JPS601124A - Coated ribosome drug containing ubidecarenone - Google Patents

Abstract

PURPOSE:To increase the rate of dissipation of ubidecarenone useful as a cardiac function improving agent from blood and the rate of transfer of the compound from blood to the organ, by coating a ubidecarenone-containing ribosome with a mixed ester of a polysaccharide fatty acid and inorganic acid. CONSTITUTION:A coated ribosome containing ubidecarenone is prepared by coating a ubidecarenone-containing ribosome with a mixed ester of a polysaccharide fatty acid and inorganic acid. The elemental substance constituting the ribosome membrane is mainly phospholipids (e.g. phosphatidylcholine, phosphatidylserine, etc.) and sterols. Sterol is preferably cholesterol. The necessary amounts of phospholipid and sterol is >=10mol and >=1mol per 1mol of ubidecarenone, respectively. The mixed ester of polysaccharide fatty acid and inorganic acid is preferably the ester of a polysaccharide such as amylopectin, pllulan, etc. with palmitic acid and phosphoric acid or sulfuric acid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a ubidecarenone-containing liposome coated with a ubidecarenone-containing liposome and a polysaccharide-layered fatty acid/inorganic acid mixed ester that coats the membrane surface of the liposome. Regarding the body.

Ubidecarenone, also known as coenzyme QIO, has recently come into widespread clinical use as a drug effective for improving cardiac function ψ.

However, there is still room for improvement in the rate of transfer from blood to target organs when this substance is administered intravenously or orally. That is, 2. For example, when the substance is formulated and administered based on conventional techniques, the rate of disappearance from the blood is quite low, and the rate and amount of transfer to target organs are also small.

In the first place, the substance is a fat-soluble substance that is solid at room temperature and has a melting point of 48 to 52°C, so in order to make it easier to administer intravenously, surfactant 1) (For example) II) (Polyoxyethylene hydrogenated castor oil) The substance must be solubilized by conventional techniques, such as using However, when ubidecarenone is administered after being solubilized in this way, the rate of disappearance of ubidecarenone from the blood is slow, as shown in the experimental examples described later.

In view of the above circumstances, the present inventor first conducted various studies on ubidecarenone-containing compositions and coatings thereof that are effective in increasing the transfer from the blood to predetermined target organs, and as a result, incorporated ubidecarenone into liposomes. Furthermore, it was discovered that the desired solution could be provided by coating with a polysaccharide layer fatty acid ester, and based on this knowledge, the invention was filed as patent application No. 56-105689 and No. 57-82993. A patent application was filed.

In other words, when administering ubidecarenone-containing liposomes or their coatings, the rate of disappearance from the blood after administration and the amount transferred to organs within a predetermined period of time are calculated. Compared to this, the rate of disappearance from the blood is significantly high, and high amounts are transferred to organs other than the lungs and kidneys.

As interest in so-called organ-directed preparations gradually increases in the medical and pharmaceutical fields, technology that utilizes liposomes has come to be seen as a means to achieve this goal. As is well known. That is, by focusing on the fact that liposomes exist as unique liposomes in each organ, by containing a prescribed drug in a prescribed liposome and administering it,
Techniques are being developed that are intended to selectively and intensively deliver drugs to target containers.

An example of such an invention is JP-A-52-143218.

Japanese Patent Publication No. 52-151718. Japanese Patent Publication No. 53-133616
．． The invention in Japanese Patent Publication No. 55-8488 can be mentioned.

All of these inventions are related to techniques for holding specific biological components or medical co-components using liposomes as carriers, and it is expected that these technologies will eventually be applied to other biological components and pharmaceutical components in medical and pharmaceutical sciences. Ru.

The invention related to patent application No. 56-105689 is based on liposomes.
Although it is used for the purpose of +ζ organ-directing, and this itself is already known, we applied liposome technology specifically to ubidecarenone to obtain a novel ubidecarenone-containing liposome, thereby solving the above-mentioned basic problem with ubidecarenone. We succeeded in solving this problem and significantly increasing the utility value of ubidecarenone.

Furthermore, the present inventors focused on the fact that organ targeting differs depending on the physical and chemical properties of liposomes themselves, and continued.

We investigated methods for selectively targeting ubidecarenone to the lungs and kidneys. As a result, it was discovered that the intended purpose could be achieved by coating ubidecarenone-containing liposomes with a polysaccharide fatty acid ester to form a ubidecarenone-containing liposome-coated body, and the invention was completed as an invention related to Japanese Patent Application No. 57-82993. .

