JPH10265383A - Medicine composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicine composition, capable of giving reaction specificity in vivo by including microspheres that contain a 2-phenyl-imidazole-1-yloxy-3- oxide (PTIO) derivative. SOLUTION: This medicine composition essentially contains a compound shown by the formula (R<1> to R<4> are each H or a 1-4C alkyl; and R<5> is amino an amino having a 1-4C alkyl or quarternary ammonium having a 1-4C alkyl) or its salt, and at least one type of cholesterol or its analogue, which are included in a spherical film containing a saturated phospholipid, e.g. hydrogenated lecithin and phosphatidyl choline, in such a way to form a microsphere. The microsphere containing a PTIO derivative is give reaction specificity in vivo, and is useful as a preparation of PTIO, because of its good preservation stability in the preparation. The medicine composition can sufficiently exhibit the inherent efficacy of the PTIO.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a preparation containing a 2-phenyl-imidazolo-1-yloxy-3-oxide derivative as an active ingredient and a pharmaceutical composition containing the same.
More specifically, the present invention relates to a microsphere-shaped preparation capable of imparting the compound with storage stability in a preparation and reaction specificity in a living body, and a pharmaceutical composition containing the same.

[0002]

2. Description of the Related Art 2-Phenyl-4,4,5,5-tetramethyl-imidazolo-1-yloxy-3-oxide (PTIO) and 2- (4-carboxyphenyl) -4,
PTIs such as 4,5,5-tetramethyl-imidazolo-1-yloxy-3-oxide (carboxyPTIO)
O and its derivatives (hereinafter, such compounds having a 2-phenyl-imidazolo-1-yloxy-3-oxide skeleton are collectively referred to as “PTIOs”) have excellent nitric oxide erasing ability, It is speculated that it will work effectively against cardiovascular diseases and inflammation that are said to be involved. However, these PTIOs react with blood components as soon as they enter the blood due to their good reactivity and lose their ability to eliminate nitric oxide, deactivating them. The reality was that it was not well documented. Therefore, in order to use these PTIOs as medicines, there has been a demand for the development of a preparation in which PTIOs are given reaction specificity in vivo.

[0003] Under such circumstances, attempts have been made to adjust the reactivity in vivo by encapsulating the above-mentioned PTIOs in liposomes, whereby a formulation in which the PTIOs effectively act in vivo is attempted. Can be obtained. However, in these conventional liposome-encapsulated PTIO preparations, PTIO is gradually reduced due to the fact that the phospholipid, which is a component of the liposome, contains an unsaturated compound phospholipid in order to maintain the membrane elasticity of the liposome. Due to inactivation, storage stability as a formulation could not be obtained. That is, the conventional liposome-encapsulated PTIO preparations have been limited to use in preparation at the time of use.

[0004] Therefore, there is a demand for the development of a formulation of PTIOs that imparts reaction specificity in vivo to the PTIOs and that the PTIOs are stably stored without being deactivated in the formulation. Was.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made from the above-mentioned viewpoints, and provides PTIOs with a reaction specificity in a living body, and furthermore, PTIOs having good storage stability of PTIOs in pharmaceutical preparations. It is an object of the present invention to provide a pharmaceutical composition and a pharmaceutical composition.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, in the conventional liposome preparation, the membrane elasticity was adjusted to adjust the reactivity of PTIOs in vivo. Retention is required, and for this purpose unsaturated phospholipids are used,
Although storage stability of the preparation was not obtained due to oxidation by the liposome, it was revealed that the membrane elasticity of the liposome is not an important factor for imparting reaction specificity to PTIOs in vivo.

[0007] Under such circumstances, the present inventors have developed a specific PTI into a spherical membrane containing only a phospholipid of a saturated compound as a phospholipid instead of the phospholipid of an unsaturated compound.
If PTIOs are formulated as microspheres by encapsulating Os, reaction specificity in vivo is imparted to the PTIOs to effectively act upon administration, and PTIOs inside the microspheres during storage of the formulation are It has been found that the cholesterol and its analogs are contained in a spherical membrane containing the phospholipid of the above-mentioned saturated compound, and PTIO is similarly stored.
It has been found that the preparation of such a preparation leads to more excellent storage stability of the preparation, thereby completing the present invention.

[0008] That is, the present invention provides a small sphere comprising a phospholipid-containing spherical membrane and a compound represented by the general formula (1) and / or a physiologically acceptable salt thereof encapsulated therein. A small sphere characterized in that the phospholipid is a saturated phospholipid and the spherical membrane contains one or more selected from cholesterol and its analogs.

