JPH07233057A - Anti-cataractous medicinal composition - Google Patents

Abstract

PURPOSE:To obtain a medicinal composition useful for treating cataract, containing a free radical capturing substance selected from a reducing thiol derivative, its disulfide derivative and its sulfide derivative. CONSTITUTION:This medicinal composition contains a free radical capturing agent selected from a compound of formula I or formula II (R<1> to R<3> each is a lower alkyl which is substitutable with a lower alkkyl with hydroxy, a lower alkyloxy or a lower alkylcarbonyloxy), a disulfide derivative of formula III and a sulfide derivative as an active ingredient. The medicinal composition can be formulated in an aqueous pharmaceutical preparation advantageous to local administration by treating the surface of fine-particle carrier (e.g. liposome) with a fat-soluble positive charge substance and using the surface- treated carrier to the medicinal composition. By sealing the anti-cataractous medicine in the interior of the treated liposome, the liposome exhibits positive charge and specifically exhibits cornea-directing property and binding roperty and the liposome is retained on a cornea and the anti-cataractous medicine in the interior of the liposome gradually reaches intraocular tissue through the cornea to carry out treatment for cataract without damaging the cornea and other eye tissue.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a pharmaceutical composition containing a reducing thiol derivative and a disulfide derivative thereof and a radical scavenging substance selected from a sulfide derivative as an anti-cataract drug, a fine particle carrier for their formulation, and It relates to a pharmaceutical preparation containing the same.

[0002]

2. Description of the Related Art In recent years, the incidence of senile cataract has been increasing in Japan as the population ages. Although the mechanism of cataract development and progression has not yet been fully elucidated, the function of the antioxidant system in vivo in the lens generally declines for some reason, causing lens proteins to aggregate and cause the lens to become cloudy. Is generally recognized. The deterioration of the function of this antioxidant system is due to (1) occurrence of a reaction system that abnormally consumes antioxidants in the body during the course of cataract development, and (2) abnormal lens membrane permeation along with other lens components. It is considered that outflow of glutathione (GSH), which is a reducing biological component, out of the lens due to diffusion. In addition, the cloudiness of the lens is Na / in the lens.
Changes in the ion balance of K and irreversible ion balance due to Ca ²⁺ are also considered.

On the other hand, there is a remarkable development of an anti-cataract drug by inhibiting aldose reductase for diabetic cataract which occurs at the time of diabetes, and a compound which finds its effectiveness in animal experiments has also appeared. However, in a clinical experiment using a powerful aldose reductase inhibitor, a death case was reported in the United States, and the application of this type of compound as an anticataract drug has almost stopped. As described above, there is no effective anti-cataract drug in combination with the fact that the mechanism of the occurrence and progression of cataract is not clearly understood, and it is expected that a drug effective as an anti-cataract drug will be developed for many years. It had been.

[0004]

[Means for Solving the Problems] The present inventors have searched for compounds that inhibit the decline of antioxidant system function as anticataract agents, but the present invention was completed based on the findings obtained in these processes. It is a thing. A first aspect of the present invention is to provide a radical scavenging substance selected from reducing thiol derivatives or their disulfide derivatives or sulfide derivatives. Diethyldithiocarbamate (D
DC) is one of reducing thiol derivatives and is a compound confirmed by the present inventors to have a therapeutic effect on cataract.
The cornea allows fat-soluble substances to pass through easily, but DDC is difficult to pass through the cornea because it is water-soluble, and therefore has little bioavailability in the aqueous humor and lens and has low bioavailability and oxidation. It has the drawback that it is easily affected and unstable, and its medicinal effect cannot be fully exerted.

As the search was continued, it was found that disulfide compounds and sulfide compounds, which are derivatives thereof which easily pass through the cornea, are lipophilic and chemically stable, are effective as anticataract agents. That is, the present inventors have further investigated the effect of treating a cataract on a radical scavenger having a stable reducing SH group with high bioavailability, and have confirmed the administration method and safety in the United States as an alcoholic drug. DSF (disulfiram (bis (diethylthiocarbamyl) disulf), which is a dimer of DDC.
ide)) has a significantly higher cataract treatment effect than DDC.

DSF (1) is easy to pass through the cornea because it is lipophilic, and (2) DSF produces 2 molecules of DDC by the reductive catalytic action of albumin, and 1 molecule of DSF DDC binds to SH group of albumin and
From the fact that it is known to release DDC of the molecule, after passing through the cornea, albumin in the aqueous humor and the lens produces DDC, which has a reducing SH group.
It is considered that C shows a therapeutic effect on cataract.

Further, the second aspect of the present invention provides a fine particle carrier for making the above-mentioned fat-soluble anti-cataract drug such as disulfide and sulfide derivative into an aqueous formulation which is advantageous for local administration at the stage of formulation. Is. That is, DS
F is fat-soluble and can easily pass through the cornea, but it is not miscible with tear fluid and is not suitable for topical administration to the eye. Therefore, it is effective to use a fine particle carrier for preparing an aqueous preparation. It raised this defect and solved this defect.

Further, a third aspect of the present invention is to provide a positively charged particulate carrier which effectively delivers an anti-cataract drug to the aqueous humor and the lens. That is, since the cornea is negatively charged, the particulate carrier was treated with a positively charged substance, and the present inventors measured the zeta potential of the positively charged membrane of the particulates and confirmed that the charged membrane was positive. did. That is, a positively-charged particulate carrier with high bioavailability has been developed, in which a therapeutic agent for cataract is surely targeted to the cornea and is delivered to the aqueous humor and the lens.

The fourth aspect of the present invention is to provide a pharmaceutical preparation containing a lipid-soluble positively charged fine particle carrier carrying the anti-cataract drug of the present invention.

"Reducing thiol derivative" is, for example,
formula:

[Chemical 3] [Wherein R ¹ , R ² and R ³ are the same or different,
A linear or branched lower alkyl group which may be substituted with a hydroxy group, a lower alkyloxy group or a lower alkylcarbonyloxy group], a derivative thereof and a pharmaceutically acceptable Examples thereof include salts thereof to be obtained.

In particular, DDC (diethylthiocarbamate), 1-methyl-1H-tetrazol-5-yl-thiol and 1- (2-hydroxyethyl) -1H-tetrazol-5-yl-thiol and their pharmaceuticals. Preferred are the salts which are acceptable.

The "disulfide derivative" is, for example,

[Chemical 4] Examples thereof include compounds represented by the formula [wherein R ¹ and R ² have the same meanings as described above], derivatives thereof and pharmaceutically acceptable salts thereof. Particularly preferred is DSF.

Examples of the "sulfide derivative" include cephem compounds, which are cephamycins closely related to cephalosporins, particularly cefamandole,
Cefopirazone, cefmenoxime hydrochloride, cefmetazole, cefotetan, latamoxef, cefbuperazone, cefpyramide, flomoxef and their pharmaceutically acceptable salts are preferred. Furthermore, more preferable are cefmetazole and salts thereof. These compounds are derivatives of 1-methyl-1H-tetrazol-5-yl-thiol and 1- (2-hydroxyethyl) -1H-tetrazol-5-yl-thiol.

The "radical scavenging substance" is a compound having a high reactivity with a radical which is an active chemical species,
It is a substance that reacts rapidly with radicals to remove them from the system and stop subsequent reactions. More specifically, it is a substance capable of scavenging radicals from active oxygen species such as singlet oxygen, hydrogen peroxide, superoxide anions, and hydroxyl radicals, and protecting against cascading biological damage due to oxidative reactions involved. . “Treatment” includes prophylaxis of cataracts and treatment of symptoms resulting from cataracts. The lower alkyloxy group and the “lower alkyl” of the lower alkylcarbonyloxy group are saturated linear or branched carbon atoms having 1 to 1 carbon atoms.
It refers to a hydrocarbon residue containing 6, preferably 1 to 5, and more preferably 1 to 4. For example, methyl, ethyl, propyl, isopropyl, butyl, t-butyl and the like are included.

