JPH09301865A - Medicine composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicine composition comprising a globule, by including a phenylimidazaloyloxy-oxide derivative in a specific spherical membrane, capable of providing the derivative with storage stability and reaction specificity in vivo. SOLUTION: This medicine composition comprises a globule, preferably a liposome, obtained by including (B) a compound of formula I (R<1> to R<4> are each independently H or a 1-4C alkyl; R<5> is amino, a 1-4C alkyl-containing secondary or tertiary amino or a 1-4C alkyl-containing quaternary ammonium) and/or its pharmacologically permissible salt in (A) a phaspholipid-containing spherical membrane composed only of a saturated compound. The compound of formula I is preferably 2-4-(trimethylamino)phenyl-4,4,5,5- tetramethylimidazalo-1-yloxy-3-oxide of formula II. The phospholipid of the component A is preferably a hydrogenated lecithin and/or dimyristoyl phosphatidylcholine.

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 a drug, it has been desired to develop a preparation in which PTIOs have 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 inventors of the present invention have conducted extensive studies to solve the above problems, and as a result, in the conventional liposome preparation, in order to adjust the reactivity of PTIOs in vivo, the membrane elasticity was adjusted. Is required, and unsaturated phospholipids are used for this purpose.
Although the storage stability of the preparation could not be obtained because it was oxidized by Os, it was clarified that the membrane elasticity of the liposome is not an important factor for imparting in vivo reaction specificity to PTIOs. . Then, a specific PTIO was formed on a spherical membrane containing saturated phospholipids and no unsaturated compounds.
By encapsulating PTIOs as microspheres by encapsulating them, the PTIOs are given the specificity of in-vivo reaction and act effectively during administration, and the PTIOs inside the microspheres are stable when the formulation is stored. The inventors have found that they can be preserved and have completed the present invention.

That is, the present invention is a microsphere containing a spherical membrane containing a phospholipid and a compound of the general formula (1) and / or a physiologically acceptable salt thereof encapsulated in the spherical membrane. It is a small sphere characterized in that the spherical film is composed only of a saturated compound.

[0008]

Embedded image

(In the formula, 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 is an amino group and 1 to 4 carbon atoms. It 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.) Specific examples of the compound represented by the general formula (1) used in the present invention Specifically, 2- (4- represented by the following formula (2)
(Trimethylamino) phenyl) -4,4,5,5-tetramethyl-imidazolo-1-yloxy-3-oxide (hereinafter abbreviated as “trimethylamino PTIO”), represented by the following formula (3): -(4- (dimethylamino) phenyl) -4,4,5,5-tetramethyl-imidazolo-1-yloxy-3-oxide (hereinafter abbreviated as "dimethylamino PTIO") and the like, In the invention preferably trimethylamino PTI
O is used.

[0010]

Embedded image

[0011]

Embedded image

In the present invention, it is also possible to use a physiologically acceptable salt thereof as in the case of the compound represented by the general formula (1). Examples of such salts include mineral acid salts such as hydrochlorides, nitrates and phosphates, and organic acid salts such as citrates, oxalates and tartrates. 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.

In the present invention, such a compound represented by the above general formula (1) and / or a physiologically acceptable salt thereof (PTIOs) is contained in a spherical membrane containing a phospholipid and other compounds. It is included with optional components.

The phospholipid used for the spherical membrane 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”.

Examples of the saturated phospholipid used in the present invention include hydrogenated lecithin, dimyristoylphosphatidylcholine, dimyristoylphosphatidylinositol, dimyristoylphosphatidic acid and hydrogenated lysolecithin. In the present invention, the saturated phospholipid is used. Is preferably hydrogenated lecithin or dimyristoylphosphatidylcholine. As a more preferable saturated phospholipid, hydrogenated lecithin can be mentioned because it is inexpensive and easily available. In the present invention, one of these saturated phospholipids may be used alone, or two or more thereof may be used in combination.

Furthermore, the globular membrane of the present invention may contain any saturated compound other than the above-mentioned saturated phospholipids. Specific examples of such microspheres of the present invention include liposomes and niosomes having the above-mentioned features of the present invention. In the present invention, liposomes are preferably used.