Thereafter, the present inventor further coated the ubidecarenone-containing liposome with a polysaccharide fatty acid inorganic acid mixed ester to obtain a ubidecarenone-containing liposome.

The inventors discovered that the bosome-coated body also increases the rate of disappearance of ubidecarenone from the blood and increases its transfer to the A organ, leading to the present invention. That is, an object of the present invention is to provide technical means for increasing the rate of disappearance of ubidecarenone from the blood.

The present invention will be explained in detail below.

In the liposome according to the present invention, ubidecarenone is contained in the membrane that constitutes the liposome.

The basic substances constituting the liposome membrane are mainly phospholipids and sterols, and ubidecarenone coexists with these and is uniformly dispersed in the membrane.

Examples of the phospholipids used in the present invention include phosphatidelcholine, phosphatidelethanolamine, phosphatidelserine, sphingomyelin, and gusetyl phosphate. Stearylamine.

Phosphatideglycerol, bosphatidic acid.

Examples include, but are not limited to, phosphatidelinositol and mixtures thereof. Cholesterol is the most preferred sterol.

Regarding the composition ratio of ubidecarenone, phospholipids, and sterols, it is sufficient that 1 molar ratio of ubidecarenone to 10 molar ratios of phospholipids or more, and 1 molar ratio of sterols to 1 molar ratio of ubidecarenone. 10 when using egg yolk phosphatidercholine
-30 molar ratio, and when cholesterol is used as the sterol, the preferred use range is 1-10 molar ratio.

Examples of preferable molar composition ratios of phospholipid and cholesterol with respect to 1 molar ratio of ubidecarenone are as shown in 1 to 7 below.

PC: phosphatidercholine DCP nidicetyl phosphate PS: phosphatiderserine SA: stearylamine The liposome according to the present invention has a molecular weight of 0.1 to
It is a sphere with a particle diameter of about 5.0μ. The freeze-dried product aggregates and has a block-like appearance.

When added to water or salt solutions, it disperses well and gives a homogeneous solution.

Next, the liposome according to the present invention can be produced by applying conventional techniques related to the production of liposomes.

For example, put the liposome membrane components and ubidecarenone in an eggplant flask, add chloroform and dissolve them once.
Next, the solvent is distilled off, the resulting membrane is peeled off by adding glass beads and a suitable buffer solution, and after ultrasonication, it is passed through a Sephadex or Sepharose column to collect the liposome fragments and remove the solvent. For example, freeze-drying treatment can be applied to remove the solvent.

The freeze-drying process is 2. If the liposomes are allowed to stand for 3 hours or more at a temperature below O Torr, the desired purpose is achieved and the liposomes can be taken out in powder form; however, the present invention is not limited to such conditions. As a desirable condition, a method of freeze-drying at 0.3 Torr for 5 hours is selected as shown in Examples below.

Next, in the polysaccharide fatty acid inorganic acid mixed ester according to the present invention, the polysaccharide refers to a polysaccharide having a molecular weight of 40,000 or more, and specifically dextran.

Amylopectin and pullulan are preferred, and others include dextran sulfate, chitic acid, pullulan sulfate, and the like. In the present invention, the polysaccharide is a combination of two or more types having different molecular weights, or a combination of amylopectin and dextran (multiple polysaccharides may be used in combination).

Further, the fatty acids in the polysaccharide fatty acid inorganic acid mixed ester according to the present invention are lauric acid and myristic acid.

Palmitic acid and stearic acid are particularly preferred, and palmitic acid is particularly preferred. When a liposome is coated with the polysaccharide fatty acid ester according to the present invention, it is presumed that the alkyl chains of the fatty acid are oriented in a wedge shape in the lipid layer of the liposome. It is therefore believed that the above fatty acids are particularly preferred because their alkyl chain lengths are suitable for wedge-shaped orientation into the liposomal lipid layer.

The inorganic acids in the polysaccharide fatty acid inorganic acid mixed ester are phosphoric acid and sulfuric acid.

Next is the production of polysaccharide fatty acid inorganic acid mixed ester.

This can be done roughly as follows. first.

