JPH0789874A - Carrier recognizing injured part of intravascular wall - Google Patents

Abstract

PURPOSE:To obtain a medicine-containing carrier having a high encapsulation ratio of a neutral or anionic substance, exhibiting a remarkably high targeting property to an injured part of the intravascular wall and also remarkably excellent in safety. CONSTITUTION:In this medicine-containing carrier, at least one of the constituting components is a compound having a part carrying positive charges within a physiological pH range and the parts carrying positive charges within a physiological pH range are partly or wholly exposed on the surface. A glucamine derivative, a galactosamine derivative, a glucosamine derivative, etc., are exemplified as the compound and a diagnostic or therapeutic agent for an injured part of the intravascular wall as the medicine.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a drug carrier containing a drug for recognizing a vascular endothelial damage site and diagnosing or treating the vascular endothelial damage site.

[0002]

2. Description of the Related Art It is widely known that damage to blood vessels, especially to the endothelium of aorta, is one of the causes of the development of arteriosclerosis by causing hyperproliferation of vascular smooth muscle cells. England Journal
Of Medicine (R. Rosset al, N. En
gl. J. Med. ), 314, 488 (198
6)). Causes of such injury include thrombus formation or endothelial cell injury due to peroxidation reaction. Recently, the stenosis site of the coronary artery is compressed by physical force, or expanded by cutting or burning. With the practical use of therapy, thickening of tissues occurs at the site injured by such physical treatment, and so-called restenosis, in which the once enlarged blood vessel lumen is again narrowed, is a problem.

In particular, the method of expanding a stenotic coronary artery by physical compression using a catheter introduced percutaneously into the blood vessel (PTCA) is extremely useful as a therapeutic method for ischemic heart disease such as angina. Widespread, 30 to 5 after treatment
Prevention of restenosis occurring in 0% of patients has become the most important issue for the treatment of the disease. Since this restenosis is mainly caused by the excessive proliferation of vascular smooth muscle cells, attempts have been made to prevent restenosis by a drug having an action of suppressing this proliferation. However, up to now, many drugs having an effect of suppressing the growth of smooth muscle cells cultured on a petri dish have been found, but none of them has clinically effective effects (Cassels, Circulation (W.
Cascells, Circulation), 86
(3), 724 (1992)).

As one of the causes of such clinically ineffectiveness, when the drug is systemically administered by intravenous administration or oral administration, the drug does not exert a systemic action. Depending on the dose, it may not be possible to ensure a sufficient concentration to suppress smooth muscle cell proliferation at the site of vascular endothelial injury. For example, heparin is a drug having such an action, but when administered systemically, it is difficult to administer a sufficient amount of the drug due to actions such as bleeding tendency. However, it has been confirmed by experiments by Okada et al. That vascular hyperplasia can be suppressed without such action by local administration (Tomohisa Okada et al., DDS, 7 (1), 51). ，
(1992)). They confirmed that a method of opening the chest and applying a gel containing heparin from the outside to the outside of the blood vessel to sufficiently raise the drug concentration in the local area of the blood vessel and resulting therapeutic effect.

However, such an administration method is too invasive for the living body, and one of the practical values thereof is that it is a particularly minimally invasive treatment method.
This is a method that cannot be recognized as a method combined with A therapy. That is, in the field, the drug can be accumulated in a specific and sufficient concentration at the site of vascular skin injury even by a less invasive administration method such as oral administration, intravenous administration, or local administration with a catheter even in the case of the highest invasiveness. There is a demand for a drug carrier capable of achieving the above, that is, a drug carrier having a high degree of specific accumulation at the site of vascular endothelial injury and a high drug-encapsulating ability, but such a drug carrier has not been realized so far.

[0006] In recent years, much research has been conducted to apply closed vesicles such as liposomes, emulsions, lipid microspheres and nanoparticles to drug delivery systems as drug carriers.

However, the biggest problem in using these closed vesicles as a drug carrier in the above fields is that it is extremely difficult to control the pharmacokinetics. In particular, closed vesicles are easily captured by the reticuloendothelial system (RES) such as the liver and spleen, so targeting to internal organs other than RES is extremely difficult, and especially targeting to sites where the vascular endothelium is damaged. It has never been taken into consideration.

Furthermore, when trying to encapsulate a substance in a closed vesicle, a closed vesicle composed of a commonly used membrane material cannot encapsulate a high concentration of a drug, and as a result, In addition to not being able to fully utilize the advantages of closed vesicles, it causes problems such as increase in manufacturing cost, stability in blood is extremely poor, and problems such as closed vesicles coagulating via proteins in blood Have made practical application of these drug carriers difficult.

Various means have been devised to overcome these problems. For example, a method of improving the retention in blood by using a charged substance such as stearylamine as a membrane constituent (JP-A-63-7782).
No. 4), a method of modifying the surface of vesicles with sugars such as sialic acid and glucuronic acid, and derivatives thereof (Biochemica Biophysical Actor (BBA), 1126, 255).
(1992), DDS, 7,345.
(1992)), a method of using a derivative of a hydrophilic polymer such as polyethylene glycol as a protein adsorption inhibitor (JP-A-2-149512, JP-A-3-14912).
-218309). Also, a membrane component using a glucosamine derivative and the like have been disclosed (JP-A-4-159216).