[0009]

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(Wherein R ¹ , R ² , R ³ , and R ⁴ each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms; R ⁵ represents an amino group; Represents a secondary or tertiary amino group having an alkyl group, or a quaternary ammonium group having an alkyl group having 1 to 4 carbon atoms.) It is represented by the general formula (1) used in the small sphere of the present invention. As the compound, specifically, 2- (4- (trimethylamino) phenyl) -4,4 represented by the following formula (2)
5,5-tetramethyl-imidazolo-1-yloxy-3
-Oxides.

[0011]

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The saturated phospholipid used in the microspheres of the present invention includes, specifically, hydrogenated lecithin,
Phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid and the like can be mentioned.

Specific examples of such microspheres of the present invention include liposomes having the above-mentioned features of the present invention. Further, the present invention provides a pharmaceutical composition containing the above-mentioned microspheres of the present invention as an active ingredient.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below. First,
The PTIOs encapsulated by the globular spherical membrane of the present invention will be described.

(1) PTIOs Used in the Present Invention The microspheres of the present invention contain one or two of the compound represented by the above general formula (1) and / or a physiologically acceptable salt thereof.
More than one species is used as PTIOs. This is the PTI
Among the Os, the compound represented by the general formula (1) and / or
Or in PTIOs other than physiologically acceptable salts thereof,
This is because it is difficult for the microspheres of the present invention to be incorporated into a phospholipid-containing spherical membrane.

As the compound represented by the above general formula (1) used in the present invention, specifically, 2- (4- (trimethylamino) phenyl) -4, represented by the following formula (2)
4,5,5-tetramethyl-imidazolo-1-yloxy-3-oxide (hereinafter referred to as "trimethylamino PTI
O "), and 2- (4-
(Dimethylamino) phenyl) -4,4,5,5-tetramethyl-imidazolo-1-yloxy-3-oxide (hereinafter abbreviated as “dimethylamino PTIO”) and the like, but in the present invention, preferred is Trimethylamino PTIO is used.

[0017]

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[0018]

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The physiologically acceptable salt of the compound represented by the above general formula (1) used in the microspheres of the present invention, specifically, the compound represented by the above general formula (1) Mineral salts such as hydrochloride, nitrate, phosphate, citrate,
Organic acid salts such as oxalate and tartrate are exemplified. Among these salts, a hydrochloride is preferably used in the present invention. Further, among the hydrochlorides, those in which R ^{5 in the} above general formula (1) is a quaternary ammonium chloride are more preferably used.

The PTIOs used in the present invention, that is, the compounds represented by the above general formula (1) and / or their physiologically acceptable salts are all known compounds, and their production methods are It is known.

For example, in order to produce the compound represented by the above general formula (1), a dihydroxylamine derivative is reacted with a para-substituted benzaldehyde as shown in the following reaction formula, and the resulting compound is converted to lead dioxide or the like. Oxidation may be performed using the metal oxide as a catalyst.

[0022]

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(However, R ¹ , R ² , R ³ , R ⁴ , and R ⁵ have the same meanings as those in the general formula (1).) The physiology of the compound represented by the above general formula (1) For example, a salt which is acceptable is a compound represented by the general formula (1) and an acid such as a mineral acid such as hydrochloric acid, nitric acid, phosphoric acid and an organic acid such as citric acid, oxalic acid and tartaric acid. It is easily obtained by mixing in a polar or non-polar solvent.

The thus-obtained compound represented by the general formula (1) or a physiologically acceptable salt thereof can be easily purified by a conventional method such as recrystallization or column chromatography.

Next, in the small sphere of the present invention, the PTI
The spherical membrane containing Os will be described.

(2) Spherical membrane of small sphere of the present invention The spherical membrane of small sphere of the present invention contains a phospholipid and one or more selected from cholesterol and analogs thereof. Is composed of only a saturated compound phospholipid.

The phospholipid used for the spherical membrane of the microsphere of the present invention is a saturated compound as described above. Here, a saturated compound refers to a compound that does not contain an unsaturated carbon-carbon multiple bond in the compound, and an unsaturated compound refers to a compound that has an unsaturated carbon-carbon multiple bond in the compound, Hereinafter, in the present specification, a phospholipid classified as a saturated compound is referred to as “saturated phospholipid”, and a phospholipid classified as an unsaturated compound is referred to as “unsaturated phospholipid”.