Examples of the particulate carrier include liposomes, emulsions, nanocapsules, albumin microspheres and the like.

In particular, liposomes formed by redispersing naturally-derived lipids in water have a very high degree of similarity as a model of artificial cells having a cell membrane structure, and can be used as a tissue-directed drug carrier (drug carrier) or artificial population. It has good biocompatibility as a medicinal material such as erythrocytes, cell modifiers and enzyme-immobilized bases, and it has been proposed that it can be used in a wide range of fields of medicine and pharmacy.

However, even if the conventional liposomes are applied to the above-mentioned purpose, it is the current situation that the results of enduring the actual use have not been obtained. The first reason is that the conventional liposome itself is originally an assembly (assembly) of natural lipids due to non-covalent interactions, and therefore is required for practical use even though it has good biocompatibility. It is pointed out that the liposome itself lacks structural stability, and the second point is that the specific target tissue or target cell orientation, which is the most important as a drug carrier, is hardly exhibited.

Therefore, the present inventors have conducted investigations to improve these above-mentioned drawbacks, and by modifying the surface of the liposome with a lipophilic positive charge-loading substance, the mechanical properties of the liposome under physiological conditions have been improved. It has been found that the strength of the liposome is improved and the ability to selectively direct the target tissue is exhibited when the liposome treated in this manner is administered to a living body.

Up to now, the technique of modifying the surface of the liposome with a certain type of lipophilic positive charge-loading substance has been basically possible, but it has been empirically carried out by adding an amount of the modifying agent. However, the relationship between the quantitative membrane charge burden and the membrane permeability has not been clarified. In the present invention, the membrane charge and the stability of the membrane at the time of adding the lipophilic positive charge-loading substance are quantitatively examined by zeta potential measurement and differential scanning calorimetry, and optimal release control of the anticataract drug from liposomes and corneal permeation are conducted. It was possible to find a formulation capable of

That is, when an anti-cataract drug is encapsulated inside the liposome modified with such a lipophilic positive charge-loading substance, the liposome-modifying compound exhibits a positive charge and specifically exhibits corneal directivity and binding properties, and the cornea The liposome is retained above and the internal anti-cataract drug can gradually reach the inside of the eye through the cornea and, at the same time, causes almost no harm to the cornea and other ocular tissues, thereby providing an effective therapeutic means for the above-mentioned cataract. Become.

In addition to the above-mentioned liposomes, emulsions, nanocapsules, albumin microspheres and the like usually used in the pharmaceutical formulation technology can be used as the particulate carrier of the present invention.

Method for preparing liposomes Lipids are dissolved in an organic solvent such as chloroform or ether and then placed in a round bottom flask, and the organic solvent is removed under a nitrogen stream or under reduced pressure to form a lipid thin film on the round bottom of the flask. Further, in order to completely remove the organic solvent, it may be left in a desiccator under reduced pressure. Next, an appropriate buffer solution is added onto the lipid thin film to hydrate the lipid, whereby an emulsion-colored liposome is obtained. In the liposome preparation method by the reverse phase method, a W / O type emulsion is prepared by adding a solution of a compound as an active ingredient to an ether solution of phospholipid and sonicating. Ether is removed from this W / O type emulsion by an evaporator under reduced pressure, then a buffer solution is added and the mixture is stirred by a vortex mixer to invert the phase into a W / O type emulsion, and a residual organic solvent is removed to form liposomes. . In any case, when the anti-cataract drug of the present invention is fat-soluble, it is dissolved in an organic solvent together with a lipid,
When water-soluble, liposomes are prepared by dissolving an anti-cataract drug in a suitable buffer. In addition to these preparation methods, a French press method, a freeze-drying method, a freeze-thawing method, etc. can also be used.

The liposome itself provided by the present invention can be produced by a conventionally known method, and as the liposome, a conventionally known liposome composed of phospholipid or phospholipid and cholesterol can be used. Such phospholipids include egg yolk phospholipids such as egg yolk lecithin, soybean phospholipids such as soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, dicetylphosphate, stearylamine, phosphatidylglycerin, phosphatidic acid, phosphatidylyl. Examples thereof include ruinositol, and these may be used alone or as a mixture of two or more kinds. The liposome of the present invention is composed of at least the above-mentioned phospholipids, and may further contain cholesterol or the like depending on the kind of the anti-cataract drug to be encapsulated inside and the fat-soluble charged compound to be charged and added.

Method for Producing Emulsion Although an emulsion is also called an emulsion, a water-oil emulsion has an oil-in-water type (W / O) and a water-in-oil type (O / O).
W), and either type can be prepared by selecting the type of emulsifier. By stirring, each phase temporarily disperses the other into fine particles, but depending on the nature of the emulsifier, it becomes one continuous phase and becomes an O / W type or W / O type emulsion. For example, a W / O / W type complex emulsion is prepared by a two-step emulsification method. That is, a W / O type emulsion that becomes composite particles is prepared in the first step, and this is again dispersed in water using an appropriate emulsifier. Also, a typical w / o emulsion is about 10 (w /
w)% soybean oil, 0.6-2.0 (w / w)% egg lecithin (phospholipid) and about 2.5 (w / w)% glycerin dissolved in sufficient water To do. An emulsifier (egg lecithin) is dissolved in the oil phase, heated to 80 ° C. in a container, and hot water previously warmed to 80 ° C. is added thereto. Mix using a high speed automatic stirrer. The temperature of the mixture is maintained at 80 ° C. for 30 minutes after starting stirring in the vessel. Stirring is performed at 10,000 rpm. Furthermore, in order to produce a fine particle emulsion, a coarse particle emulsion is further added at 4500 ps.
Apply to the operating two-stage pressure homogenizer. The resulting dispersion can be used as the emulsion of the present invention.

Method for producing microcapsule A particle system in which fine particles of a drug are used as a core substance and the surface of which is coated with a polymer substance is called a microcapsule. The microcapsules are produced by a coacervation method, an interfacial polymerization method, or the like. The coacervation method is
For example, the core substance is dispersed in a solution of the coating substance to cause coacervation of the coating substance. Then, the coacervate is condensed on the surface of the core substance, and then the condensed coacervate is cured. As the coating material, ethyl cellulose, polylactic acid, polyvinyl acetate, gelatin, starch and the like are used.

Method of Producing Microspheres Microspheres refer to a particle system in which the microcapsules are one type of coating, whereas the microspheres are dispersed in a polymeric carrier in a dissolved state or as fine crystals. Basically,
An aqueous albumin solution (in which a drug is dissolved or suspended therein) is made into a W / O type emulsion with cottonseed oil or an organic solvent, and solidified by heat change, chemical crosslinking, in-liquid drying, radiation polymerization and the like. Examples of carriers include albumin, gelatin, dextran, polylactic acid, and polyethylene carbonate, which are biodegradable materials, and non-degradable polystyrene, agarose, polyacrylamide, and the like. Depending on the use of surfactants, 0.3-50
It is possible to change the size to 0 μm.

Manufacturing method of nanocapsules Particles having a size of nanometer. Similar to microcapsules, there are coacervation method and micellar polymerization method. As polymeric materials, albumin, gelatin,
Alkyl cyanoacrylate or the like is used.