However, none of these methods has realized a drug carrier having a high specific accumulation on the damaged site of vascular endothelium and a high drug-encapsulating ability.

[0011]

SUMMARY OF THE INVENTION Therefore, the object of the present invention is to provide a high drug encapsulation rate, excellent stability in the body, and a site where the vascular endothelium is damaged, such as after PTCA or atherosclerotic lesion. To provide a recognizable drug carrier.

[0012]

The above object can be solved by the present invention described below.

(1) A carrier for recognizing a damaged site of vascular endothelium, which comprises at least one component of a compound having a site having a positive charge in a physiological pH range.
A drug carrier, wherein a site having a positive charge in the H range is present on the surface of the carrier.

(2) In the above (1), wherein the carrier constituent component further comprises a hydrophilic polymer derivative, and at least a part of the hydrophilic polymer moiety of the hydrophilic polymer derivative is present on the surface of the carrier. The described drug carrier.

(3) The drug carrier according to the above (1) or (2), which has a drug for diagnosing and / or treating a vascular endothelial damage site encapsulated therein.

(4) The carrier has a diameter of 0.02 to 250 μm.
The drug carrier according to any one of (1) to (3) above, which comprises fine particles having a size of

(5) The carrier is a macromolecule, a fine aggregate,
The above (1) comprising at least one of fine particles, microspheres, nanospheres, liposomes and emulsions
The drug carrier according to any one of (4) to (4).

(6) The carrier comprises a phospholipid or a derivative thereof, and / or a lipid other than a phospholipid or a derivative thereof, and / or a stabilizer, and / or an antioxidant, and / or another surface modifier. The drug carrier according to any one of (1) to (5) above, which is contained.

(7) The compound having a site having a positive charge in the physiological pH range contains at least one or more aliphatic primary / secondary amino group, amidino group, aromatic primary / secondary amino group. The drug carrier according to any one of (1) to (6) above, which comprises a compound having the compound and a compound having the compound further bound to a residue having one or more hydroxyl groups.

(8) The drug carrier according to the above (1) to (6), wherein the compound having a positively charged site in the physiological pH range is an amino sugar.

(9) The drug carrier according to the above (8), wherein the amino sugar comprises a monosaccharide such as glucosamine, galactosamine, mannosamine, inoramic acid and inoramic acid ester, and a free form or various glycosides thereof.

(10) The drug carrier according to (8) above, wherein the amino sugar comprises an oligosaccharide such as chitin, a polysaccharide, and a free form or various glycosides thereof.

(11) The hydrophilic polymer derivative is a derivative of polyethylene glycol with a long-chain aliphatic alcohol, a sterol, a polyoxypropylene alkyl, or a glycerin fatty acid ester, (2) to (1).
The drug carrier according to 1).

(12) The drug carrier according to the above (3) to (11), wherein the drug for diagnosing and / or treating the vascular endothelial damage site is electrically neutral or anionic.

(13) The drug for diagnosing and / or treating the vascular endothelial damage site is an anti-inflammatory agent, anti-cancer agent,
Enzymes, antibiotics, antioxidants, lipid uptake inhibitors, hormones, angiotensin converting enzyme inhibitors, smooth muscle cell proliferation / migration inhibitors, platelet aggregation inhibitors, chemical mediator release inhibitors, or vascular endothelium The drug carrier according to the above (3) to (12), which is a cell growth or inhibitor.

(14) The drug carrier according to the above (3) to (12), wherein the drug for diagnosing and / or treating the vascular endothelial damage site is glycosaminoglycan and its derivative.

(15) The drug carrier according to the above (3) to (12), wherein the drug for diagnosing and / or treating the vascular endothelial damage site is an oligo- and / or polysaccharide and a derivative thereof.

(16) Various in-vivo diagnostic agents, such as X-ray contrast agents, radioisotope-labeled nuclear medicine diagnostic agents, and nuclear magnetic resonance diagnostic diagnostic agents, are used for diagnosing and / or treating the vascular endothelial damage site. The drug carrier according to (3) to (12) above, which is

(17) The drug for diagnosing and / or treating the vascular endothelial damage site is at least one of heparin, theopherin, cyclic AMP (cAMP), captopril, papaverine, and forskolin. The drug carrier according to 3) to 12).

In the process of studying the factors that control the pharmacokinetics of various drug carriers, the present inventors have surprisingly found that the surface of the drug carrier is positively charged in the physiological pH range. It was found that when at least a part of the site of the compound having the positive charge in the physiological pH range is present, the drug carrier accumulates at the site of vascular endothelial damage.

Further, surprisingly, by further adding a hydrophilic polymer to the surface of the drug carrier in which the positively charged site is present, the carrier is accumulated at the damaged site on the vascular endothelium. It was found that the stability of the carrier in the blood can be secured by preventing the carrier from aggregating in the blood and the retention in the blood can be improved by avoiding RES supplementation. Was completed.