The microspheres of the present invention are usually prepared by using an encapsulating component containing the compound represented by the general formula (1) and / or a physiologically acceptable salt thereof and a spherical membrane component containing a saturated phospholipid. It can be manufactured by the method of.

Further, the present invention provides a pharmaceutical composition containing the above-mentioned microsphere as an active ingredient. The microsphere of the present invention has a form in which a spherical membrane containing a phospholipid and composed only of a saturated compound encapsulates the above 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. Here, when the spherical membrane is composed of only a saturated compound, the spherical membrane does not contain an amount of the unsaturated compound that causes an oxidative reaction with the encapsulated PTIOs to substantially deactivate the medicinal effect. It means that.

When the microspheres of the present invention are administered as a pharmaceutical composition or the like, the PTIOs have a reaction specificity imparted by a spherical membrane, and therefore are transferred to the affected area in vivo without losing their reactivity. It is possible to exert various pharmacological actions by eliminating a specific substance, specifically nitric oxide, through the spherical membrane in the affected area in need thereof.

[0020]

Embodiments of the present invention will be described below. First, the PTIOs encapsulated by the spherical spherical membrane of the present invention will be described. (1) PTIOs used in the present invention The microspheres of the present invention include the compound represented by the general formula (1) and / or one or two physiologically acceptable salts 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.

The compound represented by the general formula (1) and / or the physiologically acceptable salt thereof used in the present invention is as described above. Such PTIOs are already known, The manufacturing method thereof is also known.

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

[0023]

[Chemical 6]

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

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

(2) Spherical membrane of microspheres of the present invention The spherical membrane of microspheres of the present invention contains saturated phospholipids and is composed only of saturated compounds. The saturated phospholipid contained in the spherical membrane of the present invention is as described above. The preferable content of the saturated phospholipid in the spherical membrane of the present invention is 10 to 10
0% by weight, more preferably 30 to 100% by weight,
50 to 100% by weight is more preferable.

The spherical membrane of the present invention may contain a saturated compound other than the above-mentioned saturated phospholipid as an optional component. Such optional components include polyhydric alcohols such as 1,3-butanediol and propylene glycol, and PO.
Examples thereof include E-cured castor oil and surfactants such as POE stearic acid ester. Components such as these polyhydric alcohols and surfactants have the advantage that the spherical film can be made flexible, but on the other hand, depending on the content, there is the disadvantage that the PTIOs that are included tend to flow out, so the content is It is necessary to make use of these advantages and set so as not to impair the effects of the invention. The content of polyhydric alcohol is 1 to 20% by weight.
Is preferred, 3 to 15% by weight is more preferred, and 5 to 10
The weight% is more preferable. Further, in the surfactant, 0.1 to 10% by weight is preferable, and 0.5 to 7%
% By weight is more preferable, and 1 to 5% by weight is even more preferable.

(3) Microsphere and Pharmaceutical Composition of the Present Invention In the microsphere of the present invention, the spherical membrane containing the saturated phospholipid and containing no unsaturated compound is a compound represented by the general formula (1), and And / or encapsulating a physiologically acceptable salt thereof.

When the compound represented by the general formula (1) and / or the physiologically acceptable salt thereof in the microspheres of the present invention is included in the spherical membrane, these PTIOs alone are included. Alternatively, it 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 PTIOs in the microspheres of the present invention is preferably 1 to 30% by weight, more preferably 5 to 20% by weight, even more preferably 7 to 17% by weight.
The microspheres of the present invention can be produced by treating a raw material component of a spherical membrane containing a saturated phospholipid, PTIOs and other optional components by a method such as a usual thin film dispersion method or reverse evaporation method. is there. 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. It is also possible to produce the microspheres of the present invention by a solution method, a solution-extrusion method or the like, and these methods are preferable production methods in the present invention. A more specific example of the manufacturing method will be described below.

Saturated phospholipids and other optional components for membrane formation and an organic solvent were sonicated with a sonicator, and PTIOs and other optional components were dissolved in a solvent such as phosphate buffered saline. Add solution 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 repeated about 4 to 5 times until the blue-purple color of the PTIO derivative disappears in the supernatant.