Method for Treating Particulate Carrier with Fat-Soluble Positive Charge A fat-soluble positive charge-loading substance is added to an organic solvent that dissolves the lipid forming the particulate carrier, and the particles are prepared, and at the same time, surface charge treatment is performed.

Examples of the fat-soluble positive charge-loading substance used in the present invention include cetylpyridinium chloride (CPC), dimethyldialkyl (C8-18) ammonium bromide (DC-1-8-18), and N-methyl-N-. (Β-hydroxyethyl) -didodecyl ammonium bromide (D
C-2-12), N- (α-trimethylammonioacetyl) -didodecyl-L-glutamate chloride (DC
-3-12L), N- (α-trimethylammonioacetyl) -didodecyl-D-glutamate chloride (DC
-3-12D), N- (α-trimethylammonioacetyl) -O, O′-bis- (1H, 1H, 2H, 2H-
Perfluorododecyl) -L-glutamate chloride (DC-5-8F2L) and the like, and as the amine salt which is a cationic surfactant, for example, an alkylamine salt,
Polyamine and alkanolamine fatty acid derivatives, alkyl quaternary ammonium salts such as alkyl trimethyl ammonium salts, dialkyl ditrimethyl ammonium salts, alkyl dimethyl benzyl ammonium salts, and cyclic quaternary ammonium salts such as alkyl pyridinium salts and alkyl isoquinone salts. Examples of the norinium salt and aromatic ammonium salt include benzethonium chloride salt and nitrogen-containing surfactants such as polyoxyethylene fatty acid amide, polyoxyethylene alkylamine, alkylolamide, and alkylamine oxide. You can The lipophilic positive charge-loading substance is added in an amount of 1 to 3 relative to the total carrier component of the fine particles, for example, the lipid component.
0 mol%, preferably 3-20%, most preferably
Add 5 to 15%.

The lipid-soluble, positively charged, charge-loaded particulate carrier containing the anti-cataract drug of the present invention prepared according to the above-mentioned method is prepared by an ordinary formulation technique such as an aqueous eye drop, an aqueous suspension eye drop, a non-aqueous eye drop or a non-aqueous suspension. It can be prepared as a cloudy eye drop or an eye ointment. The dose per dose is the severity of cataract symptoms,
Although it varies depending on the age of the patient, etc., the toxicity of the drug used in the present invention is extremely low, and therefore there is no particular limitation, but it is 1 μg to 1 mg. The daily dose is 1 μg to 50 mg.

[0031]

【Example】

Example 1 Method for Preparing DSF-Containing Liposomes A method in which the reverse phase solvent evaporation method was partially modified was used. In other words, 10 mg of dichloromethane and 10 mg of DSF and DPPC
(Dipalmitoylphosphatidylcholine), DMPC
(Dimyristoylphosphatidylcholine) and CPC
(Cetylpyridinium chloride) was dissolved to obtain a solvent phase. This solvent phase and a buffer solution (A: Hind-Goryan buffer solution)
pH 6.5, B: Gifford buffer pH 8.0) 10 ml
Was placed in a three-necked flask and subjected to ultrasonic treatment for 10 minutes in a nitrogen stream using a bath-type sonicator. Next, the mixture was stirred for 20 minutes to form an emulsion, and then decompressed with an aspirator for 30 minutes to remove dichloromethane. The preparation temperature was 37 ° C. The liposomes thus prepared were 1.0, 0.0
Each of the particles was passed 10 times through an extruder equipped with a membrane filter having four different pore sizes of 6, 0.2, and 0.1 μm to obtain a singlet liposome (average particle diameter of about 80 nm) having a uniform particle diameter. Preparation of Cefmetazone-Containing Liposomes Cefmetazone-containing liposomes were prepared in the same manner as in Example 1 except that cefmethasone was used instead of DSF.

Measurement of Encapsulation Rate of DSF-Containing Liposomes The concentration of DSF in liposomes was measured by the HPLC method by adding methanol to the liposome preparation obtained by the above-mentioned preparation method and adding the internal standard solution to the disrupted lipid membrane. did. The liposomes were diluted 5 times with a buffer solution (A: Hind-Goryan buffer solution pH 6.5B: Gifford buffer solution pH 8.0) and subjected to ultrafiltration with Centrisalto I to obtain the liposome external solution. An internal standard was added, and the drug concentration in the external solution was measured by the HPLC method. It was confirmed that no drug was detected in the external liquid, and DS added at the time of liposome preparation
The encapsulation rate was calculated with the F amount as 100%.

DSF Content Encapsulation Rate of Liposomes Liposomes were prepared with lipid compositions having different phase transition temperatures shown in Table 1 and a total of five kinds of preparations with liposome external pH values of 6.5 and 8.0. The encapsulation rate of all liposomes is about 9
It was as good as 0% or more. In addition, no drug was detected in the external liquid of the liposome.

DSF-Containing Liposome Membrane Zeta Potential Measurement For the zeta potential measurement, a laser rotating prism type zeta potential measuring device LAZAR ZEE METER type 501 manufactured by PEN KEM, USA was used. Liposomes were added to the sample as potassium phosphate buffer (5 mM K ₂ H
PO ₄ , 5 mM KH ₂ PO ₄ pH 6.5) was used and diluted 150 times, and the voltage was measured at 150V. The correction of the zeta potential due to the temperature change was performed as follows. Zeta potential (correction value) = Zeta potential (measurement value) x (1-0.0
2T) T: Measurement temperature -20 ° C

DSF content liposome membrane zeta potential measurement results CPC was added for the purpose of giving a positive charge to the membrane in order to improve the retention efficiency of the liposome and the membrane permeability.
From the result of zeta potential measurement, CP of 1 molar ratio to lipid
It was found that the liposome membrane to which C was added had a positive charge (40 to 50 mV), and the one without addition had a negative charge (about -13 mV).

[Table 1]

Release test of DDC from DSF-containing liposomes Liposomes were buffered ((1) Gifford buffer pH 8.0,
(2) 2.5 times diluted with Gifford buffer pH 6.5) and a solution of bovine serum albumin (BSA) dissolved in the same buffer were mixed in 0.25 ml at 0 ° C for 6 hours at 35 ° C. After heating, cool with ice. Ultrafiltration was performed using Centrisart I, an internal standard was added, and the DDC release amount was measured by the HPLC method.

Releasability of DDC from DSF-containing liposomes (1) DPPC / DMPC / CPC (2/8/1) external liposome solution: Gifford buffer solution, pH 8.0 The external liposome solution was adjusted to pH 8.0 in FIG. The amount of DDC released is shown. Compared to the case of pH 9.5, the release amount decreased, but DPPC / DMPC / CPC (2/8/1) and
Both liposomes (5/5/1) showed the highest DDC release amount when 3.5% BSA was added.

(2) DPPC / DMPC / CPC (2/8/1) Liposome external solution: Hind-Gouyan buffer solution, pH 6.5 As a result of a release test when the liposome external solution was pH 6.5, DDC was obtained. Was not observed. The BSA solution used had three concentrations of 1.0, 1.75, and 3.5%.

In Vitro Corneal Permeation Test A male white rabbit having a body weight of about 2.0 kg was euthanized by injecting a lethal dose of pentobarbital injection solution through an ear vein, and then the eyeball was carefully removed and the cornea was removed. The part was excised so as not to damage, leaving 1-2 mm of the sclera. This cornea was attached to an acrylic resin corneal permeable cell as shown in FIG. 2, and an isotonic HEPES buffer solution (10 mM HE was used on the aqueous humor side).
PES, 136.2 mM NaCl, 5.3 mM KCl, 1.
0 mM K ₂ HPO ₄ , 1.7 mM CaCl ₂ · 2H ₂ O, 5.5
mM glucose (pH 7.4), a DSF liposome / BSA mixed solution was added to the tear side, and the mixture was heated at a corneal surface temperature of 35 ° C. 50 μl over time from the start of the experiment to 6 hours later
Aqueous humor side liquid was collected and the amount of permeated drug was measured by the HPLC method.