Therefore, according to the present invention, which has the above-mentioned object, a surface has a site having a positive charge in a physiological pH range, and a drug for diagnosing and / or treating a vascular endothelial damage site is enclosed therein. It is a drug carrier that recognizes the damaged site of vascular endothelium. Further, it is a drug carrier having a hydrophilic polymer further present on its surface.

The carrier of the present invention has a particle size of 0.02 to 250 μm.
The size of m, especially 0.05 to 0.4 μm is preferable.

Various forms of the structure are conceivable, and there is no need to limit the structure. In particular, a macromolecule, a microaggregate, a fine particle, which has a potential function of encapsulating a drug therein at a high concentration, Most preferably, it comprises at least one or more of microspheres, nanospheres, liposomes and emulsions.

In the present invention, a compound having a site having a positive charge in a physiological pH range as a constituent of the drug carrier, and a hydrophilic polymer derivative having a hydrophilic polymer site may be used. The constituents other than these are not particularly limited to the combination as long as they can form the above-mentioned form, but considering their safety and stability in vivo, phospholipids or their derivatives, phospholipids It is desirable to add lipids other than lipids or derivatives thereof, or stabilizers, antioxidants, and other surface modifiers.

As the phospholipid, natural or synthetic phospholipids such as phosphatidylcholine (= lecithin), phosphatidylglycerol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin and cardioribine, or a conventional method thereof is used. And the like, which are hydrogenated according to the above.

Examples of the stabilizer include sterols such as cholesterol, which reduce membrane fluidity, and sugars such as glycerol and sucrose.

Examples of antioxidants include tocopherol homologues such as vitamin E. Tocopherol has four isomers of α, β, γ and δ, but any of them can be used in the present invention.

Other surface modifiers include derivatives of water-soluble polysaccharides such as glucuronic acid, sialic acid and dextran.

The compound having a site having a positive charge in the physiological pH range is not particularly limited as long as it does not impair the structural stability of the drug carrier, but at least one or more aliphatic first, second Primary amino group, amidino group,
Examples thereof include compounds having aromatic primary and secondary amino groups, and compounds obtained by combining the above compounds with a residue having one or more hydroxyl groups.

Specifically, the compound and palmitic acid,
Long-chain fatty alcohols such as stearic acid, sterols,
Derivatives with polyoxypropylene alkyl or hydrophobic compounds such as glycerin fatty acid esters are preferred. These derivatives can stably insert a hydrophobic compound site into the membrane of a drug carrier (for example, liposome), and allow a site having a positive charge in the physiological pH range to exist on the surface of the drug carrier.

The compound having a site having a positive charge in the physiological pH range may be an amino sugar,
Examples of the amino sugars include monosaccharides such as glucosamine, galactosamine, mannosamine, inoramic acid and inoramic acid ester, oligosaccharides such as chitin and polysaccharides, and free or various glycosides thereof.

The amino sugar can stably insert the hydrophobic compound site into the membrane of the drug carrier (for example, liposome), and the site having a positive charge in the physiological pH range can be present on the surface of the carrier. it can.

In the present invention, the physiological pH range refers to the pH range in the living body, mainly in the blood, the cytoplasm and the inside of the organelles present therein, and specifically, pH 4 to 10.
And more preferably pH 6-8.

In the present invention, the hydrophilic polymer moiety present on the surface is not particularly limited as long as it does not impair the structural stability of the drug carrier. Various hydrophilic polymers can be considered. For example, polyethylene glycol, dextran, pullulan, ficoll,
Polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, synthetic polyamino acid, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan and the like. Among them, polyethylene glycol is most preferable because it has a remarkable effect of improving blood retention.

The hydrophilic polymer may be a long-chain aliphatic alcohol, a sterol, a polyoxypropylene alkyl, or a derivative in which a hydrophobic compound such as glycerin fatty acid ester is bound, so that the hydrophobic compound site is a drug. It can be stably inserted into the membrane of a carrier (eg, liposome). Thereby, the hydrophilic polymer can be present on the surface of the drug carrier.

Examples of the hydrophilic polymer derivative that can be specifically used in the present invention include polyethylene glycol-phosphatidyl ethanolamine.

In the present invention, the drug to be encapsulated in the drug carrier is a pharmacologically active substance, physiologically active substance or diagnostic substance which is pharmaceutically acceptable depending on the purpose of diagnosis and / or treatment of the endothelial injury site. Can be used.

The nature of the drug to be encapsulated is basically no problem with any substance, but the electrically neutral or anionic substance has a higher encapsulation rate because the surface of the carrier has a positive charge. Can be expected.

The kind of the drug to be encapsulated is not particularly limited as long as it does not impair the formation of the drug carrier, and it is a drug that suppresses various reactions at the site of vascular endothelial damage and induces normal vascular tissue. It can be used without any restrictions. Specifically, anti-inflammatory agents, anti-cancer agents, enzyme agents, antibiotics, antioxidants, lipid uptake inhibitors, hormone agents, angiotensin converting enzyme inhibitors, smooth muscle cell proliferation / migration inhibitors, platelet aggregation inhibitors, Examples thereof include those that can be used as a chemical mediator release inhibitor, or a vascular endothelial cell growth promoter or inhibitor.

