was found that sulfated cellulose normalizes the cornea when the proteoglycan or the like is changed and damaged in a dry state.

That is, according to the present invention, O- in the sugar repeating unit
A sulfated cellulose having a sulfate group of 2.6 or more or a pharmacologically acceptable salt is provided. The sulfated cellulose of the present invention can be obtained by reacting insoluble cellulose and sulfur trioxide at a temperature of 30 to 50 ° C. in an amount of 10 to 15 times.

The effect of such sulfated cellulose was obtained using human corneal epithelial cells and human conjunctival cells. That is, even if the corneal epithelial cells or the conjunctival cells were brought into contact with the sulfated cellulose and then the corneal epithelial cells or the conjunctival cells were dried, the survival rate of these cells was significantly improved.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION As described above, the O-sulfated cellulose of the present invention is one in which the hydroxyl groups of cellulose are sulfated, and cellulose has D-glucopyranose β-1,
It has a repeating unit of sugar linked by four bonds. When all the hydroxyl groups in the sugar unit are sulfated, the number of sulfate groups in the sugar unit is three. In the O-sulfated cellulose of the present invention, the number of sulfate groups in the sugar unit is 2.6 or more on average, more preferably 2.7 or more, further preferably 2.8 or more,
Most preferably, it is 3.0.

The molecular weight of the O-sulfated cellulose of the present invention is not particularly limited, but is usually 5,000 to 1,000,000, preferably 5,000 to 200,000, and more preferably 10,000 to 100,000.

The sulfated cellulose of the present invention may be a pharmacologically acceptable salt. Examples of the salt include, but are not limited to, alkali metal salts such as sodium salt and potassium salt, calcium salt and the like.

When the above-mentioned sulfated cellulose is used as an active ingredient for the treatment of corneal diseases, the concentration range of sulfated cellulose is not particularly limited, but it is preferably 1-5000 μg, more preferably 50-1000 μg, and most preferably 100-5.
It is 00 μg.

Treatment or prevention of corneal or conjunctival diseases with the sulfated cellulose of the present invention (hereinafter abbreviated as "treatment of corneal diseases")
When used in the above, it is preferable that it is in the form of a topical administration agent, particularly an eye drop, an eye ointment or a lyophilized agent, but is not limited thereto. It is preferable to use eye drops, eye ointments, creams and gelling agents, but not limited to these. Eye drops, eye ointment, when used as a cream, sodium dehydroacetate as an additive, preservatives such as methyl paraoxybenzoate, sodium chloride, isotonic agents such as glycerin, carboxymethyl cellulose, thickeners such as polyvinyl alcohol, Common stabilizers such as sodium edetate and polysaccharides can be used, but the stabilizers are not limited to these and may be in the physiologically acceptable range. As the buffer solution added to the above-mentioned various dosage forms, it is preferable to use an isotonic buffer solution having a pH of 4-9 and a pH of 5-9.

Further, the therapeutic agent for corneal diseases of the present invention is used in combination with various ophthalmic physiologically active substances such as fibronectin, hyaluronic acid and chondroitin sulfate as long as the function of sulfated cellulose as an active ingredient is not hindered. It can be used as a therapeutic agent for corneal diseases. Furthermore, it can be used in combination with an antibiotic, a steroidal anti-inflammatory drug or a non-steroidal anti-inflammatory drug. The usage and dose of the therapeutic agent for corneal disease of the present invention varies depending on the symptoms, age, etc. of the patient, but it is usually preferable to apply the eye drops 1 to 5 times a day and 1 to 5 drops per time. In the case of eye ointment, usually 1-
Use 3 times by applying an appropriate amount into the keratoconjunctival sac.

The present invention will be specifically described below based on examples. However, the present invention is not limited to the following examples.

[0024]

Examples [Preparation of Sulfated Cellulose and Structural Analysis] (Reagent) 1. Raw material SIGMA cellulose (Fibrous, medium) was treated with trifluoroacetic acid and used. 2. Reagents dimethyl sulfoxide (dehydrated), sodium hydride (oil-based), and N, O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) were purchased from Wako Pure Chemical Industries, Ltd. 3. Column TSKgel Sugar AXI (4.0 mm i.
d. x 30 mm) as a column for sulfate ion analysis using TSKgel
IC-Anion-PW (4.6 mm id x 50 mm) to Tosoh Corporation
Purchased from. Asahipak GF510HQ (7.6 mm)
id x 300 mm) was purchased from Asahi Kasei Corporation.