The pharmaceutical composition of the present invention contains the above-obtained 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 dosage, administration method, etc. of the pharmaceutical composition of the present invention are appropriately selected in consideration of the kind of disease, symptoms, age of patient, body weight 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.

Containing such PTIOs in a stable manner,
The above-mentioned pharmaceutical composition of the present invention containing the microspheres of the present invention capable of effectively acting is specifically used in a pharmaceutical composition for cardiovascular disease as a vascular protective agent and as a respiratory protective agent. It can be used as a pharmaceutical composition for organ diseases or as an anti-inflammatory agent.

[0035]

Embodiments of the present invention will be described below.

[0036]

[Example 1] 44 mg of hydrogenated lecithin and 3 m of ether
1 was weighed into a test tube, sonicated for 5 minutes with a sonicator, and trimethylamino PTI was added thereto at a concentration of 2 mM.
O-containing 10 mM phosphate buffered saline (pH 7.
4) (hereinafter abbreviated as “PBS”) (1 ml) was added, 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.

0.6 ml of PBS was added to the obtained microspheres, shaken well, and then centrifuged at 14000 G for 15 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.

Further, the microspheres of Comparative Example 1 were produced by the same method as described above except that lecithin was used instead of hydrogenated lecithin in the production of the above-mentioned microspheres. When the electron spin resonance (ESR) of the small spheres obtained in Example 1 and Comparative Example 1 was measured, an absorption spectrum of a nitroxide radical peculiar to PTIOs was detected in both small spheres. The line widths of these spectra were wide, and it was confirmed that the above-mentioned trimethylamino PTIO was encapsulated in the microspheres obtained in Example 1 and Comparative Example 1.

[0039]

Example 2 Microspheres were produced in exactly the same manner as in Example 1 except that dimyristoylphosphatidylcholine was used in place of hydrogenated lecithin in the production of the microspheres of Example 1 above. When the ESR measurement was performed on the obtained microspheres, the results were the same as in Example 1 above, and it was confirmed that trimethylamino PTIO was encapsulated in the microspheres.

[0040]

Example 3 Microspheres were produced in the same manner as in Example 1 except that dimyristoylphosphatidylinositol was used in place of hydrogenated lecithin in the production of the microspheres of Example 1 above. ESR of the obtained microspheres
When the measurement was carried out, the result was the same as in Example 1 above, and it was confirmed that trimethylamino PTIO was encapsulated in the microsphere.

[0041]

Example 4 Microspheres were produced in the same manner as in Example 1 except that dimyristoylphosphatidic acid was used in place of hydrogenated lecithin in the production of the microspheres of Example 1 above. When the ESR measurement was performed on the obtained microspheres, the results were the same as in Example 1 above, and it was confirmed that trimethylamino PTIO was encapsulated in the microspheres.

[0042]

Example 5 Microspheres were produced in exactly the same manner as in Example 1 except that hydrogenated lysolecithin was used instead of hydrogenated lecithin in the production of the microspheres of Example 1 above. When the ESR measurement was performed on the obtained microspheres, the results were the same as in Example 1 above, and it was confirmed that trimethylamino PTIO was encapsulated in the microspheres.

[0043]

[Example 6] 44 mg of hydrogenated lecithin and 4 mg of 1,3-butanediol were weighed into a test tube and dissolved in a solution heated at 80 ° C to a solution of trimethylamino PTIO at a concentration of 2 mM.
1 ml of PBS containing PBS heated to 80 ° C. was gradually added. This was applied to an extruder equipped with a 100 mm micron filter for extrusion treatment. The obtained dispersion liquid of small spheres is 14000G
After centrifuging for 15 minutes, the supernatant was discarded leaving the microspheres.

After 0.6 ml of PBS was added to the obtained microspheres and shaken well, the microspheres were pelletized by centrifugation at 14000 G for 15 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. When the ESR measurement was performed on the obtained microspheres, the results were the same as in Example 1 above, and it was confirmed that trimethylamino PTIO was encapsulated in the microspheres.

<Evaluation of Microspheres of the Present Invention> With respect to the microspheres encapsulating trimethylamino PTIO obtained in each of the above Examples, a storage stability test of PTIOs in a preparation, a nitric oxide scavenging and erasing activity test, and Drug efficacy test (PTI in vivo
The reaction specificity of O's was evaluated).