In particular, sulfated glycosaminoglycans such as heparin, oligo- and polysaccharides and their derivatives are effective as substances that inhibit the proliferation and migration of smooth muscle cells, which is the cause of vascular hypertrophy, at the site of vascular endothelial damage. In addition, theopherin, cyclic AMP (cAM
P), captopril, papaverine, forskolin and the like.

However, since the carrier of the present invention basically has the property of accumulating at the site of vascular endothelium damage, what kind of drug should be selected depending on the characteristics of the disease at that site? There is no limit. Especially at the injury site,
Platelets are easily activated and various leukocytes are accumulated, and various chemical mediators are released to cause an inflammatory reaction. It is also well known that lipid peroxidation and enhancement of lipid uptake occur in these tissues, and it is considered that these reactions progress in a complex manner to cause arteriosclerotic lesions. Since the contribution of these phenomena to lesion formation is expected to differ from case to case, it goes without saying that all drugs corresponding to these symptoms can be the subject of the present invention.

It is important to identify the lesion site prior to drug treatment, and various internal diagnostic agents (X-ray contrast agents, radioisotope-labeled nuclear medicine diagnostic agents, nuclear magnetic resonance) that can be used for this purpose. It is needless to say that a diagnostic agent for diagnosis, etc.) enables specific diagnosis of a vascular lesion by encapsulating it in the carrier of the present invention, and the diagnostic agent can also be a target of the present invention.

The drug carrier of the present invention can be easily obtained by a conventional method, one example of which is shown below. The inner wall of the flask is prepared by mixing a compound having a site having a positive charge in the physiological pH range in the flask and other carrier constituents such as phospholipid with an organic solvent such as chloroform, distilling off the organic solvent and vacuum drying. A thin film is formed on. next,
A liposome dispersion is obtained by adding a drug into the flask and stirring vigorously. The drug carrier can be obtained as a dispersion by centrifuging the obtained liposome dispersion and decanting the supernatant to remove the drug not encapsulated. It can also be obtained by mixing the above-mentioned respective components and discharging the mixture under high pressure using a high-pressure discharge type emulsifier.

The drug carrier of the present invention can be administered by intravenous injection or the like, but a particularly preferred method is to insert a catheter into a blood vessel, guide its tip to the vicinity of the vascular endothelial damage site, and administer through the catheter. Is preferred.

The drug carrier of the present invention has an excellent retention property in the blood and the site of vascular endothelial damage, and is retained in the blood for 6 hours or more and for 3 days or more after the administration of one time. Stays stable in.

Further, it is effective to administer the drug carrier of the present invention within 1 hour after the occurrence of vascular damage due to PTCA surgery or the like. This is because when the drug is administered about 1 hour after the occurrence of vascular damage, the accumulation of the drug carrier of the present invention at the site of vascular damage is temporarily reduced. This is considered to be due to the effect of adhesion of blood platelets or the like on the site of blood vessel damage. Therefore, the drug carrier of the present invention is preferably administered within 1 hour after the occurrence of vascular damage.

However, after a certain amount of time has passed, the drug carrier of the present invention has a re-improved property of accumulating on the damaged blood vessel. Therefore, the administration after 1 hour after the occurrence of the blood vessel damage is not invalidated, and the administration thereafter is also effective.

[0059]

The present invention will be described in more detail with reference to the following examples.

(Example 1) 6-o showing the structure in Formula I
-Palmitoyl-methyl-D-galactosaminide (6
-O-palmitoyl-methyl-D-gal
A liposome containing actosaminide) as a membrane constituent was prepared as follows.

The following three types of membrane constituents were added as chloroform solutions to an eggplant type flask having a volume of 50 ml and mixed.

Phosphatidylcholine (concentration 100m
M): 840 μl Cholesterol (concentration 100 mM): 240 μl 6-o-palmitoyl-methyl-D-galactosaminide (10 mM): 1200 μl

[0063]

[Chemical 1]

Further, 10 ml of chloroform was added.
After the chloroform was distilled off, it was vacuum dried overnight to form a lipid thin film on the inner wall of the flask. Then 100
300 mM sorbitol / μg of heparin dissolved
A liposome (MLV) dispersion was obtained by adding 4 ml of 10 mM Tris-HCl buffer into the flask and vigorously stirring and stirring.

This dispersion was centrifuged (120,000
g, 70 minutes). The supernatant was decanted and the unencapsulated heparin was removed to obtain a liposome pellet. This pellet is 300 mM sorbitol /
Dispersion in 10 mM Tris-HCl gave the title liposome dispersion in which heparin was encapsulated.

Reference Example 6-o-palmitoyl-methyl-D-galactosaminide used in this example was prepared as follows.

N-benzyloxycarbonyl-methyl-D-from D-galactosaminide according to known procedures.
Galactosaminide was obtained, and the N-benzyloxycarbonyl-methyl-D-galactosaminide (9.3 g) and palmitoyl chloride (8 ml) were added to pyridine (50 ml), and the mixture was stirred at room temperature under a nitrogen gas atmosphere at room temperature. Stir for 24 hours. After the reaction is complete, the reaction mixture is poured into 10% ice-cold hydrochloric acid and extracted with ethyl acetate. Then, the extract was washed with saturated sodium hydrogen carbonate (NaHCO ₃ ) and brine, dried over anhydrous sodium sulfate (Na ₂ SO ₄ ),
The solvent was removed to obtain a crude product, which was further recrystallized from an ethyl acetate solution to give N-benzyloxycarbonyl-6-o.
-Palmitoyl-methyl-D-galactosaminide (7.65 g) was obtained.