(Device) 1. GC and GC-MS The device (gas chromatograph) used in GC is G-3000 type gas chromatograph, and the column is J & W Scientific (Folsom, C
A) Capillary column DB-1 type (0.25 μm film thickne
ss, 0.25 mm id x 30 m), a hydrogen flame ionization detector was used as the detector. GC-MS is Hewlett-Packard gas chromat
ograph Model 5890 seriesII, column is capillary column DB-1 type, detector is GC-MS 5971A mass spectrometer, C
The hemstation data system was used.

2. The HPLC separating and delivering liquid pump is Intel by JASCO Corporation.
ligent HPLC Pump PU-980, Hitachi L-6000 pump,
Shimamura Mfg. Double plunger pump SPU-2.5NP type, detector is Tosoh Conducto Monit
or CM-8, Shimadzu Fluorescene Spectromonitor R
F-530, Hitachi Fluorescence Spectromonitor F-
1050, reactor is Dry Re manufactured by Seishin Pharm. Co., Ltd.
actionBath DB-2, Shimamura Keiki Seisakusho Dry Reaction Bat
h DB-5, Column bath is Yamato Scientific Co., Ltd. Water Bat
h Incubater BT-22, recorder is Chromato I made by Hitachi Ltd.
ntegrator D-2500 was used.

3. Absorptiometer UV-VIS Spectrophotometer UV-1200 made by Shimadzu, TCC-240A electronic thermostatic cell holder, Kinetics Program
I used Pack. 4. Polarimeter Spectropolarimeter manufactured by JASCO Corporation (JASCO)
DIP-140 was used. 5. IR equipment Hitachi FT / IR-230 was used. 6. NMR equipment JNM-GSX400A or JNM manufactured by JEOL Ltd.
-The GSX 500A was used. 7. Electrophoresis Cellulose Acetate Membrane Electrophoresis: The cellulose acetate membrane is SEPARAX-SP manufactured by JOKO, and the constant current voltage supply device for electrophoresis is PAV-20, an automatic constant current voltage device manufactured by JOKO.
0. As the migration tank, a micro-analysis migration device manufactured by Tsuneko was used. Polyacrylamide gel electrophoresis (PAGE): As a polyacrylamide gel, ATTO's Pagel NPG-520L
Type (concentration gradient 5-20%) and NPG-1020L type (concentration gradient 10-
20%) was used.

1. experimental methodPreparation of sulfated cellulose The complete O-sulfated cellulose was prepared by a conventional method. You
In other words, water-insoluble cellulose treated with trifluoroacetic acid.
Suspension in dimethylformamide, and add sulfur trioxide.
Add 10 to 15 times the calculated amount of lysine complex and
And reacted for 6 hours. Add water to stop the reaction and
Precipitated with ethanol and desalted with cetylpyridinium chloride
(CPC) After precipitation treatment, finally ethanol precipitation against water
Sulfated cellulose was prepared by subjecting to dialysis and dialysis. Finally
The sulfated cellulose obtained is freely soluble in water (Fig. 1).

[0029]Cellulose acetate membrane electrophoresis Cellulose acetate membrane in 25% aqueous methanol solution (v / v)
After soaking, 1.0 M acetic acid / pyridine (pH 3.5) running solution [27]
Soak for 30 minutes. Perform pre-electrophoresis and sample
Spot on the membrane with a probe and run at 0.5 mA / cm for 20 minutes.
It was. For staining, use 0.5% Alcian blue / 0.3% acetic acid solution
I shook it for 20 minutes. After decolorizing with 1% acetic acid, wash with water
It was