(1) Storage stability test The small spheres obtained in Examples 1 to 6 and Comparative Example 1 were treated at 4 ° C.
It was left to stand for 1 month under the temperature condition of. When ESR was measured for one month from the start of the test for these small spheres, all of the small spheres obtained in the examples showed ESR immediately after production in the ESR measurement result after one month. Almost the same peak as when measured was observed. On the other hand, in the comparative microspheres, a decrease in the absorption peak of the nitroxide radical was already observed 7 days after the production.

From these results, it was found that the PTIOs-containing microspheres of the present invention can stably store PTIOs for a long term.

(2) Nitric oxide trapping and scavenging activity test The PTIO-encapsulating microspheres of the present invention were tested for nitric oxide trapping and scavenging activity using the microspheres obtained in Example 1.

A) Measurement Using ESR Regarding the nitric oxide trapping / eliminating action of the small spheres obtained in Example 1, trimethylamino PTIO in the small spheres when nitric oxide was supplied using ESR. ESR
It was measured by quantifying the change in signal (absorption spectrum of nitroxide radical). That is, nitric oxide was added to the microspheres obtained in Example 1 and reacted in PBS, and ESR measurement was performed before and after the reaction to compare the signals. As a result, a reduction in the ESR signal of trimethylamino PTIO in the same amount as the added nitric oxide was observed, and it was found that nitric oxide was almost 100% captured and eliminated by the microspheres.

B) Extinction activity against endogenous nitric oxide released from vascular endothelial cells of rabbit vascular annulus. A ring-shaped specimen of rabbit aorta was used as an organ bath.
When stimulated with acetylcholine in h), a relaxation reaction of blood vessels is observed with the release of nitric oxide from vascular endothelial cells. When this test was conducted in the presence of the microspheres obtained in Example 1, no relaxation reaction of blood vessels was observed. This indicates that the PTIO-encapsulating microspheres of the present invention efficiently eliminate nitric oxide even in a biological system.

(3) Drug efficacy test The microspheres encapsulating the PTIOs of the present invention were subjected to a drug efficacy test using the microspheres obtained in Example 1, and the living body (tissue) was tested.
It was evaluated whether the PTIOs of the microspheres of the present invention had reaction specificity.

A) IL-8 production inhibitory effect The microspheres obtained in Example 1 are treated with trimethylamino PTIO.
The amount of 10% fetal bovine serum (F
Human bronchial epithelial cells were cultured in MEM supplemented with (BS). When the amount of IL-8, which is a proinflammatory cytokine, was measured under these conditions, it was suppressed in the presence of microspheres.

B) Effect on pneumonia A mouse was infected with influenza virus to prepare a pneumonia model, and the microspheres obtained in Example 1 were converted into the amount of trimethylamino PTIO and administered in an amount of 1 mg. The pathological tissues of the lungs were observed when microspheres were not administered to the pneumonia model mice prepared as described above. Compared with mice that did not receive microspheres, mice that received microspheres had less lymph infiltration and edema in the pathological tissues of the lung, and the survival rate was also significantly higher.

From these results, in the PTIO-encapsulating microspheres of the present invention, the PTIOs are encapsulated in the above-mentioned specific spherical membrane so that they have reaction specificity in a living body or in a living tissue. It was found that various medicinal effects can be sufficiently exerted.

[0055]

INDUSTRIAL APPLICABILITY The microspheres of the present invention contain P
Since TIOs are endowed with reaction specificity in a living body and the storage stability of PTIOs in the microspheres is good, the microspheres of the present invention are useful as a preparation of PTIOs. In addition, the pharmaceutical composition of the present invention can sufficiently exhibit the medicinal properties of PTIOs.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display area A61K 9/127 A61K 9/127 R // C07D 233/24 C07D 233/24

Claims (5)

[Claims] 1. A microsphere comprising a spherical membrane containing a phospholipid and a compound represented by the general formula (1) and / or a physiologically acceptable salt thereof encapsulated therein, comprising: A small sphere, wherein the spherical film is composed only of a saturated compound. 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 phospholipid is hydrogenated lecithin and / or dimyristoylphosphatidylcholine.
Or the small sphere described in 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.