The polysaccharide is dissolved in anhydrous dimethylformamide by heating at 60 to 70°C. A pre-dissolved fatty acid chloride was added to anhydrous pyridine and anhydrous dimethylformamide, and 60
Stir for several hours at ~70°C, followed by 24 hours at room temperature. After the reaction is completed, 9. When ethanol is added to the reaction mixture, a white precipitate is precipitated, which is collected in i5, washed with ethanol, further dispersed in ether, and collected again in f. Dry under reduced pressure to obtain polysaccharide fatty acid ester. Next, the obtained polysaccharide fatty acid ester was dissolved in anhydrous formamide or pyridine for 6 hours.
The mixture is dissolved by heating at 0 to 70°C, polyphosphoric acid or chlorosulfuric acid is added, and the mixture is stirred at room temperature for 24 hours or at 60 to 70°C for 4 hours. After the reaction is completed, methanol ester is obtained in the reaction mixture.

The ester obtained here can be identified by IR spectrum (KBr method) and H'-NMR spectrum (solvent d'-DMSO, internal standard TM8).

Furthermore, the degree of fatty acid substitution and the degree of inorganic acid substitution can be increased.

The degree of fatty acid substitution refers to the number of fatty acids introduced per 100 sugar units, and this is measured by H'-NMR as follows. For example, when a valmitoyl group is introduced, the amount of introduction is) ('-The peak area of the proton of the valmitoyl group appearing at 0.9 ppm and 1.28 ppm in the NMR spectrum and 3.5 to 5.2 ppm)
It is given by the ratio of the broadening appearing in m to the proton peak area. That is, when X valmitoyl groups are substituted in 100 sugar units, the number of protons of the sugar is 9X + 10 (100-x).

Further, the number of protons of the valmitoyl group is 31X.

Therefore, let the number of protons of the sugar determined from the integral curve of the l'-NMR spectrum be y, and the number of protons of the valmitoyl group be 2.

It is. When the degree of substitution, which can be determined by be.

The degree of inorganic acid substitution refers to the number of fatty acids per 100 sugar units, and can be determined according to the description for the degree of fatty acid substitution.

Furthermore, in order to coat liposomes with the ester according to the present invention, the ester-containing aqueous solution may be added to the aqueous solution in which the liposomes are formed and stirred.

For coating, it is sufficient to coat a single ester, but
Two or more esters according to the present invention may be combined and coated as a so-called composite ester.

The effects of the ubidecarenone-containing liposome-coated body of the present invention will be explained using the following experimental examples.

Experimental Example 1 14CC0QI instead of COQ, o in the descriptions of Sample Examples 1 to 3. The following test samples a to d were prepared in the same manner as in Examples 1 to 3 except that the following samples were used.

a, "CC0Qto containing'J ho7 Ab,'4C-
COQto-containing liposomal coating, however, the coating ester is O-balmitoyl amylopectin phosphate (molecular weight 112,000, degree of fatty acid substitution 4.9 degree of phosphoric acid substitution 1)
．． 6) C, "C-C0QIo-containing liposomal coating, where the coating ester is 0-balmitoylamylopectin sulfate (molecular weight 112,000, degree of fatty acid substitution 4.9, degree of sulfuric acid substitution 2.1) d," C -C0Qto-containing liposomal coated body, however, the coating ester is 0-palmitoylburlanphosufunito (molecular weight 230,000, degree of fatty acid substitution 1.0, degree of phosphoric acid substitution 4.2). It is radiolabeled ubidecarenone represented by the following formula.

In addition, this specific radioactivity is 7°95μCi/nLg,
Radiochemically, it was found to be single by thin layer chromatography using two types of developing solvents (chloroform/benzene = 1/1 and acetic acid/benzene = 1/9).

Methods Animal Experiments A sample of 0°6 my14C-COQl was injected into the left femoral vein of a male guinea pig (body weight 300-350 g). 7 kg was injected and sutured. After that, the two guinea pigs were left in the breeding cage.

Blood was collected from the ear vein at predetermined intervals. The concentration of "CC0Qto" in the collected blood was measured by the method described below.

Measurement of radioactivity in blood Collect 20 μt or 50 μL of blood from the ear vein.
Solubilize with 75 ml of 5oluene 350/isopropyl alcohol (1/1) and decolorize by adding a few drops of hydrogen peroxide.

Instagram/0.5N I(Of(9/1)
) 5+rLL was added and radioactivity was measured using a liquid scintillation counter.

14CQoQt when the resultant sample was administered. Figure 1 shows a graph showing the time course of the radioactivity concentration in the blood.

Explanation of Figure 1: The ○ line, the O line, the 9 circle line, and the Δ line indicate sample a and sample A, respectively. Sample C0 Sample d. The graph shows the transition (average value of 3 cases) when administered.