This N-benzyloxycarbonyl-6-
o-Palmitoyl-methyl-D-galactosaminide (1.35 g) was dissolved in methanol (50 ml), a catalytic amount of 5% Pd-C was added, and catalytic reduction was carried out at room temperature and atmospheric pressure for 24 hours to react. After the completion, this was filtered to remove the solvent, and the residue was purified by silica gel column chromatography to obtain 6-o-palmitoyl-methyl-D-of the target compound.
Galactosaminide (874 mg) was obtained. The data presented below supports the structure of Formula I.

M. p. : 122-124 ° C Elemental analysis: Actual value C = 64.24%, H = 10.84
%, N = 3.04% Calculated value C = 64.0%, H = 10.51%, N = 3.
25% (calculated for C ₂₃ H ₄₃ O ₆ N)

IR (KBr): 3353 cm ^-1 , 291
^{7cm -1, 2854cm -1, 1728cm -1} , 1462
cm ^-1 MS (FAB): 435 (M + 1) ⁺

¹ H-NMR (DMSO-d ₆ ) δ (pp
m): 5.15 (n, 1H), 5.00 (n, 1H),
4.51 (d, J = 3.4 Hz, 1 H), 4.30 (d,
J = 10.6 Hz, 1H), 4.04 (dd, J = 6.6
Hz, J = 6.8 Hz, 1H), 3.53 (m, 1H),
3.26 (s, 3H), 3.10 (m, 2H), 2.40
(M, 1H), 2.29 (t, J = 7.2Hz, 2H),
1.51 (m, 2H), 1.24 (b, 24H), 0.8
6 (t, J = 6.0 Hz, 3H).

Example 2 6-o-palmitoyl-methyl-D-galactosaminide and a polyethylene glycol-phosphatidylethanolamine polymer (PEG-PE) whose structure is represented by formula II are used as membrane constituents. The liposomes containing were prepared as follows.

The following four types of membrane constituents were added as chloroform solutions to an eggplant type flask having a volume of 50 ml and mixed.

Phosphatidylcholine (concentration 100m
M): 840 μl Cholesterol (concentration 100 mM): 240 μl 6-o-palmitoyl-methyl-D-galactosaminide (10 mM): 1200 μl PEG-PE (concentration 0.4 W / V%): 1000 μ
l.

[0075]

[Chemical 2]

Thereafter, the same procedure as in Example 1 above was carried out to obtain the above-mentioned liposome dispersion retaining heparin.

Example 3 A liposome containing glucamine palmitoyl ester having the structure represented by the formula III as a membrane constituent was prepared as follows.

The following four types of membrane constituents were added as chloroform solutions to an eggplant type flask having a volume of 50 ml and mixed.

-Phosphatidylcholine (concentration 100m
M): 840 μl Cholesterol (concentration 100 mM): 240 μl Glucamine palmitoyl ester (concentration 10 mM):
1200 μl ・ PEG-PE (concentration 0.4 W / V%): 1000 μ
l.

[0080]

[Chemical 3]

Thereafter, the same procedure as in Example 1 was carried out to obtain the above-mentioned liposome dispersion retaining heparin.

(Test Example 1: Measurement of Heparin Encapsulation Rate) The encapsulation rate of heparin used in Examples 1 and 2 in liposomes was determined as follows. First, the amount of heparin in the supernatant after centrifugation was determined by the carbazole method to determine the amount of heparin not encapsulated in the liposome. The encapsulation rate of heparin in the liposome was determined by comparing this with the total amount of heparin added initially in the liposome preparation.

For comparison, a liposome dispersion prepared in the same manner as in the above Example without using 6-o-palmitoyl-methyl-D-galactosaminide and PEG-PE was prepared. Then, the encapsulation rate of heparin was determined by the same method as described above. The results are shown in Table 1.

[0084]

[Table 1]

As is clear from the above results, physiological p
A liposome having a compound having a positively charged portion in the H range, or a compound having a positively charged portion in a physiological pH range and a hydrophilic polymer derivative has a higher heparin content than a neutral liposome not containing the compound. The encapsulation rate was shown.

Although not shown in the table, liposomes containing a compound having a site having a positive charge in another physiological pH range of the present invention also showed similar excellent results.

Test Example 2: Pharmacokinetics 1 Carboxyfluorescein (Carb) prepared from the femoral vein of SD male rats anesthetized with urethane according to Examples 1 and 2
Oxyfluorescein) (100 mM) -encapsulated liposome dispersion (500 μl) was injected intravenously. Blood was collected in Eppendorf tubes over time. Pharmacokinetics was measured by measuring the fluorescence intensity of the collected blood.

For comparison, 6-o-palmitoyl-methyl-D-galactosaminide and PEG-PE were also used.
Without using, the in vivo kinetics of the liposome dispersion prepared according to the above-mentioned Example was measured by the same method as described above. The results are shown in Fig. 1.