The saturated phospholipid contained in the spherical membrane constituting the microsphere of the present invention is not particularly limited as long as it is a phospholipid classified as a saturated compound. Specifically, hydrogenated lecithin, dimyristoyl phosphat Fatidylcholine,
Phosphatidylcholine such as distearoyl phosphatidylcholine and dipalmitoylphosphatidylcholine, and similar phosphatidylinositol, phosphatidylethanolamine and phosphatidylglycerol such as dimyristoyl, distearoyl, and dipalmitoyl. Phosphatidic acid, and their lyso forms. These saturated phospholipids can be produced by a usual method, and there are also commercially available products. Therefore, these commercially available products can be used in the present invention.

In the present invention, among these saturated phospholipids, hydrogenated lecithin, distearoylphosphatidylcholine, distearoylphosphatidylglycerol and the like which are inexpensive and easily available are preferably used. One type of these saturated phospholipids may be used alone or in combination of two or more types in the spherical membrane constituting the microspheres of the present invention. The content of the saturated phospholipid in the spherical membrane constituting the microspheres of the present invention is preferably 1 to 90% by weight, more preferably 10 to 70% by weight, and still more preferably 20 to 60% by weight.

The spherical membrane constituting the microsphere of the present invention contains one or more selected from cholesterol and cholesterol analogs in addition to the above-mentioned saturated phospholipid. Specific examples of the cholesterol analog used in the present invention preferably include hydroxycholesterol. These cholesterol and its analogs can be obtained from various base organisms by a usual method, but it goes without saying that cholesterol and its analogs used in the present invention are not limited to any source. Since these compounds are commercially available, they can be used in the present invention.

Here, unsaturated compounds such as cholesterol and analogs thereof are liable to be oxidized by PTIOs. Therefore, the coexistence of these unsaturated compounds with PTIOs impairs the stability of PTIOs. It has been estimated by the present inventors that cholesterol and its analogs do not impair the stability of PTIOs even if they are unsaturated compounds, but rather improve the stability, as shown in the Examples below. Has been.

In the present invention, only one kind of cholesterol and cholesterol analogs (hereinafter, also referred to as “cholesterols”) may be used, or two or more of these may be used in combination.
The preferable content of one or more selected from cholesterol and its analogs in the spherical membrane constituting the microspheres of the present invention is 1 to 50% by weight, more preferably 5 to 40% by weight, More preferably, it is 10 to 20% by weight.

The spherical membrane constituting the microspheres of the present invention contains various optional components other than the above-mentioned saturated phospholipids and cholesterol for the purpose of improving the membrane properties and the like within a range that does not impair the effects of the present invention. It is possible. Examples of such optional components include polyhydric alcohols such as 1,3-butanediol and propylene glycol, and surfactants such as POE hardened castor oil and POE stearic acid ester. These components, such as polyhydric alcohols and surfactants, have the advantage of being able to impart flexibility to the spherical membrane, but have the disadvantage that, depending on the content, they have the disadvantage that the contained PTIOs are more likely to flow out. The amount needs to be set so as to take advantage of these advantages and not to impair the effects of the present invention. Such a content is preferably 1 to 20% by weight, more preferably 3 to 15% by weight, and more preferably 5 to 1% by weight in a polyhydric alcohol.
0% by weight can be more preferably exemplified. In the surfactant, 0.1 to 10% by weight is preferable, and 0.5 to 10% by weight.
7% by weight is more preferred, and 1-5% by weight is even more preferred.

(3) Microspheres and Pharmaceutical Composition of the Present Invention The microspheres of the present invention contain a phospholipid-containing spherical membrane, a compound represented by the general formula (1) and / or a compound thereof. And a physiologically acceptable salt, comprising:
The phospholipid is a saturated phospholipid, and the spherical membrane contains one or more selected from cholesterol and its analogs.

When the compound represented by the general formula (1) and / or a physiologically acceptable salt thereof in the microspheres of the present invention are encapsulated in the above-mentioned spherical membrane, the PTIOs alone are encapsulated. Or may be included as a suitable solution. Further, it may be included together with other optional components. P
When the TIOs are encapsulated in the spherical membrane as a solution, water is preferably used as a solvent, and more preferably, physiological saline, phosphate buffer, or phosphate buffered saline prepared by adding various components to water. Water or the like is used.