Results of in vitro corneal permeation test (1) DPPC / DMPC / CPC (2/8/1) Liposomal external solution: Gifford buffer, pH 8.0 Liposomal external solution was pH 8.0, lipid composition DPPC / D
FIG. 3 shows the corneal permeation amount when the MPC / CPC (2/8/1) liposome was used. Tests were performed with and without addition of 1.75% BSA to the external liquid. Both of them showed the same permeation of DDC.
It was about twice as much as the permeation amount of / DMPC / CPC (5/5/1).

(2) DPPC / DMPC / CPC (2/8/1) Liposome External Solution: Hind-Gouyan Buffer, pH 6.5 FIG. 4 shows the amount of DDC corneal permeation when the liposome external solution is pH 6.5. Indicates. The test was conducted with and without addition of 1.75% BSA. Corneal permeation of DDC was observed regardless of the presence or absence of BSA, but the permeation amount decreased to about 1/2 of that at pH 8.0.

Example 2 Method for Preparing DSF Microspheres Using Lactic Acid-Glycolic Acid Copolymer LGA-7515 (lactic acid-glycolic acid copolymer, Wako Pure Chemical Industries, Ltd.) 850 mg, DSF 100 mg, cetylpyridinium chloride (CPC) 50 mg Was dissolved in 2.5 ml of methylene chloride and stored in a 1-liter three-necked flask at 0.5%.
The total amount is added in 150 ml of polyvinyl alcohol (PVA). While maintaining the temperature of the solution at 25 ° C., stirring is performed for 2 hours at a rotation speed of 500 rpm using a Teflon rotor.
The above solution is depressurized for 2 hours using a water jet pump to remove methylene chloride. The microspheres thus prepared are passed through a sieve having a mesh of 297, 177 and 75 μm, and the microspheres in the obtained filtrate are collected by a glass filter (17G4). The microspheres on the glass filter are washed with purified water until PVA is completely removed. The microspheres collected on the glass filter are dried under reduced pressure in a desiccator. The encapsulation rate of DSF in the microspheres thus prepared was about 90%. The particle size of this microsphere was about 1 μm.

Example 3 Method for Preparing DSF-Containing Albumin Microspheres 250 mg of bovine serum albumin is dissolved in 1 ml of purified water, and 250 mg of DSF is suspended therein. Next, this albumin-DSF suspension was added to cottonseed oil (100 ml) containing 10% (v / v) of Span 85 and stirred with a glass stirrer at a rotation speed of 250 rpm for 10 minutes, and then an ultrasonic generator (Shimadzu US
P-600) and emulsify (100w, 30 minutes),
A water-in-oil type (W / O type) microemulsion is used.
This is added to a glycerin oil bath at 100 ° C. to form microspheres. After that, the oil content is removed using an organic solvent such as ether and dried under reduced pressure in a desiccator. The encapsulation rate of DSF in the microspheres thus prepared was about 95%. This DSF-containing albumin
The particle size range of the microspheres was 0.6-1.5 μm, and the average particle size was about 1 μm.

Example 4 Formulation Example The DSF-encapsulated liposome produced in Example 1 can be used as it is as an aqueous dispersion formulation, but if necessary, a commonly used additive such as a preservative and a tonicity agent. To prepare a formulation for eye drops.

In vitro cataract induction and lens GS
Anti-cataract drug activity test by H measurement (1) Test method After dissecting the neck of a 5-6 week old ddY mouse (male) and euthanizing it, the lens is removed from the eyeball under a stereomicroscope, and then D Pre-warmed in MEM (low glucose) medium for 2 hours at 37 ° C. Then, the intact lens was selected under a stereomicroscope, and a glucose-free HEPES buffer solution containing 1 mM diamide (composition: 10 mM HEPES, 13
8.85 mM NaCl, 5.4 mM KCl, 1 mM K ₂ H
PO ₄ , 1.7 mM CaCl ₂ 2H ₂ OpH 7.4 37
In order to induce the cataract by heating at 37 ° C. for 1.5 hours in 3 ml of 3 ml, in order to see the drug effect, H containing 1 mM glutathione isopropyl ester (GE) or DDC-Na.
EPES buffer (composition: 10 mM HEPES, 136.
2 mM NaCl, 5.3 mM KCl, 1.0 mM K ₂ HP
O ₄ , 1.7 mM CaCl ₂ .2H ₂ O, 5.5 mM glucose pH 7.4) 3 ml in a group heated at 37 ° C. for 5 hours,
3 in 3 ml HEPES buffer to see cataract progression
The treatment was carried out separately to a group (daiamide (+) group) heated at 7 ° C. for 5 hours. Control group is HEPES without glucose
Warm in 3 ml of buffer at 37 ° C for 1.5 hours, then HEP
The mixture was heated at 37 ° C. for 5 hours in 3 ml of ES buffer (Daiamide (−) group). Wet weights were measured on all of the lenses treated so far.

(2) Quantification of GSH in lens A. DTNB method The treated lens and 0.5 ml of purified water were added and homogenized with a Teflon homogenizer. This homogenate 10
0 μl was put into a sample tube, 150 μl of metaphosphoric acid solution was further added, and the mixture was left for 5 minutes and then subjected to low temperature centrifugation at 12000 × g for 15 minutes. 200 μl of the supernatant was 0.3 M.
Na ₂ HPO ₄ solution 800 μl, DTNB solution (DTNB was proposed by BEUTLER, and the reagent itself is water-soluble, easy to handle, and good in benignity. In the presence of DTNB, reduced glutathione becomes oxidized glutathione, while DTN
B was quantitatively reduced to become a dye having an absorption at 412 nm), and 100 μl thereof was added to quantify the GSH amount in the lens. The quantification was carried out using a Shimadzu UV-2200 absorptiometer at an absorption wavelength of 412 nm. The calibration curve is obtained by diluting GSH, which is a GSH standard product, with purified water to obtain a concentration of 0 to 100 μg / m.
Created in the l range. The sample was quantified by setting it within the concentration range of the calibration curve. The GSH amount per one lens was calculated by the following formula). GSH amount in lens (μg / lens) = (absorbance value at 412nm /
Slope of calibration curve) x 0.5

B. High Performance Liquid Chromatography (HPLC method) The outline of this measurement method is shown below. The cataract-induced lens and purified water by the in vitro method were added to the rat in an amount of 1 ml and in the mouse to 0.5 ml, and homogenized with a Teflon homogenizer. 100 μl of the internal standard was added to 50 μl of this homogenate, low-temperature centrifugation was performed at 12,000 × g for 15 minutes, and the supernatant was filtered through a chromatodisc 4N with a pore size of 0.45 μm and then quantified by HPLC. The HPLC apparatus was Shimadzu LC-10AD. A visible-ultraviolet detector SPD-10A was connected and C-R5A was used for data processing. The mobile phase was 5% methanol containing 0.1% TFA, the detection wavelength was 210 nm, and the flow rate was 1.0 ml / min. The separation column to which the card column was connected was a Merck Supersphere 100 RP-18 (4 μm, 4.0 ×).
250 mm) was used. 100 μg / ml as internal standard
A solution of sodium pantothenate in methanol was prepared and mixed with the collected solution: internal standard at a ratio of 1: 2 to give 10 μlH
It quantified by injecting into PLC. The calibration curve was prepared in the concentration range of 10-500 μg / ml under the same conditions as the sample.
The GSH amount was calculated from the slope of the calibration curve obtained from this. In the HPLC method, the peaks of the crystalline lens preparation and the GSH standard preparation were separated cleanly, so that accurate and stable measurement was possible.