[0030]Composition analysis by methanolysis Sample is P₂O_FiveDry in desiccator in presence for 24 h with vacuum pump
Then, the reaction was carried out in 1 M hydrochloric acid-methanol at 80 ° C. for 24 hours. Raw
The monosaccharides formed are converted to TMS by BSTFA and then measured by GC or GC-MS
did. The GC conditions are shown below. Column, DB-1 type (0.25 μ
m film thickness, 0.25 mm i.d. x 30 m); Carrier
Gas, helium; flow rate, 50 cm / s; column temperature, initial
Temperature: 100 ℃, Final temperature: 260 ℃, Temperature rising rate: 20 ℃ / min;
Detector, flame ionization detector (FID); detection temperature, 280
° C. The conditions of GC-MS are shown below. Detector is GC-MS 5971A
A mass spectrometer, Chemstation data system was used. Measurement
EI (electron impact ionization), ionization voltage
Was performed at 70 eV. Other conditions are the same as GC.

[0031]Molecular weight measurement Gradient polyacrylamide gel electrophoresis (Gradie
nt PAGE) is a polyacrylamide gel from ATTO Co., Ltd.
Pagel NPG-520L type (concentration gradient 5-20%), and PAJI
NPG-1020L type (concentration gradient 10-20%)
Squirrel-at 1.25 M glycine buffer (pH 8.3) at 200 V
It was Staining was performed with Alcian blue [16]. Gel filtration
The chromatographic conditions are shown below. Column, Asah
ipakGF510HQ (7.6 mm i.d. x 300 mm); eluent, 50 mM
Ammonium bicarbonate; flow rate, 0.7 ml / min; column temperature,
Room temperature; detection reagent 1, 50 mM guanidine (flow rate, 0.25 ml / mi
n); detection reagent 2, sodium hydroxide (flow rate, 0.25 ml / mi
n); Reaction coil (0.5 mm i.d.x 10 m); Reaction temperature, 11
0 ℃; cooling coil (0.25 mm i.d. x 3 m); detection, excitation
Wavelength 320 nm, fluorescence wavelength 420 nm. Agarose gel electrophoresis
Is a 1% agarose gel and TBE buffer (0.045 M Tri
s-borate, 0.001 M EDTA) at room temperature and 80 V for 1 hour.
It was Staining was performed with 0.5% AzureA / 1% acetic acid.

[0032]Neutral sugar analysis by fluorescence HPLC After acid hydrolysis of the sample, the resulting monosaccharide is separated and determined by HPLC.
WeighedSulfate ion measurement After acid hydrolysis of the sample, the sulfate ion produced is
It was quantified by chromatography.

[0033]Methylation analysis According to the Hakomori method [20], the hydroxyl groups of sugars are
Lucanbanion (MSC) ie DMSO and NaH, CH₃I (You
Methylated). Then acid hydrolysis
Then reduced, re-O-acetylated and partially methylated.
Lactate The formed derivative is GC or GC
-Measured by MS.

[0034]Optical rotation Using DIP-140 manufactured by JASCO Corporation (JASCO),
It was measured with sodium D line. Weigh the dried sample and
Dissolve in double-distilled water to a concentration of 0.5 mg / ml or 1 mg / ml.
Optical rotation was measured.

[0035]Infrared absorption spectrum Using FT / IR-230 manufactured by JASCO Corporation (JASCO)
It was measured. The measurement conditions are as follows. Total number of times, 64
 times; disassembled, 4 cm^-1; Gain, 32; speed, 2.0 mm /
sec; light source, standard; apodization, Cosine;
Zero Filling, x2; Beam Splitter, KBr.

[0036] ¹ H-NMR spectrum Heavy water used was Aldrich (purity 99.96%). NMR device
Is JNM-GSX 400A or JNM- made by JEOL Ltd.
GSX 500A was used. Dissolve 2 mg of sample in 0.5 ml of heavy water,
After repeated freeze-drying to remove exchangeable protons,
P at reduced pressure₂O_FiveDry in desiccator in presence for 24 hours. Complete
Dissolve the completely dried sample in 0.5 ml of heavy water, and use Wilmard N
MR measurement tube pp-528 (5.0 mm o.d.x 25 cm)
Using. Measurements are taken at resonance frequencies of 400 Mz or 500 Mz.
It was. The measurement conditions for the one-dimensional spectrum are as follows.
It Observation width, 8 kHz; pulse width, 45 ° (4.90? Sec); Sun
Pulling point, 32 K; pulse interval, 2 sec; integration times
Number, 400 times; temperature, 318 K. NOESY, TOCS for confirmation
Two-dimensional spectra such as Y and DQF-COSY were measured. use
As for the pulse sequence, the standard equipment
Used (Fig. 4, Fig. 5, Fig. 6).