As shown in FIG. 1, the fact that ubidecarenone rapidly disappears from the blood and is transferred to organs by being contained in liposomes is a phenomenon already known in the invention of Japanese Patent Application No. 105689/1983.

This phenomenon does not disappear even when the membrane surface of the liposome is coated with a mixed ester of polysaccharide and fatty acid inorganic acid (in fact, it is observed that the rate of disappearance from the blood is accelerated by coating with the ester).

The present invention will be specifically explained with reference to Examples described below.

Example 1 Egg yolk phosphatidino and choline 54mp (6,75X
10'mol).

Cholesterol 8.78 mg (2,25X10-'m
ol) and ubidecarenone or CoQl, 4.
6 mg (5,32XIQ-'mol) was dissolved in 4 ml of chloroporm and the chloroform was removed under reduced pressure using a rotary evaporator.

The obtained thin film was dried overnight in a vacuum desiccator.

Chloroform was completely removed. To the thin film remaining at the bottom, a total of 4° Oml of buffer A and one glass bead were added twice at 2°0ral intervals, and the mixture was shaken with a Portex φ mixer until the film was completely peeled off.

Next, this solution was transferred to a test tube with a branch, and a probe-type sonicator was used at 0° C. under an argon atmosphere.

When ultrasonication was performed intermittently every 30 seconds, a yellowish-white solution was obtained. This solution contains C0Qto-containing liposomes.

Next, add O-balmitoylamylopectin phosphate (molecule ff1ix2.oo) to 2.2 ml of this yellow-white solution.
o, fat'f+&fi'i conversion rate 4.9. ! A solution (10.3 mg) of I phosphate substitution degree 1.6 dispersed in buffer A
10.6nLL) were mixed and stirred at room temperature for 30 minutes to obtain 9 coated bodies of the present invention.

Example 2 In the description of Example 1, 0-palmitoyl amylopectin sulfate) (molecule 1112) was used instead of 0-balmitoyl amylopectin phosphate (molecular weight 112,000, degree of fatty acid substitution 4.9, degree of phosphoric acid substitution 1.6). ，
000° Fatty acid substitution degree 4.9. A ubidecarenone-containing liposome/coated body was obtained in the same manner as in Example 1 except that the degree of sulfuric acid substitution was 2.1).

Example 3 ' In Cζ described in Example 1, 0-palmi ψ toyl pullulan was used instead of 0-balmitoyl amylopectin phosphate (molecular weight 112,000, degree of fatty acid substitution 4.9, 17, degree of phosphoric acid substitution 1.6). Phos7ate (molecular weight 230,000.
A ubidecarenone-containing liposome-coated body was obtained in the same manner as in Example 1, except that the degree of fatty acid substitution was 1.0.9 and the degree of phosphoric acid substitution was 4.2.

[Brief explanation of the drawing]

Figure 1 shows experimental example 1. This drawing corresponds to FIG. 1 described in the Results section, and shows the rate of disappearance of ubidecarenone in the blood. Patent application artificial - Zai Co., Ltd.

Claims (1)

[Scope of Claims] (1) A ubidecarenone-containing liposome coated body consisting of a polysaccharide-layered fatty acid inorganic acid mixed ester that coats the membrane surface of the ubidecarenone-containing liposome and the ribosome (2) The membrane of the liposome is made of phospholipids and sterols. Claim 1, item 1, which is a film formed by <7'i
Ubidecarenone-containing liposome-coated body of fj'+ (31
The phospholipids are phosphatidercholine, phosphatibril ethanolamine, phosphatiderserin, sphingomyelin, dicetyl phosphate, stearylamine, phosphatideglycerol, phosphatidic acid, and phosphatidelinositol. and the ubidecarenone-containing liposome-coated body according to claim 2 which is a mixture thereof (the feature ff which is 41xf chlorcholesterol) Claim 2 ↑
or the ubidecarenone-containing liposome coated body (5) according to item 3, in which the phospholipid is 10 molar ratio to 1 molar ratio of ubidecarenone.
The ubidecarenone-containing liposome coating (6) according to claims 2 to 4, in which the ubidecarenone-containing liposome coating (6) polysaccharide layer fatty acid inorganic acid mixed ester has a molar ratio of amylopectin, pullulan, and dextran, wherein the sterol has a molar ratio of at least 1 molar ratio. The ubidecarenone-containing liposome coating according to claims 1 to 5, which is an ester of any polysaccharide and palmitic acid and phosphoric acid or sulfuric acid.