(3) Anticataract Drug Activity Effect in GSH Measurement in Lens The amount of GSH in the lens by the DTNB method is shown in Table 2 and FIG.

[Table 2] GSH amount in lens by DTNB method GSH amount per lens (μg / lens) Deviation Diamide (-) 10.3 0.5 Diamide (+) 7.6 0.3 mM GE 10.3 .8 1mM DDC-Na 9.8 0.8 Comparison between diamide (-) and diamide (+) was 10.
A decrease of 3 ± 0.5 μg / lens and 7.6 ± 0.3 μg / lens was observed. By treatment with 1 mM GE and DDC-Na, respectively, 10.3 ± 0.8 μg / lens and 9.8 ± 0.0.
It became 8 μg / lens and recovered to the value of diamide (-).

The amount of GSH in the lens by the HPLC method is shown in Table 3 and FIG.

[Table 3] GSH amount in lens by HPLC method GSH amount per lens (μg / lens) deviation Diamide (-) 4.4 0.3 Diamide (+) 3.1 0.2 1 mM GE 4.0 .2 1 mM DDC-Na 3.9 0.4 When comparing diamide (-) and diamide (+), 4.4
A decrease of ± 0.3 μg / lens and 3.1 ± 0.2 μg / lens was observed. It is clear that lens opacification is significantly induced. By treatment with 1 mM GE or DDC-Na, 4.0 ± 0.2 μg / lens and 3.9 ±
The value was 0.4 μg / lens, and the value was restored to the value of diamide (−).

[0050] In vitro cataract experimental anticataract agent activity tests due to changes in Na / K ratio and Ca ² + content in mouse extraction lens in (1) the lens of Test Method 5 week old ddY mice under a stereomicroscope After extraction, 24-well tissue culture calstar (24-WELL TISSUE CULT
URE CLUSTERS) and put them individually into low glucose DM
Pre-warm in EM medium for 18-20 hours at 37 ° C. in a CO ₂ incubator. Then, the one in which no suspension was observed in the lens was selected and heated at 37 ° C. for 1.5 hours in 3 ml of a 1 mM diamide HEPES (without glucose) buffer to induce cataract. 1m to see the therapeutic effect of the drug
HEPES as a therapeutic drug for M (addition of glucose (10 mM HEP
ES, 136.2 mM NaCl, 5.3 mM KCl, 1.0
mMK ₂ HPO ₄ , 1.7 mM CaCl ₂ · 2H ₂ O, 5.5 m
M glucose, pH 7.4) in 3 ml of buffer, while in order to see the progression of cataract, it was heated in 37 ml of HEPES (glucose added) buffer for 5 hours at 37 ° C. This is Daiamide
(+) The control group was treated with diamide (-). After measuring the wet weight of all the lenses treated up to this point, they are dried under reduced pressure at 100 ° C. for 7 hours or more using a vacuum dryer, and the dry weight is measured. Add 100 μl of 60% nitric acid, incinerate at 80 ° C for about 20 minutes until the lens dissolves, add 1 ml of purified water, and centrifuge at 3000 xg for 10 minutes.
200 μl of the supernatant was collected, and 3 ml of purified water was added to add Na-
Use as a K measurement sample. 100 μl of lanthanum chloride was added to the remaining whole amount of the supernatant, and the supernatant was centrifuged at 3000 × g for 10 minutes to obtain a Ca measurement sample.

(2) C. Atomic absorption measurement of Na, K, and Ca ions Quantitative determination of Na, K, and Ca ion content is Hitachi 180-80.
It was measured by a type atomic absorption spectrometer. Atomic absorption measurement conditions Na K Ca Lamp current 10 mA 10 mA 10 mA Wavelength 589.0 nm 766.5 nm 422.7 nm Slit 0.4 nm 2.6 nm 2.6 nm Burner head Standard type Standard type Standard burner height 7.5 mm 7.5 mm 12 .5mm frame Air-acetylene Air-acetylene Air-acetylene Combustion gas pressure 1.6kg / cm ² 1.6kg / cm ² 1.6kg / cm ² Fuel gas pressure 0.25kg / cm ² 0.3kg / cm ² 0.0 4 kg / cm ²

The calibration curve was prepared by using Na standard products, K standard products, 6
Purified water is added to 0% nitric acid to make a Na-K stock solution, which is diluted with Na
It was prepared so that the concentration was in the range of 0 to 2.5 μg and the K concentration was in the range of 0 to 5 μg. Similarly, use Ca as a stock solution of Ca, dilute it, and add 100 μl of lanthanum chloride to add Ca concentration: 0 to 25 μm.
Created in the range of g. The amounts of Na, K and Ca in the lens were determined by the following formula. Ion amount in lens = (absorbance / gradient of calibration curve) x (1
100/200) ÷ atomic weight ÷ water content The water content shown here is the water content of the lens obtained by subtracting the dry weight from the wet weight.

[0053] In vitro mouse extraction lens during the cataract therapy experiments Na / K ratio and Ca ² + content variation due to anti-cataract agents active effect Daiamide treated mice after excision lens vitro cataract treatment experiments using the results Na The / K ratio is shown in Table 4 and FIG.

[Table 4] Na, K and Ca contents in lens and Na / K ratio Na K Ca Na / K ratio Diamide (−) 22.03 ± 3.39 95.90 ± 3.49 1.20 ± 0.27 0.23 ± 0.04 Diamide (+) 60.38 ± 13.49 61.29 ± 13.78 1.98 ± 0.27 1.11 ± 0.04 DDC-Na 36.56 ± 4.38 86.25 ± 4.02 1.09 ± 0.33 0.43 ± 0.07 GE 51.60 ± 9.57 79.67 ± 8.63 1.39 ± 0.30 0.67 ± 0.20 CMZ 40.59 ± 10.16 84.91 ± 10.06 1.11 ± 0.36 0.50 ± 0.19

Diamide (-) showed a value similar to the intracellular Na / K ratio and was 0.23 ± 0.04 mmol / kg H ₂ O, and 1.11 ± for cataract-induced diamide (+).
Increased to 0.56mmol / kg H ₂ O. The therapeutic drug is SH
A GE, CMZ and DSF reductant having a group D
Three kinds of DC-Na drugs and DTT (dithiothreitol) having a strong reducing action were used as comparative substances.
All of these drug treatments showed a significantly lower Na / K ratio than diamide (+), and in particular, a remarkable therapeutic effect was observed with DDC-Na, and 0.43 ± 0.07 mmol / kg H ₂
O and the control value, then CMZ, DTT,
The effects were recognized in the order of GE.

FIG. 7 shows the results of examining the behavior of Ca ions. Diamide (-) 1.20 ± 0.27 mmol /
kg H ₂ O, 1.98 ± 1.00 mmol for diamide (+)
The Ca content increased up to / kgH ₂ O. DDC-Na also showed the most excellent lowering effect on this Ca content, and
A significant Ca ion lowering effect was also observed in CMZ next to DC-Na. On the other hand, the other two drugs also showed a Ca ion lowering effect, but no significant difference was observed.