The content of the above-mentioned PTIOs in the small spheres of the present invention is preferably 1 to 30% by weight, more preferably 5 to 20% by weight, and still more preferably 7 to 17% by weight.
The microspheres of the present invention comprise a raw material component of a spherical membrane containing cholesterols and a saturated phospholipid as a phospholipid, PTI
It can be produced by treating Os and other optional components by a method such as a normal thin film dispersion method or a reverse evaporation method. However, since the phospholipids used in the present invention are all saturated phospholipids and do not contain unsaturated phospholipids, the solubility in the solvent used in these methods is low, so that the ultrasonic waves before It is preferable to carry out a dispersion process using the method described above. Further, it is also possible to produce the small spheres of the present invention by a solution method, a solution-extrusion method, a thin film dispersion-freeze-thaw-extrusion method, etc., and these methods are preferred production methods in the present invention. is there. Among these production methods, it is particularly preferable to produce the small spheres of the present invention by a thin film dispersion-freeze-thaw-extrusion method. A more specific example of the manufacturing method will be described below.

The saturated phospholipids, cholesterols, and other optional components for forming a film and an organic solvent are sonicated with a sonicator, and PTIOs and other optional components included therein are mixed with a solvent such as a phosphate buffered saline. And sonicate. The obtained dispersion is subjected to an evaporator to slowly remove the organic solvent under reduced pressure to obtain small spheres. After adding phosphate-buffered saline to the mixture and shaking well, centrifugation is performed to pellet the small spheres. Discard the supernatant and wash the pelleted pellet well. This shaking-centrifugation-washing operation is performed for about 4 to 4 until the blue-violet color of the PTIO derivative disappears from the supernatant.
Pelletized small spheres are obtained by repeating the process five times.

Alternatively, a saturated phospholipid, cholesterol and other optional components for forming a film are dissolved in an organic solvent and then dried to form a thin film. A solution dissolved in a solvent such as acid-buffered saline is added, sonicated and freeze-thawed. This is extruded to obtain a uniform particle size, and then centrifuged to take out the obtained sediment (small sphere) to obtain a small sphere.

Specific examples of preferred forms of the thus obtained microspheres of the present invention include liposomes and niosomes. In the present invention, liposomes are more preferable. The preferable average particle diameter of the small spheres of the present invention is, for example, about 10 to 500 nm. Further, the average particle size is more preferably about 10 to 300 nm, and still more preferably about 10 to 200 nm.

The microspheres of the present invention comprise cholesterols,
A spherical membrane containing a saturated phospholipid as a phospholipid has a form containing the above-mentioned PTIOs. Thus, when the microspheres are stored as a preparation, the highly reactive PTIOs are protected by the spherical membrane, so that there is no inactivation due to the reaction with various substances, and the reactive PTIOs react with the spherical membrane components. It is stored stably without being deactivated.

The pharmaceutical composition of the present invention contains the microspheres of the present invention thus obtained. The dosage form of the pharmaceutical composition of the present invention is not particularly limited. For example, a commonly used dosage form such as an injection, a powder, a granule, a tablet, a capsule, a liquid, an external preparation, or the like can be used as the pharmaceutical composition of the present invention. Can be mentioned as a dosage form. When formulating, the above-mentioned PTIO
Excipients, binders, disintegrants, lubricants, flavoring agents, bulking agents, coatings, sugar coatings, emulsifying / solubilizing / dispersing agents, stabilizers, pH adjusters, Any components commonly used in pharmaceuticals such as isotonic agents and external preparation bases can be used in any amount, and the pharmaceutical composition of the present invention can be prepared by formulating these components and the above-mentioned PTIOs according to a conventional method. You can get things.

The dose, administration method and the like of the pharmaceutical composition of the present invention are appropriately selected in consideration of the type and symptom of the disease, the age and weight of the patient, and the like. Examples of the method for administering the injection include intravenous administration, intraarterial administration, intraportal administration, intraperitoneal administration, intramuscular administration, and subcutaneous administration.

Such PTIOs are stably contained,
The pharmaceutical composition of the present invention containing the microspheres of the present invention, which can be effectively acted on, stores the PTIOs stably at the time of storage, and at the time of administration, the PTIOs have reactivity in the living body up to the affected site. It is transported without losing it, and it is possible to exert various pharmacological actions by erasing a specific substance, specifically, nitric oxide, through a spherical membrane in an affected area that requires it. The pharmaceutical composition of the present invention is specifically, as a vascular protective agent, a pharmaceutical composition for cardiovascular diseases such as myocardial infarction, a respiratory protective agent for a pharmaceutical composition for respiratory diseases such as pneumonia, or It can be used as an anti-inflammatory agent, a therapeutic agent for atopic dermatitis, and the like.

[0044]

Embodiments of the present invention will be described below.