As is apparent from FIG. 1, the liposome of the present invention containing a compound having a site having a positive charge in the physiological pH range as a membrane constituent component is retained in blood as compared with a drug liposome containing no compound. It was confirmed that the property is high. Further, it was confirmed that the liposome surface-modified with a hydrophilic polymer (PEG-PE) has a higher retention in blood than that of only the compound having a site having a positive charge in the physiological pH range. It was

Although not shown in the figure, liposomes containing a compound having a site having a positive charge in another physiological pH range of the present invention also showed similar excellent results.

(Test Example 3: Organ distribution 1) A 2 French balloon catheter was inserted into the femoral vein of an SD male rat anesthetized with urethane, and the carotid artery was rubbed 5 times to prepare a vascular endothelial injury model. .

Immediately after rubbing, carboxyfluorescein (Ca) prepared according to Examples 1 and 2 from the femoral vein was used.
(rboxyfluorescein) 100 mM encapsulated liposome dispersion (500 μl) was injected intravenously. The carotid arteries were collected 3 hours, 16 hours, 24 hours, and 3 days later. Carboxyfluorescein was extracted from the collected carotid artery, and the fluorescence intensity was measured to measure the amount of liposomes accumulated in the carotid artery.

Furthermore, after injecting 500 μl of a liposome dispersion liquid encapsulating 100 mM of carboxyfluorescein prepared according to Examples 1 and 2 from a femoral vein of a rat which does not scratch the carotid artery, the same method as above The distribution of liposomes to the carotid artery was measured by.

For comparison, 6-o-palmitoyl-methyl-D-galactosaminide and PEG-PE were used.
Without using, the amount of liposomes accumulated in the carotid artery of the vascular endothelial injury model was measured in the same manner as above for the liposome dispersion prepared according to the above example.

The results of Examples 1 and 2 and the comparative control after 3 hours in the vascular endothelial damage model are shown in FIG. 2, and the results of Example 2 measured over time for 3 days are shown in FIG. Although not shown, the accumulation amount when the vascular endothelium was not damaged was almost the same as the accumulation amount of liposomes in the carotid artery of the comparative vascular endothelial injury model.

As is clear from the above results, physiological p
The liposome of the present invention containing a compound having a site having a positive charge in the H range, or a compound having a site having a positive charge in the physiological pH range and a hydrophilic polymer derivative as a membrane constituent does not contain them. Compared with liposomes, it has a higher accumulation property on a vascular damage site, and has a higher accumulation property on a vascular damage site compared to a blood vessel whose endothelium is not damaged. Therefore, it has excellent targeting property on a vascular damage site.

It was also found that when administered immediately after rubbing, it was retained in the vascular injury site for 3 days.

Furthermore, a similar rat test was carried out in Example 3.
As a result of carrying out similarly for the liposome prepared according to the above, as shown in FIG. 4, the same excellent effect as that of the other examples was shown.

Although not shown in the figure, the carrier containing a compound having a site having a positive charge in another physiological pH range of the present invention also showed similar excellent results.

(Test Example 4: Pharmacokinetics 2) The same experiment as in Test Example 2 was conducted using castrated pigs (30 kg) instead of rats. That is, Carboxyfluorescein 1 prepared according to Example 2 from the femoral vein of a pig anesthetized with ketamine.
30 ml of the liposome dispersion liquid encapsulating 00 mM was intravenously injected. Blood was collected over time. Plasma was obtained from the collected blood and the in vivo kinetics was measured by measuring the fluorescence intensity of the plasma.

For comparison, 6-o-palmitoyl-methyl-D-galactosaminide and PEG-PE were also used.
Without using, the in vivo kinetics of the liposome dispersion prepared according to the above-mentioned Example was measured by the same method as described above.

As a result, the same excellent blood retentivity as in Test Example 2 was exhibited, indicating that the present invention is effective for large animals such as pigs. The results of the comparative control were similar to those of Test Example 2.

(Test Example 5: Organ distribution 2) A 7.5 French balloon catheter was inserted into the femoral artery of a castrated pig (30 kg) anesthetized with ketamine, and the carotid artery was rubbed 5 times to cause vascular endothelial damage. I created a model.

From the femoral vein, 30 ml of a liposome dispersion liquid encapsulating 100 mM of carboxyfluorescein prepared according to Example 2 was intravenously injected. The pig carotid artery was harvested after 3 hours. Carboxyflorescene was extracted from the collected carotid artery and the distribution of liposomes in the carotid artery was measured by measuring the fluorescence intensity.

Furthermore, for pigs not scratching the carotid artery, 30 ml of a liposome dispersion liquid encapsulating 100 mM of carboxyfluorescein prepared according to Examples 1 and 2 was intravenously injected from the femoral vein. The distribution of liposomes to the carotid artery was measured by the same method.

For comparison, a liposome dispersion prepared according to the above example without using PEG-PE, 6-o-palmitoyl-methyl-D-galactosaminide was prepared in the same manner as above for the carotid artery. The distribution of liposomes in the liposome was measured.