Anticataract drug activity test by inhibition of aldose reductase (AR) (1) Test method The lens of the rat was extracted and weighed, and 20 times amount of 5 mM sodium phosphate buffer (5 mM NaH ₂ PO ₄ ·.
200 ml of 2H ₂ O, 5 mM Na ₂ HPO ₄ , pH 7.4)
Is homogenized and centrifuged at 18,000 xg for 5 minutes to give a supernatant. To this, 760 g / l (100% saturated) ammonium sulfate was added to collect a 30 to 75% saturated ammonium sulfate fraction, which was finally suspended in 3.2 M ammonium sulfate to prepare an AR enzyme solution (about 45 units / ml). The reaction system concentration was 0.1 M NaH ₂ per 1.0 ml.
PO ₄ buffer (pH 6.2), 0.4M NH ₄ SO ₄ , 10 mM
DL-glyceraldehyde, 0.16 mM NADP
H and AR enzyme solutions were included. Therefore, the reagent is 0.8M (N
H ₄ ) ₂ SO _4, including 0.2 M NaH ₂ PO ₄ (pH 6.2), 10
0 mM DL-glyceraldehyde (dissolved in purified water), 1.
6 mM NADPH (dissolved in 5 mM sodium phosphate buffer) and AR enzyme solution were prepared. The concentration of AR enzyme solution is NA
Since the molar absorption coefficient of DPH is 6220 OD / M, it is diluted with 5 mM sodium phosphate buffer so as to be in the range of 0.0625-0.1 OD / min, and the control is taken. I adjusted it.

(2) Method of measuring absorbance of NADPH The rate of decrease of NADPH was measured using a Shimadzu spectrophotometer UV-2200 at an absorption wavelength of 340 nm. The measurement conditions are as follows. Parameters in time Measurement mode: ABS Sampling pitch (delta t): Automatic (0.5 sec) Wavelength: 340.0 nm Slit width: 2.0 nm Measurement time: 300 sec In a quartz cell (1.0 ml), 0.8M ( 0.5 ml of 0.2M NaH ₂ PO ₄ (pH 6.2) buffer containing NH ₄ ) ₂ SO ₄ , AR
Enzyme solution 0.1 ml, 1.6 mM or 2 mM NADPH solution 0.1 ml, purified water 0.1 ml, 5% DMSO or purified water 0.1 ml, 100 mM MDL-glyceraldehyde solution 0.1.
1 ml was added in this order, and the mixture was quickly stirred and measured. Using this as a control, quercitrin as the search drug and 5% for DSF
DMSO and other drugs were dissolved in purified water and the concentration was changed. At this time, the temperature is kept at 30 ° C. in a thermostat and the reaction is observed for 5 minutes.

(3) Effect of aldose reductase (AR) inhibitory activity All drugs used in the AR inhibitory experiment and the measured concentration range are shown in Table 5. FIG. 8 shows the inhibitory profile of the drugs that showed strong AR inhibitory activity. The% inhibition is plotted on the vertical axis and the drug concentration μM is plotted on the horizontal axis.
The concentration corresponding to 50% inhibition was determined from this graph and the IC ₅₀
Expressed as Quercitrin was used as a standard compound for AR inhibitors, and pyrenoxine was used as a comparative substance in the currently used eye drop component. A drug having an S—S or SH group showed a strong AR inhibitory activity in the order of DSF>CMZ> GE. In the S—S and SH compounds of fursultiamine to GSSG, no more remarkable inhibitory activity than that of the above-mentioned drugs was observed, and thiamine hydrochloride and glutathione thiol ester S-methylglutathione to S were obtained.
No inhibitory activity was observed up to-(n-propyl) glutathione.

[Table 5]

In vivo anti-cataract activity test of liposomes encapsulating disulfiram In vivo, the liposomes encapsulating disulfiram (tetraethylthiuram = disulfide: DSF) prepared in Example 1 were artificially induced by X-ray irradiation and selenite. The effectiveness for cataracts was evaluated. The cataract induced by X-ray irradiation has a pathological condition similar to that of senile cataract, and the cataract caused by selenite is a cataract induced only in young rats before 3 weeks of age. However, as shown in the results below, in the cataract caused by selenite, GSH was decreased and Ca amount was increased, and the nuclear cataract developed. Since GSH levels and Ca levels are also decreased in senile cataract, it can be used as one of the animal experimental models for senile cataract. To determine the effectiveness, the Na / K ratio, the Ca amount, and the GSH amount in the lens are measured, and the anterior segment image diagnostic apparatus EAS-1000
By quantifying the images taken by (Nidek), the progression status of cataracts was determined over time without killing the animals.

Cataract induction by X-ray irradiation and various drug administration tests (1) Cataract induction method A 9-week-old (male) BN rat was fixed on a prone position and 10 G
The head was irradiated with y-rays of y. At this time, in order to prevent X-ray damage to the whole body, the parts other than the head were covered with lead clothes.

(2) Image diagnosis (i) DSF (100 mg / dl) suspension, DDC-Na (sodium N, N-diethyldithiocarbamate) (using administration method and image diagnosis method in rats treated in (1)) ( 100 mg / dl) solution, GE (glutathione isopropyl ester) (200 mg / dl) solution, CM
Z (sodium cefmetazole: cefmethasone) (10
0 mg / dl) ophthalmic solution was administered once to both eyes of the rat 3 times a day for 6 months, and slits and transillumination photography were performed with EAS-1000 every month for the determination of the state of cataract progression. An image diagnosis was performed.

(Ii) Image analysis method Slit images 6 months after administration were taken by EAS-1000 (Dens
Itmetory Integrated area), and the degree of turbidity was quantified for the three parts of the anterior pole, nucleus and posterior pole of the lens.

(Iii) Effect of DSF administration by image analysis FIG. 9 shows the results of analysis by Macintosh software NIHI Image for image diagnosis of DSF-administered rats 6 months after X-ray irradiation. By X-ray irradiation, cataract progressed in the order of anterior pole, posterior pole, and finally nucleus of drug-untreated animals. As is clear from FIG. 9, in the DSF-administered rat, turbidity at the posterior pole was not observed, and turbidity at the anterior pole was also suppressed.

(Iv) Judgment of drug effect by quantifying image analysis result A graph of the analysis result by EAS-1000 of the slit image 6 months after administration is shown below. FIG. 10-A and Table 1 show a comparison of turbidity in the anterior segment of the lens. Compared with the drug-untreated animal group, the turbidity was slight, but slight in the DSF and GE administration groups. However, no significant difference was observed. Similarly, FIG. 10-B and Table 1 show the comparison at the core. As is clear from FIG. 2-B and Table 6, no effect of the drug on turbidity was observed in the nucleus. Next, FIG. 11 and Table 6 show comparison at the rear pole portion. At the posterior part, a decrease in turbidity was observed in the DSF, DDC-Na, and GE administration groups, and the DSF administration group showed the lowest apparent value. However, there was no significant difference here either. When cataracts are induced by X-ray irradiation, there is a problem that it is difficult to immobilize the animal during X-ray irradiation, and it is not possible to always irradiate a fixed position, which often causes data variations.