[0045]

Example 1 33 mg of hydrogenated lecithin, 11 mg of cholesterol and 3 ml of ether were weighed into a test tube, sonicated for 5 minutes with a sonicator, and then added to a 10 mM phosphate buffered saline solution containing trimethylamino PTIO at a concentration of 2 mM ( pH 7.4, hereinafter "PBS"
Was omitted, and sonication was further applied for 15 minutes. This was subjected to an evaporator, and ether was slowly distilled off under reduced pressure to obtain small spheres.

After adding 0.6 ml of PBS to the obtained microspheres and shaking well, the microspheres were pelleted by centrifugation at 15,000 G for 10 minutes. The supernatant of the centrifugation was discarded, and the pelleted pellet was washed with PBS. further,
The above-mentioned operations of shaking, centrifugation and washing were repeated 4 to 5 times until the blue-purple PTIO disappeared from the supernatant of the centrifugation. The average particle size of the pelletized small spheres thus obtained was 300 nm.

Also, except that hydrogenated lecithin was used instead of cholesterol in the production of the above-mentioned microspheres,
The microspheres of Comparative Example 1 were produced in exactly the same manner as described above.
Furthermore, the microspheres of Comparative Example 2 were manufactured in exactly the same manner as described above, except that lecithin was used instead of hydrogenated lecithin in the production of the above microspheres.

Electron spin resonance (ESR) measurements were performed on the small spheres obtained in Example 1 and Comparative Examples 1 and 2. Absorption spectra of nitroxide radicals specific to PTIOs were obtained for both small spheres. Was detected. These spectra have a broad line width, and it was confirmed that the trimethylamino PTIO was included in the microspheres obtained in Example 1 and Comparative Examples 1 and 2.

[0049]

Example 2 Dimyristoyl phosphatidylcholine 2
4mg and cholesterol 20mg, chloroform 30m
and dried to form a thin film, containing 10 mM PB containing trimethylamino PTIO at a concentration of 2 mM.
1 ml of S (pH 7.4) was added, sonicated and freeze-thawed. This was subjected to an extruder (filter size: 200 nm, 5 times), and the particle diameter was adjusted.
Centrifugation was performed three times at 00G for 30 minutes. The centrifuge (small sphere) obtained by this centrifugation is taken out,
The suspension was re-dispersed in 1 l of PBS to obtain a dispersion of small spheres.

Comparative Example 3 was prepared in exactly the same manner as described above, except that dimyristoylphosphatidylcholine was used in place of cholesterol in the production of the above microspheres.
Was prepared.

When ESR measurement was performed on the small spheres obtained in Example 2 and Comparative Example 3 in the state of a dispersion, the results were the same as in Example 1 above. Trimethylamino PTIO was contained in these small spheres. It was confirmed that it was included.

[0052]

Example 3 Distearoylphosphatidylglycerol (20 mg), distearoylphosphatidylcholine (19 mg) and cholesterol (5 mg) were added to chloroform 3
Dissolve it in 0 ml and dry it to make a thin film.
10 mM containing trimethylamino PTIO at a concentration of
1 ml of PBS (pH 7.4) was added, sonicated and freeze-thawed. After applying this to an extruder (filter size; 100 nm, 5 times) and adjusting the particle size,
Centrifugation was performed three times at 0000 G for 30 minutes. The centrifuged sediment (small spheres) was taken out and redispersed in 1 ml of PBS to obtain a small sphere dispersion.

A dispersion of the microspheres of Comparative Example 4 was produced in the same manner as described above except that distearoylphosphatidylglycerol was used in place of cholesterol in the production of the microspheres of Example 3 above. .

When the ESR measurement was carried out on the small spheres obtained in Example 3 and Comparative Example 4 in the state of a dispersion, the results were the same as in Example 1 above, and trimethylamino PTIO was contained in these small spheres. It was confirmed that it was included.

[0055]

Example 4 A thin film was prepared by dissolving 18 mg of dipalmitoyl phosphatidic acid, 15 mg of distearoyl phosphatidylethanolamine, and 11 mg of cholesterol in 30 ml of chloroform and drying to dryness. One ml of 10 mM PBS (pH 7.4) was added, sonicated and freeze-thawed. This was subjected to an extruder (filter size; 200 nm, 5 times) to adjust the particle diameter, and then centrifuged at 150,000 G for 30 minutes three times. The centrifuged sediment (small spheres) was taken out and redispersed in 1 ml of PBS to obtain a small sphere dispersion.

A dispersion of the microspheres of Comparative Example 5 was manufactured in the same manner as described above, except that dipalmitoyl phosphatidic acid was used instead of cholesterol in the manufacture of the microspheres.

When the ESR measurement was performed on the small spheres obtained in Example 4 and Comparative Example 5 in the state of a dispersion, the results were the same as in Example 1 above. Trimethylamino PTIO was contained in these small spheres. It was confirmed that it was included.