As shown in FIG. 5 together with the results of the rat performed in Test Example 3, excellent accumulation at the site of blood vessel damage was exhibited. In addition, the liposome as a comparative control did not show the accumulation property at the site of blood vessel damage.

From the above results, it was shown that the present invention is said to have blood vessels closer to humans, and is also effective for pigs, which are large animals. Although not shown in the figure, liposomes containing a compound having a site having a positive charge in other physiological pH range of the present invention also showed similar excellent results.

(Test Example 6: Organ Accumulation Property) A 2-French balloon catheter was inserted from the femoral artery of an SD male rat anesthetized with urethane, and the carotid artery was rubbed 5 times to give 6 groups of vascular endothelial injury models. Minutes created.

Immediately after rubbing, 500 μl of a liposome dispersion prepared by encapsulating 100 mM of carboxyfluorescein prepared according to Example 2 from the femoral vein of each rat group,
It was intravenously injected after 0.1, 0.5, 1, 3, 6, and 24 hours. The carotid artery was collected 3 hours after administration in each group.
Carboxyfluorescein was extracted from the collected carotid arteries, and the fluorescence intensity was measured to measure changes in the distribution of liposomes to the carotid arteries of each group. Results are shown in FIG.

As a result, it was found that a temporary decrease in the accumulation property of liposomes was observed in the vicinity of 1 hour after rubbing, but it was constantly accumulated thereafter. That is, 1
It was found that the administration within the time period is particularly effective, but it is also effective 1 hour after the blood vessel damage.

Further, the liposome containing the compound having a site having a positive charge in the other physiological pH range of the present invention showed similar results.

Test Example 7: Inhibitory Effect of Liposomal Formulation by Encapsulated Drug on Vascular Thickening A liposome formulation in which cyclic AMP (cAMP), captopril, papaverine, and forskolin are encapsulated in the same manner as in the heparin-containing liposome of Example 2. It was created. As a control, liposome without drug was used.

A 2-French balloon catheter was inserted from the femoral artery of an SD male rat anesthetized with urethane, and the carotid artery was rubbed 4 times to prepare a vascular endothelial injury model. 7 liposome formulations containing each drug from the tail vein
After administration over days, the carotid arteries were harvested. The degree of vascular hyperplasia was determined by the area ratio between the media and the intima of the collected carotid arteries. The calculation method is shown in FIG. 7, and the results are shown in Table 2.

[0115]

[Table 2]

As shown in Table 2, each drug-containing liposome preparation showed an excellent effect of suppressing vascular hypertrophy.

(Test Example 8: Acute toxicity) The purpose of this test is to find out the degree of toxicity of the drug carrier of the present invention as compared with that of conventional ones. For that purpose, a lethal toxicity test for rats was carried out for each of the drug carrier of the present invention prepared without drug encapsulation and the conventional drug carrier.

(Preparation of test solution) 1) Liposome dispersion of the present invention containing 6-o-palmitoyl-methyl-D-galactosaminide The same method as in Example 1 except that heparin was not added. Then, a liposome dispersion liquid containing 6-o-palmitoyl-methyl-D-galactosaminide and PEG-PE was obtained. This was concentrated using an ultrafiltration membrane and further diluted with sterile distilled water for injection to obtain a test solution.

2) 6-o-palmitoyl-methyl-D-
Liposomal dispersion of the present invention containing galactosaminide and PEG-PE 6-o-palmitoyl-methyl-D-galactosaminide was prepared in the same manner as in Example 2 except that heparin was not added. A liposome dispersion liquid containing PEG-PE was obtained. This was concentrated using an ultrafiltration membrane and further diluted with sterile distilled water for injection to obtain a test solution.

3) Conventional liposome dispersion liquid For comparison, neutral liposome dispersion liquid was prepared in the same manner as in Example without using 6-o-palmitoyl-methyl-D-galactosaminide and PEG-PE. Got This was concentrated using an ultrafiltration membrane and further diluted with sterile distilled water for injection to obtain a test solution.

(Method) Quarantined 5-week-old SD male rats were divided into 3 groups, and 2.0 ml of the above test solution was intraperitoneally injected into each group.
Was administered once. On the other hand, as a solvent control group, 2.0 ml of sterile distilled water was administered.

After the administration of the test solution, the general condition is carefully observed at least once a day for 16 days to show signs of toxicity,
The death status was recorded.

(Results) As shown in Table 3.

[0124]

[Table 3]

As shown in the above test results, no death occurred during the observation period for the drug carrier of the present invention. However, in the conventional drug carriers, the body weight drastically decreased from the first day after the start of administration, and most of them died by the 15th day as shown in the table. This is due to the subsequent organ findings that conventional drug carriers cause aggregation in the blood due to association with blood cell components and the like or association between liposomes in the blood, and embolism in the lungs and the like. It was confirmed that there is. From these results, it can be said that the drug carrier of the present invention has extremely low toxicity and high safety as compared with conventionally known drug carriers.

Although not shown in the table, the carrier containing a compound having a site having a positive charge in the other physiological pH range of the present invention also showed similar excellent results.