[Table 6] Drug effect by quantifying image analysis results Front pole Nuclear rear pole control 1206 ± 759 203 397 ± 37889 1818 ± 1728 X-ray irradiation control 4630 ± 1736 259396 ± 25752 12720 ± 9449 DSF administration 3515 ± 1656 279951 ± 32227 5716 ± 3798 DDC-Na administration 4707 ± 1494 295824 ± 42134 9347 ± 5658 GE administration 3897 ± 2299 249074 ± 55147 7787 ± 6704 CMZ administration 4578 ± 2214 264612 ± 52206 12304 ± 10843

(3) Method for Measuring Na / K Ratio and Ca Content in Extracted Lens The lens of the BN rat after 6 months of breeding described above was extracted under chloroform anesthesia, and the following operation was performed using the right eye lens. . The wet weight was measured and dried under reduced pressure at 100 ° C. for 7 hours or more using a vacuum dryer, and the dry weight was measured. 100 μl of 60% nitric acid was added to each lens, and the crystals were ashed in a dryer at 80 ° C. for about 30 minutes to dissolve the lenses. To this, 2 ml of ultrapure water was added and centrifuged at 3000 rpm for 15 minutes. 200 μl of the supernatant obtained here was collected and
ml was added to make a Na and K measurement sample. To the residue, 100 μl of lanthanum chloride (Ca: 10 ± 0.3 w / v%) was added, and the mixture was centrifuged at 3000 rpm for 15 minutes, and the supernatant was used as a Ca measurement sample. This sample was measured with a Hitachi 180-80 type atomic absorption spectrometer.

(4) Method for measuring the amount of reduced glutathione (GSH) in the isolated lens. G was measured by HPLC using the above-mentioned left lens in the isolated lens.
The SH amount was measured. 0.1% per extracted lens
TFA in 5% MeOH 0.5 ml was added and homogenized with a Teflon homogenizer. 50 μl of this homogenate
100 μg / ml sodium pantothenate MeOH solution 1
00 μl was added as an internal standard, and the mixture was subjected to low temperature centrifugation at 12,000 g for 15 minutes. The supernatant was then filtered through Chromatodisc 4A with a pore size of 0.45 μm and measured by HPLC. As for the HPLC device, a visible ultraviolet detector SPD-10A was connected to the Shimadzu LC-10AD, and C was used for data processing.
-R5A was used. Column is Superspher 10 from Merk
Using 0RP-18 (4 μm, 4 × 250 mm), eluent 0.1% TFA in 5% MeOH, stop time 19 min, flow rate 1 ml / min, wavelength 210 nm, pressure 150 kg / cm ² did. The calibration curve was prepared in the concentration range of 10 to 500 μg / ml under the same conditions.
The GSH amount was calculated from the slope of this calibration curve.

(5) Na / K ratio in extracted lens, Ca amount, G
Effect of drugs on SH amount FIG. 12-A and Table 7 show the effects of each drug on the Na / K ratio by X-ray irradiation. The Na / K ratio of the drug-untreated animal group was 0.43 ± 0.11, which was 0.26 ± 0.0 of that of the normal rat.
It increased compared to 6. DSF, DDC-N in drug administration group
In a and GE, a tendency to suppress the increase in Na / K ratio was observed. FIG. 12-B and Table 7 show the effect of each drug on the Ca amount by X-ray irradiation. The amount of Ca of drug-untreated animals was 4.2 ± 2.3, 1.2 ± 0 mmol / kgH ₂ O in normal rats.
Therefore, the amount of Ca is increased by the X-ray irradiation. GE in the drug administration group tended to suppress the increase in Ca content, but it was not observed in the other drug groups. FIG. 13, Table 7
The effect of each drug on the GSH amount by X-ray irradiation is shown in FIG. The amount of GSH was reduced from 135.2 ± 12.0 to 100.1 ± 12.0 μmol / lens by X-ray irradiation.
By administration of F, DDC-Na, and GE, this decrease could be significantly suppressed. Moreover, the decrease could not be suppressed by CMZ administration.

[Table 7] Na / K ratio in lens, Ca amount, GSH amount Na / K ratio Ca amount GSH amount (mmol / kg H ₂ O) (μmol / lens) Control 0.26 ± 0.06 1.2 ± 0 135.2 ± 12.0 X-ray irradiation Control 0.43 ± 0.11 4.2 ± 2.3 100.1 ± 12.0 DSF administration 0.35 ± 0.08 4.4 ± 1.6 137.3 ± 22.6 DDC-Na administration 0.37 ± 0.15 4.2 ± 1.3 126.7 ± 25.3 GE administration 0.30 ± 0.06 2.7 ± 1.3 130.6 ± 29.9 CMZ administration 0.47 ± 0.20 4.0 ± 2.3 99.7 ± 21.7

Cataract induction by sodium selenite and various drug eye drop test methods (1) Cataract induction method Sodium selenite was subcutaneously injected (3.28 mg / kg) into the neck of 3-star Wistar rats to induce cataract. Let

(2) Image diagnosis (i) Eye drop method and image diagnosis method DSF encapsulated DMPC / DPPC / CPC (8/2/1)
Liposomes (sodium phosphate buffer, pH 6.5, 0.
65 mg / ml DSF), DSF suspension (sodium phosphate buffer, pH 6.5, 1 mg / ml DSF), CMZ enclosed D
MPC / DPPC / CPC (3/7/1) liposome (Hind-Goyan buffer, pH 6.31, 100 mg / mlC
Using 3 kinds of preparations of MZ), 10 μl of each selenite was instilled into both eyes 4 times a day for 1 week. EAS-1
After taking a slit image and a transillumination image with 000, the lens was extracted under anesthesia with chloroform, and the opacity was observed with a stereomicroscope.

(Ii) Image analysis method In order to determine the effect of instillation of each preparation one week after the treatment with selenious acid, slit images were taken with Macintosh software NIH Image.
The EAS-1000 (Retroilluminati
on image). (iii) Macroscopic determination on drug effect FIG. 14 is a photograph of a rat head after instillation for 1 week. In the untreated rats, it was found that the central part became cloudy and nuclear cataract occurred. On the other hand, no turbidity was observed in the DSF liposome or DSF suspension eye-drop rat. In addition, slight turbidity was observed when the CMZ liposome was instilled.
FIG. 15 shows a stereoscopic microscope image of a lens extracted from the above animal. In the untreated rat, marked turbidity occurred in the nucleus, whereas in the eye drop of DSF, the opacity was obviously suppressed. On the other hand, in the DSF suspension and CMZ liposome, the effects were found to vary, and there were animals that showed marked effects and those that did not.

(Iv) Effect of Drug on Image Analysis FIG. 16 shows the results of image diagnosis of DSF and CMZ liposome instilled rats 1 week after the treatment with selenious acid. In untreated rats, marked opacity of nuclei was observed in both slit and transillumination images, but opacity of the nuclei was suppressed by instilling DSF liposomes. In addition, even with CMZ liposome eye drops, DSF
Although not so remarkable as liposomes, it was observed that turbidity in the nucleus was suppressed.

(V) Judgment of Drug Effect by Quantifying Image Analysis Results FIG. 17-A and Table 8 show the results of slit image opacity analysis by Macintosh after 1-week instillation. Both formulations suppress lens opacity due to selenite treatment.
The effect was recognized as a significant difference in the liposome. Figure 17-B, Table 8 shows the transillumination image EA after instillation for 1 week.
The comparison of the transmittance analyzed by S-1000 is shown. The selenite treatment reduced the transmittance by about 40%.
It was found that the reduction was about 8% for F liposome, and about 25% for DSF suspension and CMZ liposome.

[Table 8] Drug effect by slit / transillumination image analysis Opacity Permeability (%) (Slit) (Retro) Control 3301 ± 1021 95.60 ± 4.01 Selenite control 20683 ± 4659 56.24 ± 17.08 DSF liposome 10170 ± 6305 87.41 ± 9.72 DSF suspension 17017 ± 7195 69.74 ± 15.26 CMZ liposomes 19617 ± 6163 70.12 ± 12.64

(3) Method for measuring Na / K ratio and Ca content in the isolated lens The measurement was carried out in the same manner as in (3) above using the right lens.