[0058]

Example 5 Distearoyl phosphatidic acid 18m
g, dimyristoyl phosphatidylinositol 15
mg, 11 mg of cholesterol and 3 ml of ether were weighed into a test tube, sonicated with a sonicator for 5 minutes, and 1 ml of 10 mM PBS (pH 7.4) containing trimethylamino PTIO at a concentration of 2 mM was added thereto.
Sonication was applied for an additional 15 minutes. This was subjected to an evaporator, and ether was slowly distilled off under reduced pressure to obtain small spheres.

After adding 0.6 ml of PBS to the obtained microspheres and shaking well, the microspheres were centrifuged at 15,000 G for 10 minutes to pellet the microspheres. The supernatant of the centrifugation was discarded, and the pelleted pellet was washed with PBS. further,
The above-mentioned operations of shaking, centrifugation and washing were repeated 4 to 5 times until the blue-purple PTIO disappeared from the supernatant of the centrifugation. The average particle size of the pelletized small spheres thus obtained was 100 nm.

The microspheres of Comparative Example 6 were produced in the same manner as described above, except that distearoylphosphatidic acid was used instead of cholesterol in the production of the above microspheres.

When the ESR measurement was performed on the small spheres obtained in Example 5 and Comparative Example 6, the results were the same as in Example 1 above, and it was confirmed that trimethylamino PTIO was included in these small spheres. Was confirmed.

[0062]

Example 6 33 mg of hydrogenated lecithin, 11 mg of hydroxycholesterol and 1,3-butanediol 4
mg in a test tube and heated and dissolved at 80 ° C.
Separately, it was heated to 80 ° C. and contained 10 mM PBS containing trimethylamino PTIO at a concentration of 2 mM (pH
After 1 ml of 7.4) was gradually added, the resulting solution was diluted with 1 ml.
Extrusion treatment was performed with an extruder equipped with a 00 nm filter. This is 15000
The mixture was centrifuged at 0 G for 10 minutes, and the obtained centrifuge (spheroid) was taken out.

After adding 0.6 ml of PBS to the obtained microspheres and shaking well, the microspheres were centrifuged at 14000 G for 10 minutes to pellet the microspheres. The supernatant of the centrifugation was discarded, and the pelleted pellet was washed with PBS. further,
The above-mentioned operations of shaking, centrifugation and washing were repeated 4 to 5 times until the blue-purple PTIO disappeared from the supernatant of the centrifugation.

The microspheres of Comparative Example 7 were manufactured in exactly the same manner as described above except that hydrogenated lecithin was used instead of hydroxycholesterol in the manufacture of the above microspheres.

When the ESR measurement was carried out on the small spheres obtained in Example 6 and Comparative Example 7, the results were the same as in Example 1 above, and the fact that trimethylamino PTIO was included in these small spheres Was confirmed.

<Evaluation of the spherules of the present invention> The spheroids or the spheroid dispersions containing trimethylaminoPTIO obtained in each of the above Examples were tested for the storage stability and the efficacy of the PTIOs in the preparations. Evaluation of reaction specificity of PTIOs in the body).

(1) Storage stability test The microspheres or microsphere dispersions obtained in Examples 1 to 6 and Comparative Examples 1 to 7 were allowed to stand at room temperature for 4 months. When the ESR of these small spheres or the small sphere dispersion was measured over a period of 4 months from the start of the test, the small spheres obtained in the examples and the small sphere dispersion were all ESR after 4 months. In the measurement results, almost the same peak was observed as when the ESR was measured immediately after production. On the other hand, in the small spheres and small sphere dispersions obtained in Comparative Example 1 and Comparative Examples 3 to 7, only weak peaks were observed in the ESR measurement results after 4 months. Furthermore, in the small spheres of Comparative Example 2, the absorption peak of the nitroxide radical could not be observed already 7 days after the production.

From these results, it was found that PTIOs can be stably stored for a long period of time in the small spheres containing PTIOs of the present invention.

(2) Efficacy test The efficacy test of the small spheres containing the PTIOs of the present invention was carried out using the small spheres obtained in Example 1 above, and the small spheres of the present invention were tested in a test tube and in a living body (tissue). It was evaluated whether the spherical PTIOs had reaction specificity or stability.