2. Results After preparation of sulfated cellulose, confirmation was made by cellulose acetate membrane electrophoresis, a single band was confirmed, and it was confirmed that significant decomposition did not occur in the trifluoroacetic acid treatment and the subsequent sulfation reaction. . Moreover, in order to confirm whether or not the prepared sulfated cellulose maintains the basic skeleton of the cellulose initially used, the following points were examined and the structure thereof was confirmed. (1) Monosaccharide composition (2) Glycoside bond position (3) Anomeric configuration (4) Monosaccharide sequence (5) Sulfation degree In general, the structure of carbohydrates includes chemical analysis including composition analysis and nuclear analysis. It is determined by instrumental analysis such as magnetic resonance method, infrared absorption spectrum, and polarimeter. Again, according to the conventional method, the monosaccharide composition analysis of the prepared sulfated cellulose was decomposed into methyl glycosides by methanolysis, derivatized with TMS, and then determined by GC and GC-MS. The bond position between monosaccharides was determined by methylation analysis. The partially methylated alditol acetate compound was analyzed and identified by GC-MS. The anomeric configuration was determined by NMR. The results of the above studies are shown below in sequence.

2-1. Composition analysis In structural analysis of polysaccharides, it is important to determine the constituent sugars. Since the limited decomposition with trifluoroacetic acid and the sulfation reaction at relatively high temperature were performed this time, the monosaccharide composition of the prepared sulfated cellulose was analyzed. First, the sample is 1.0 M
Methanolysis decomposition with dry HCl / MeOH, silylating agent BSTFA
The hydroxyl groups were converted to TMS with and the monosaccharide composition was analyzed by GC and GC-MS. For confirmation, the sample was hydrolyzed with 2.5 M trifluoroacetic acid and then subjected to monosaccharide composition analysis under HPLC conditions. The results obtained by both methods were in good agreement, and only glucose was detected as a constituent. Next, the sulfated degree of the prepared sulfated cellulose was measured. That is, the sample was hydrolyzed with 2.5 M trifluoroacetic acid, and the inorganic ion was measured by ion chromatography.
As a result, it was estimated that the prepared sulfated cellulose had an average of 2.95 (2.75-3.12) sulfuric acid bonds per glucose residue. Ions other than sulfate groups were not observed.

[0039]

[Table 1]

2-2. Viscometric studies from the Mark-Houwink rela have been attempted since ancient times.
tionship, Ultracentrifugation, Light scattering
Methods, gel filtration chromatography, gel electrophoresis, etc. have been reported. However, there is still no method for determining the molecular weight of a polysaccharide having a linear structure as compared with that of a protein, and it is no exaggeration to say that it is a state of dark groping.

Therefore, in the present study, the following three methods were used to measure the molecular weight of the prepared sulfated cellulose. (1). Gel filtration chromatography (2). Gradient polyacrylamide gel electrophoresis
(Gradient PAGE) (3). Unsaturated disaccharide of agarose gel electrophoresis standard and commercially available standard glycosaminoglycan
From the calibration curve using (GAGs), the molecular weight of sulfated cellulose by gel filtration chromatography was about 24,000 Da.
Is estimated to be In addition, the molecular weight was measured by gradient polyacrylamide gel electrophoresis (Gradient PAGE) using a 5-20% gel in Tris-glycine buffer at 200 V.
The following voltages were used. When a calibration curve was prepared using a commercially available GAG as a standard product and examined, the molecular weight of sulfated cellulose was estimated to be about 24,000 Da, which was in good agreement with the result obtained by gel filtration chromatography.