(4) Method for measuring GSH amount in isolated lens A sample was prepared by the method described in the item 1E using the lens of the left eye.

(5) Na / K ratio in extracted lens, Ca amount, G
Effect of drug on SH amount FIG. 18-A and Table 9 show the results of measuring the Na / K ratio in the isolated lens. No significant change was observed in the Na / K ratio. FIG. 18-B and Table 9 show the results of measuring the amount of Ca in the extracted lens. Ca by selenite treatment
The amount increased from 0.3 ± 0.1 to 1.0 ± 0.5 mmol / kg H ₂ O, but the increase was significantly suppressed in any of the DSF liposome, DSF suspension, and CMZ liposome instillation. FIG. 19 and Table 9 show the results of measuring the GSH amount in the extracted lens. The normal rats had 125.1 ± 10.1 and the non-treatment group had 99.0 ± 5.4 μmol / lens, but the drop of GSH was significantly suppressed in each instillation of each preparation.

[Table 9] Na / K ratio in lens, Ca amount, GSH amount Na / K ratio Ca amount GSH amount (mmol / kg H ₂ O) (μmol / lens) Control 0.11 ± 0.01 0.3 ± 0.1 125.1 ± 10.1 Selenious acid Control 0.16 ± 0.05 1.0 ± 0.5 99.0 ± 5.4 DSF liposome 0.13 ± 0.03 0.5 ± 0.2 111.0 ± 11.3 DSF suspension 0.16 ± 0.02 0.5 ± 0.1 113.0 ± 14.2 CMZ liposome 0.14 ± 0.02 0.5 ± 0.1 110.7 ± 8.3

[Brief description of drawings]

FIG. 1 is a line graph showing the release properties of DDC from DSF-containing liposomes at pH 8.0.

FIG. 2 is a corneal permeability experimental apparatus used.

FIG. 3 DSF-containing DPPC / DMPC / CPC (2
/ 8/1) is a line graph showing the permeability (pH 8.0) of the rabbit cornea of DDC released from the liposome.

FIG. 4 DSF-containing DPPC / DMPC / CPC (2
/ 8/1) is a line graph showing the permeability (pH 6.5) of the rabbit cornea of DDC released from the liposome.

FIG. 5 is a bar graph showing the results of GE and DDC recovery experiments on the content of reductive glutathione (GSH) in the mouse lens after diamide treatment.

FIG. 6 is a bar graph showing the effect on Na / Ca ratio in the lens isolated from the mouse with and without anticataract drug treatment.

FIG. 7 is a bar graph showing the effect on Ca content in the lens isolated from the mouse with and without anticataract drug treatment.

FIG. 8 is a graph showing the inhibitory activity of drugs on aldose reductase purified from rat lenses.

FIG. 9 shows a slit image and a transillumination image showing the effect of DSF administration on a cataract caused by X-ray irradiation.

FIG. 10 is a bar graph showing the effect of various drugs on the opacity of the anterior pole (A) and nucleus (B) of the lens of X-ray-irradiated rats.

FIG. 11 is a bar graph showing the effect of various drugs on the opacity of the posterior pole of the lens of X-ray-irradiated rats.

FIG. 12 is a bar graph showing the anti-cataract effect of various drugs on the Na / K ratio (A) and Ca amount (B) in the lens of X-ray-irradiated rats.

FIG. 13 is a bar graph showing the anti-cataract effect of various drugs on the amount of GSH in the lens of X-ray-irradiated rats.

FIG. 14 is an ocular photograph of a selenite-treated rat by instillation of DSF liposome, DSF-suspension and CMZ-liposome.

FIG. 15 is a lens photograph of a selenite-treated rat by instillation of DSF liposome, DSF-suspension and CMZ-liposome.

FIG. 16 shows slit images and transillumination images of selenite-induced cataract rats by administration of various drugs.

FIG. 17 is a bar graph showing the effect on opacity (slit image, A) and transmittance (transillumination image, B) in the lens of selenite-treated rats with various drugs.

FIG. 18 is a bar graph showing the anti-cataract effect on the Na / K ratio (A) and the Ca amount (B) in the lens of selenite-treated rats with various drugs.

FIG. 19 is a bar graph showing the anti-cataract effect on the amount of GSH in the lens of selenite-treated rats with various drugs.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A61K 31/41 // C07C 333/14 7106-4H C07D 257/04 H

Claims (21)

[Claims] 1. A pharmaceutical composition for the treatment of cataract, which comprises a radical scavenger selected from reducing thiol derivatives and their disulfide derivatives and sulfide derivatives. 2. The reducing thiol has the formula: [Wherein R ¹ , R ² and R ³ are the same or different,
The pharmaceutical composition according to claim 1, which is a linear or branched lower alkyl group which may be substituted with a hydroxy group, a lower alkyloxy group or a lower alkylcarbonyloxy group]. . 3. The reducing thiol derivative is a derivative of diethyldithiocarbamate, 1-methyl-1H-tetrazol-5-yl-thiol and 1- (2-hydroxyethyl) -1H-tetrazol-5-yl-thiol. The pharmaceutical composition according to claim 1, which is selected from the group consisting of: 4. The disulfide derivative has the formula: The pharmaceutical composition according to claim 1, which is represented by the formula: wherein R ¹ and R ² have the same meanings as described above. 5. The pharmaceutical composition according to claim 4, wherein the disulfide derivative is disulfiram. 6. The pharmaceutical composition according to claim 1, wherein the sulfide derivative is a cephem compound. 7. The cephem compound is cefamandole, cefoperazone, cefmenoxime hydrochloride, cefmetazole, cefotetan, latamoxef, cefbuperazone,
Which is selected from cef-pyramide and flomoxef,
The pharmaceutical composition according to claim 6. 8. The composition according to claim 7, wherein the cephem compound is cefmetazole. 9. A particulate carrier for carrying a therapeutic agent to be administered to the eye. 10. The particulate carrier according to claim 9, wherein the treatment agent to be administered to the eye is a cataract treatment agent. 11. The particulate carrier according to claim 10, wherein the agent for treating cataract is the pharmaceutical composition according to any one of claims 1 to 8. 12. A surface having a lipophilic / positively charged film,
The particulate carrier according to claim 9. 13. A carrier component and a lipophilic positive charge-loading substance are dissolved in an organic phase. When the cataract treating agent is liposoluble, the cataract treating agent is further dissolved in the organic phase, and the cataract treating agent is water-soluble. 13. The carrier according to claim 12, which has a lipophilic / positively charged film on the surface, which is obtained by dissolving a cataract treating agent in an aqueous phase to form fine particles. 14. The particulate carrier according to claim 13, wherein the lipophilic positively charged substance is about 1 to 30 mol% of the carrier constituent. 15. The particulate carrier according to claim 9, which is selected from emulsions, nanocapsules, albumin microspheres and liposomes. 16. The particulate carrier according to claim 9, which is a liposome. 17. The particulate carrier according to claim 16, wherein the liposome comprises a natural or semi-synthetic phospholipid. 18. The pharmaceutical composition according to claim 1,
A pharmaceutical preparation carried on a fine particle carrier having a fat-soluble, positively charged film on the surface. 19. A fine particle carrier having a fat-soluble / positively charged film on the surface, which is obtained by dissolving a carrier component, a fat-soluble positively charged substance and a cataract treating agent in an organic phase to form fine particles. The pharmaceutical preparation according to claim 18, which is 20. The lipophilic positive charge-loading substance is about 1 to 30 mol% of the carrier constituents.
9. The pharmaceutical preparation according to 9. 21. The pharmaceutical preparation according to claim 18, wherein the particulate carrier is a liposome.