A) Nitrogen monoxide scavenging activity in a test tube The nitric oxide scavenging activity of the PTIO-encapsulated microspheres of the present invention was measured by adding nitric oxide to the trimethylamino PTIO-encapsulated microspheres obtained in Example 1 above in a test tube. Was evaluated by a method of analyzing the ESR measurement result when directly reacted. This is because when PTIOs react with nitric oxide at a molar ratio of 1: 1, the reaction product derived from PTIOs, 2-phenyl-4,4,5,5-tetramethylimidazolo-1-
Oxyl (PTI) is generated and the signal of PTIO obtained by ESR measurement and the signal of PTI, for example, the signal of sodium salt of carboxy PTI shown in FIG. 1 a) and the sodium salt of carboxy PTI shown in FIG. 1 b) This is an evaluation method that makes use of the fact that the signals are completely different.

Specifically, the trimethylamino PTIO-encapsulated microspheres obtained in Example 1 were diluted with PBS and placed in each test tube so that the concentration of trimethylamino PTIO became 100 μM. The nitric oxide releasing reagent propylamine NONOate was added in various amounts so that the added amount of nitric oxide became 0 to 40 μM. After the reaction was sufficiently performed, ESR measurement was performed for each test tube (for each addition amount), and the results were analyzed to calculate the amount of trimethylamino PTI produced at each addition amount of nitric oxide. The erasure amount (μM) was determined.

For comparison, the same test was conducted except that the trimethylamino PTIO-encapsulated microspheres obtained in Example 1 in the above test were replaced with PTIO. The results are shown in FIG.

From the above results, it can be seen that the trimethylamino PTIO-encapsulated microspheres obtained in Example 1 were more in vitro than the nitric oxide releasing reagent propylamine NONOate as compared with the conventional PTIO without any protection. It can be seen that the generated nitric oxide is quantitatively and efficiently eliminated, and that it is effectively reacting with nitric oxide.

B) Eradication activity of nitric oxide produced from rabbit aortic endothelial cells In general, nitric oxide produced from vascular endothelial cells functions as an endogenous vasorelaxant that relaxes smooth muscle in vascular tissue. ing. Thus, the rabbit aorta was stimulated with acetylcholine, and the inhibitory effect on the relaxation reaction by nitric oxide released from the endothelium was examined. The trimethylamino PTIO-encapsulating microspheres obtained in Example 1, PTIO, and the nitric oxide synthesis inhibitor Nω Comparative tests were performed using -nitro-L-arginine (L-NNA).

All three groups of nine rabbit aortas were stimulated with acetylcholine. Thereafter, the rabbit aorta of each group was isolated from the trimethylaminoP obtained in Example 1.
TIO-encapsulated microspheres, PTIO, L-NNA
9 at each concentration of 0 μM, 100 μM and 300 μM
Each sample was treated and the degree of the relaxation reaction was measured. The above-mentioned concentration of the specimen containing trimethylamino PTIO-containing microspheres obtained in Example 1 is the concentration as trimethylamino PTIO. As a control, the degree of relaxation of three rabbit aortas stimulated with acetylcholine and untreated was measured as described above. Based on the average of the rabbit rabbit aortic relaxation response of the control, the above-mentioned rabbit rabbit aortic relaxation inhibition rate was calculated. The average and standard deviation of the relaxation reaction inhibition rate were determined for each test group. The results are shown in FIG.

From these results, it can be seen that the nitric oxide synthesis inhibitor L
-Compared to NNA and conventional PTIO without any protection, trimethylamino PTIO-encapsulated microspheres obtained in Example 1 were found to strongly suppress the vasorelaxation reaction in rabbit aorta. Showed strong nitric oxide scavenging activity.

C) Elimination reaction of nitric oxide generated from macrophages Macrophage cells were transformed into interferon γ (IFN-
γ) and bacterial lipopolysaccharide (LP)
When stimulated by S), inducible nitric oxide synthase is expressed, and a large amount of nitric oxide is produced from macrophages. Thus, the trimethylamino PT obtained in Example 1 above was added to the induction system of macrophages which were stimulated by IFN-γ and LPS and expressed nitric oxide synthase.
After addition of IO-encapsulated microspheres, the same ESR as in a) above
The measurement was performed, and the chart was analyzed to evaluate the nitric oxide eliminating action of the microspheres of the present invention. FIG. 4 shows the ESR measurement results.

From the results, it was found that the trimethylamino PTIO-encapsulated microspheres obtained in Example 1 were
By capturing nitric oxide in macrophage cells stimulated by PS, trimethylamino-P
It was found that TIO was converted to trimethylamino PTI. Therefore, the small spheres containing the PTIOs of the present invention are:
It is considered that nitric oxide released from cells can be efficiently eliminated.