2-3. On the other hand, IR analysis and NMR analysis, which are nondestructive analysis methods, are powerful means for structural analysis of sugar chains. FIG. 3 shows the IR spectrum of the obtained sulfated cellulose measured by KBr tableting, and FIG. 2 shows the NMR spectrum measured in heavy water. 124
Stretching vibrations due to S = O at 0 cm ^-1 and stretching vibrations due to equatorial sulfate at around 825 cm ^-1 were observed, confirming the presence of sulfate groups. Absorption was also observed at 850 cm ^-1, and it is estimated that some axial sulphate groups were present,
Charge repulsion between adjacent sulfate groups also suggested that the sugar ring might be inverted. Also, around 900 cm ^-1 ?
? Absorption of the configuration is observed. From the NMR spectrum, it is not possible to make a comparison because the raw material cellulose is insoluble in the solvent, but it can be seen that all ring protons are shifted to the low magnetic field due to sulfation. Also,
Since only 7 kinds of signals were observed, all the hydroxyl groups of glucose were completely sulfated, indicating that sulfated cellulose having a very high degree of uniformity could be prepared. Further, when it was dissolved in water and the optical rotation was measured, it was -9.0 °. The physical properties of sulfated cellulose are shown in FIG.

[Examples of Drought Injury] Example 1. SV40 immortalized human corneal epithelial cells (HCEC) suspended in CEM medium (10% FBS SHEM) containing 10% fetal bovine serum in a 48-well multiplate. 3 × 10 ⁴ cells (0.3 mL) were seeded per well and cultured under humidified conditions of 5% CO ₂ and 37 ° C. until almost confluent. After culturing, wash the cells with 0.5 mL of PBS per well and add 0.25 mL / well of the test drug such as sulfated cellulose (20 to 500 μg / mL) dissolved in DMEM / F12 medium to the well. The contact culture was carried out for 16 to 20 hours under the conditions. After this, DMEM / F12 containing the test drug
Remove medium from cell wells and place on a hot plate.
It was dried at ℃ for 14 minutes. After drying, add 0.25 mL / well of 10% FBS SHEM again to the cell well, and add 5% C
O _2, 37 After culturing 16-20 hours ℃ humid, respiratory chain enzymes activity assay reagent (redox enzymes NADPH dehydrogenase activity) WST
-8 (Note: Dojindo Laboratories) 25 μL / well, 5% CO
_{2. After} culturing for 2 hours at 37 ℃ in a humid environment, mitochondrial dehydrogenase in living cells reduces the tetrazolium salt of WST-8 to produce yellow formazan. The number of viable cells was measured by measuring the amount of formazan produced using the absorbance at 450 nm (Japanese Patent Application No.
8-121134). The results are shown in Table 2. Note; WST-8: 2-
(2-Methoxy-4-nitrophenyl) -3- (4-nitrophenyl)
-5- (2,4-disulfophenyl) -2H-tetrazolium
Sodium salt

[0044]

[Table 2]

FIG. 8 shows the protective effect of sulphated cellulose against desiccation damage of SV40 immortalized human corneal epithelial cells.

It was found that the low-molecular-weight sulfated cellulose has a remarkable protective effect when the survival rate due to the drying disorder is low, that is, when the drying disorder is severe. Currently, 500 of hyaluronic acid is applied as a therapeutic drug for dry eye.
μg / mL and 100 μg / mL of sulfated cellulose showed similar effects.

Example 2. 10% in 48 well multiplate
Human conjunctival cancer cells (CCL20.2) suspended in CEEM medium (10% FBS SHEM) containing fetal bovine serum were seeded at 3 x 10 ⁴ cells per well (0.3 mL), 5% CO ₂ , 37 ° C. The cells were cultured under moist conditions until they became almost confluent. After culturing, wash the cells with 0.5 mL of PBS per well and add 0.25 mL / well of the test drug such as sulfated cellulose (20 to 500 μg / mL) dissolved in DMEM / F12 medium to the well. The contact culture was carried out for 16 to 20 hours under the conditions. After that, DMEM / F12 containing test drug
Remove medium from cell wells and place on a hot plate.
It was dried at ℃ for 12 minutes. After drying, add 0.25 mL / well of 10% FBS SHEM again to the cell well, and add 5% C
O _2, 37 After culturing 16-20 hours ℃ humid, respiratory chain enzymes activity assay reagent (redox enzymes NADPH dehydrogenase activity) WST
-8 (Note: Dojindo Laboratories) 25 μL / well, 5% C
After culturing in O ₂ at 37 ° C. for 2 hours, the tetrazolium salt of WST-8 is reduced by mitochondrial dehydrogenase in living cells to produce yellow formazan. The number of viable cells was determined by measuring the amount of formazan produced using the absorbance value at 450 nm (Japanese Patent Application No. 8-121134). The results are shown in Table 3. Note; WST-8: 2
-(2-Methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium sodium salt