[0127]

INDUSTRIAL APPLICABILITY As described above in detail, the drug carrier of the present invention has a higher encapsulation rate of a neutral or anionic substance as compared with the conventional drug carrier, and further, it can reach the site of vascular endothelial damage. Has very high targeting ability. It is also very safe.

From these characteristics, the novel drug carrier of the present invention encapsulating a pharmaceutically acceptable pharmacologically active substance, physiologically active substance or diagnostic substance is used for the purpose of diagnosis and treatment at the site of vascular endothelial damage. Very effective against. For example, heparin, polysaccharides and their sulfated products,
And theopherin, cyclic AMP (cAM
When P), captopril, papaverine, forskolin, etc. are encapsulated, it is useful as a blood vessel thickening preventive agent that suppresses migration of smooth muscle cells at a site where the vascular endothelium is damaged.

[Brief description of drawings]

FIG. 1 shows the blood retention of the liposome of the present invention and that of a comparative liposome in rat.

FIG. 2 shows the accumulation property of the liposome of the present invention and the liposome of the present invention on the vascular endothelial damage site in rat.

FIG. 3 shows the time-dependent changes in the retention of the liposome of the present invention at the site of vascular endothelial damage in rats.

FIG. 4 shows time-dependent changes in retention of other liposomes of the present invention at the site of vascular endothelial damage in rats.

FIG. 5 shows the accumulation property of the liposome of the present invention and the liposome of the present invention at the site of vascular endothelial damage in pig and rat.

FIG. 6 shows the time from vascular injury to administration and the accumulation property of the liposome of the present invention at the site of vascular injury.

FIG. 7 shows how to determine the degree of blood vessel thickening in Test Example 7.

Claims (17)

[Claims] 1. A carrier for recognizing a damaged site of vascular endothelium, which comprises at least one compound having a site having a positive charge in a physiological pH range, wherein the site having a positive charge in the physiological pH range is A drug carrier, which is present on the surface of the carrier. 2. The carrier component further comprises a hydrophilic polymer derivative, and at least a part of the hydrophilic polymer moiety of the hydrophilic polymer derivative is present on the surface of the carrier. Drug carrier. 3. A drug for diagnosing and / or treating a vascular endothelial damage site is encapsulated inside.
The drug carrier according to 1. 4. The drug carrier according to claim 1, which comprises fine particles having a diameter of 0.02 to 250 μm. 5. The drug carrier according to claim 1, wherein the carrier is composed of at least one of macromolecules, microaggregates, fine particles, microspheres, nanospheres, liposomes and emulsions. . 6. The carrier is a phospholipid or a derivative thereof,
6. The drug carrier according to claim 1, further comprising a lipid other than phospholipid or a derivative thereof, and / or a stabilizer, and / or an antioxidant, and / or another surface modifier. 7. The compound having a site having a positive charge in the physiological pH range is at least one or more aliphatic first,
A compound having a secondary amino group, an amidino group, an aromatic primary or a secondary amino group, and a compound obtained by further combining the compound with a residue having at least one hydroxyl group.
7. The drug carrier according to claim 6. 8. The drug carrier according to claim 1, wherein the compound having a site having a positive charge in the physiological pH range is an amino sugar. 9. The drug carrier according to claim 8, wherein the amino sugar comprises a monosaccharide such as glucosamine, galactosamine, mannosamine, inoramic acid, or inoramic acid ester, and a free form or various glycosides thereof. 10. The drug carrier according to claim 8, wherein the amino sugar comprises an oligosaccharide such as chitin, a polysaccharide, and a free form or various glycosides thereof. 11. The hydrophilic polymer derivative is polyethylene glycol, a long-chain aliphatic alcohol, a sterol,
The drug carrier according to claim 2, which is a derivative of polyoxypropylene alkyl or glycerin fatty acid ester. 12. The drug carrier according to claim 3, wherein the drug for diagnosing and / or treating the vascular endothelial damage site is electrically neutral or anionic. 13. The drug for diagnosing and / or treating the vascular endothelial damage site is an anti-inflammatory agent, anti-cancer agent, enzyme agent, antibiotics, antioxidant, lipid uptake inhibitor, hormone agent, angiotensin converting enzyme inhibition. The drug carrier according to claim 3, which is an agent, a smooth muscle cell proliferation / migration inhibitor, a platelet aggregation inhibitor, a chemical mediator release inhibitor, or a vascular endothelial cell proliferation or inhibitor. 14. The drug carrier according to claim 3, wherein the drug for diagnosing and / or treating the vascular endothelial damage site is glycosaminoglycan and a derivative thereof. 15. The drug for diagnosing and / or treating the vascular endothelial damage site is an oligo- and / or polysaccharide, or a derivative thereof, according to claim 3.
The drug carrier according to item 2. 16. A drug for diagnosing and / or treating a site of vascular endothelial damage is various in-vivo diagnostic agents such as an X-ray contrast agent, a radioisotope-labeled nuclear medicine diagnostic agent, and a nuclear magnetic resonance diagnostic diagnostic agent. The drug carrier according to any one of claims 3 to 12. 17. The drug for diagnosing and / or treating the vascular endothelial injury site is heparin, theopherin,
The drug carrier according to claim 3, which is at least one of cyclic AMP (cAMP), captopril, papaverine, and forskolin.