D) Stability in vivo of PTIO-encapsulated microspheres In order to examine the in vivo stability of the microspheres of the present invention, the trimethylamino-PTIO encapsulation obtained in Example 1 above was incorporated into blood collected from rats. The microspheres and PTIO were separately added, and the change in the remaining amount of added PTIOs in each added blood was measured over time by ESR. FIG. 5 shows the results. The vertical axis in FIG. 5 shows the residual amount of PTIOs in the blood as a percentage of the initial concentration.

From these results, it can be seen that when conventional PTIO, which is not protected at all, is added to blood, the PTIO is rapidly eliminated by the reducing substances in the blood. When PTIO-encapsulated microspheres were added, it was found that the ESR signal indicating PTIOs was very stable and hardly changed even after 24 hours.

Further, the trimethylamino-PTIO-encapsulated microspheres obtained in Example 1 and PTIO were intravenously administered to each of two rats, and the PTI in blood was subsequently administered.
The change in the residual amount of Os was measured over time by ESR,
The in vivo stability was evaluated. FIG. 6 shows the results. The vertical axis in FIG. 6 represents the residual amount of PTIOs in the blood as a percentage of the initial concentration.

From the above results, it was found that the trimethylamino PTIO obtained in Example 1 was also compared with the unprotected conventional PTIO in the body of a rat as well as in the blood collected in the test tube. It was found that trimethylamino PTIO of the encapsulated microspheres was retained for a long time.

From the above results, in the small spheres encapsulating PTIOs of the present invention, PTIOs have both reaction specificity and stability in a living body or a living tissue by being encapsulated in the specific spherical membrane. Therefore, it was found that various medicinal effects can be sufficiently exhibited.

[0084]

EFFECTS OF THE INVENTION The small spheres of the present invention are provided with PTIOs contained therein with a reaction specificity in vivo, and furthermore, have good storage stability of PTIOs in the small spheres.
It is useful as a formulation for IOs. In addition, the pharmaceutical composition of the present invention can sufficiently exhibit the medicinal properties of PTIOs.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a view showing the difference between ESR measurement signals of PTIOs and PTIs. a) shows an ESR measurement signal of a Na salt of carboxyPTIO which is a kind of PTIO, and b) shows an ESR measurement signal of a Na salt of carboxy PTI which is a kind of PTI.

FIG. 2 shows trimethylamino PTI obtained in Example 1.
It is a figure which shows the relationship between the amount of nitric oxide addition and the amount of nitric oxide elimination when nitric oxide is made to directly react with each of O-containing microspheres and PTIO.

FIG. 3 shows the percentage of inhibition of the relaxation response to rabbit aorta stimulated by acetylcholine, based on the trimethylamino PTIO-encapsulated microspheres obtained in Example 1, PTIO and L-
It is the figure compared with NNA.

FIG. 4 is a view showing ESR measurement results when the trimethylamino PTIO-encapsulated microspheres obtained in Example 1 were added to macrophage cells stimulated with IFN-γ and LPS.

FIG. 5: Trimethylamino PTI obtained in Example 1
It is a figure which shows the time-dependent change of the residual amount of PTIOs when O inclusion microsphere and PTIO are added to rat blood, respectively.

FIG. 6: Trimethylamino PTI obtained in Example 1
It is a figure which shows the time-dependent change of the amount of PTIOs remaining in the blood when O-encapsulated microspheres and PTIO are each administered intravenously to rats.

Claims (5)

[Claims] 1. A small sphere comprising a phospholipid-containing spherical membrane and a compound represented by the general formula (1) and / or a physiologically acceptable salt thereof encapsulated therein. The phospholipid is a saturated phospholipid, and the spherical membrane is one or two selected from cholesterol and analogs thereof.
A small sphere characterized by containing at least one species. Embedded image (Wherein, R ¹ , R ² , R ³ , and R ⁴ each independently represent a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and R ⁵ represents an amino group or an alkyl group having 1 to 4 carbon atoms. Grade 2 or 3
Represents a quaternary ammonium group having a primary amino group or an alkyl group having 1 to 4 carbon atoms. ) 2. The compound represented by the general formula (1) is a compound represented by the following formula (2): 2- (4- (trimethylamino) phenyl) -4,4,5,5-tetramethyl-imidazolo The small sphere according to claim 1, which is -1-yloxy-3-oxide. Embedded image 3. The method of claim 1, wherein the phospholipid is selected from hydrogenated lecithin, phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid. Or the small sphere according to 2. 4. The microsphere according to claim 1, which is a liposome. 5. A pharmaceutical composition containing the microspheres according to any one of claims 1 to 4.