[0048]

[Table 3]

FIG. 9 shows the protective effect of sulfated samples against desiccation injury of SV40 immortalized human corneal epithelial cells. The protective effect of other sulfated polysaccharides was also examined. The protective effect against desiccation injury is specific to sulfated cellulose. It was target.

Example 3 Effect on Extra-orbital Lacrimal Excision Rat Dry Injury Model Experimental Materials and Methods Preparation of extraorbital lacrimal excision rat: Tsutomu Fujihara (Inves)
Ophthalmol Vis Sci 42: 96-100, 2001). SD male rats (6.5 weeks old, Charles River Japan) were given pentobarbital sodium (Nembutal
(Registered trademark), Dainippon Pharmaceutical Co., Ltd. was intraperitoneally administered (40 mg / kg) for general anesthesia, and the extraorbital lacrimal gland was surgically excised. The rat from which the extraorbital lacrimal gland was removed was kept in the breeding room until the instillation was started.

Test substance: For the test substance, sulfated cellulose (average molecular weight 2.4 × 10 ⁴ ) was dissolved in physiological saline (JP Pharmacopoeia, Otsuka Pharmaceutical Co., Ltd.) at a concentration of 0.1% (w / v), and then a Millipore filter (0.2
A solution sterilized with μm pore size, Millipore GV) and a solution obtained by diluting it with a physiological saline solution to a concentration of 0.02% (w / v) were used.
Control substances include saline and hyalerin (registered trademark).
Mini 0.3 (Lot.3HB0251, Santen Pharmaceutical Co., Ltd.) was diluted with physiological saline to use a 0.1% (w / v) sodium hyaluronate (HA) solution. The prepared eye drops were stored at 4-8 ° C. and used within 5 days.

Method of administration of test substance: Three weeks after removal of extraorbital lacrimal gland, physiological saline solution, sulfated cellulose (hereinafter referred to as SC) 0.02% solution, sodium hyaluronate solution 5 times once
μL / eye was instilled 4-6 times a day. The eye drops were applied for 4 weeks.

Measurement of corneal epithelial barrier function: The corneal epithelial barrier function was measured using an anterior fluorometer FL-500 (Kowa). The measurement was performed before the instillation of the test substance (3 weeks after the extraorbital lacrimal gland removal), and the value was used as the previous value.
After the start of instillation, the measurement was performed every week (4, 5, 6, and 7 weeks after the removal of extraorbital lacrimal gland). The rat corneal epithelial barrier function was measured as follows. Fluorescein sodium (hereinafter referred to as FS: Wako Pure Chemical Industries) dissolved in physiological saline was used as a tracer substance for measuring the corneal barrier. FL-5
The 00 measurement mode is a cornea (wide area) mode that can measure a relatively wide range of fluorescence that is set for the human cornea.
[Measurement openings were 0.1 mm in diameter (37 holes) were evenly arranged within 2 mm in diameter]. Pentobarbital sodium was intraperitoneally administered to rats (40 mg / kg) for general anesthesia. After anesthesia, fix the rat vertically to the microscope part of FL-500, open the eyelids sufficiently, adjust the position of the microscope part in front of the eye to be examined, and further illuminate at 30 ° to the microscope part. After fixing, the measurement windows were aligned with the central part of the rat cornea, and further with the upper part of the ear and the lower part of the nose, and the background value was measured at each position. Next, 0.5% FS (3 μL) was instilled into the eye, and after 5 minutes, the eye was washed with an excess amount (6-7 mL) of physiological saline, and immediately the fluorescence of the cornea at 3 points was measured as described above. 10 for each measurement
The measurements were repeated, and the average value was used as the measured value. The value obtained by subtracting the background value from the fluorescence measurement value after FS instillation and washing was taken as the FS amount taken into the cornea, and the corneal epithelial barrier function was quantitatively evaluated from the amount.

Effect on Rats with Extraorbital Lacrimal Gland Removal Instillation was started 3 weeks after the removal of extraorbital lacrimal gland, and the corneal epithelial barrier function test was conducted on the rats instilled with the test substance for 4 weeks. The corneal epithelial barrier function test was performed before the instillation and every 4 weeks after the instillation was started, and the change with time was observed. The amount of FS uptake into the cornea of the unextracted lacrimal gland group (blank group) was almost constant at any measurement site. The FS uptake amount in the lacrimal extirpated group (control group) did not change with time, and was significantly higher than that in the blank group at any measurement time and measurement site. SC (0.02
%) In the eye drop group, in the central cornea and lower nasal side,
The value was almost the same as that of the control group until 2 weeks after instillation, but after 3 weeks, a difference became apparent, and the value was significantly low 4 weeks after instillation. The HA (0.1%) eye drop group showed no difference from the FS value of the control group at any measurement site (Fig. 10).

According to the present invention, a novel O-sulfated cellulose having a higher degree of sulfation of free hydroxyl groups than ever before is provided. The O-sulfated cellulose of the present invention has an excellent healing effect on corneal disorders, and is useful for treating corneal diseases, particularly preferably for treating corneal disorders and corneal diseases based on lacrimal gland disorders, dry eye, and the like. .

[Brief description of drawings]

FIG. 1 is a diagram showing steps of a method for producing O-sulfated cellulose of the present invention.

FIG. 2 is a diagram showing a 1 H-NMR spectrum of sulfated cellulose.

FIG. 3 is an IR spectrum of cellulose and sulfated cellulose.

[Figure 4] NOESY: Nuclear Overhauser Enhancement
Spectroscopy

[Figure 5] TOCSY: Total (HOHAHA; Homonuclear Har
tmar Hahn Spectroscopy) Connectivity Spectroscopy

FIG. 6 DQF-COSY: Double Quentum Filtered
Chemical Shift Correlation Spectroscopy

FIG. 7 shows the physical properties of sulfated cellulose.

FIG. 8 is a graph showing the protective effect of sulfated cellulose against desiccation injury of SV40 immortalized human corneal epithelial cells.

FIG. 9 is a diagram showing the protective effect of a sulfated sample against desiccation injury of SV40 immortalized human corneal epithelial cells.

FIG. 10 shows the effect of the O-sulfated cellulose of the present invention on extraorbital lacrimal extirpated rats (4 weeks after instillation).

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Takeo Omura             1-5-3 Nihonbashi Muromachi, Chuo-ku, Tokyo             Kamoto Pharmaceutical Co., Ltd. F term (reference) 4C086 AA01 AA02 AA03 EA24 MA01                       MA04 NA14 ZA33                 4C090 AA02 AA09 BA27 BB12 BB29                       BB33 BB36 BB54 BB63 BB95                       BD36 BD37 DA09 DA23

Claims (3)

[Claims] 1. An O-sulfated cellulose in which the hydroxyl group of cellulose is sulfated, wherein --SO in the monosaccharide repeating unit is used.
Sulfated cellulose having an average number of _three groups of 2.6 or more, or a pharmaceutically acceptable salt thereof. 2. An average molecular weight of 5,000 to 500,000.
The sulfated cellulose according to claim 1, which is 0, preferably 10,000 to 100,000, or a pharmaceutically acceptable salt thereof. 3. A therapeutic or prophylactic agent for corneal or conjunctival diseases, or a therapeutic agent for dry eye or a therapeutic agent for dry cornea, which comprises the sulfated cellulose according to claim 1 or 2 or a pharmacologically acceptable salt thereof as an active ingredient.