JPH0827030A - Medicine carrier recognizing kidney - Google Patents

Abstract

PURPOSE:To obtain a medicine carrier having high including ratio of a neutral or anionic medicine and extremely high targeting property to glomerulus existing in kidney, especially cortex resin and excellent in safety and capable of recognizing kidney. CONSTITUTION:This medicine carrier has at least one shape among macromolecule, fine aggregate, fine particles, minute globules, nanoglobule, lipozome and emulsion and has a residue having positive charge in physiological pH range on the surface and as necessary, bonds a hydrophilic polymer on the surface and encloses a medicine for diagnosing and treating kidney or intrarenal minute organ therein. The carrier has preferably 0.02-250mum, especially 0.05-0.4mum particle diameter. An aliphatic or aromatic primary, secondary, tertiary or quaternary amino group, amidino group or a residue binding the group to a residue having hydroxy group is preferably used as a residue having positive charge. Polyethylene glycol having remarkable effect improving blood residence is most preferably used as the water soluble polymer.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a drug carrier that recognizes the kidney.

[0002]

2. Description of the Related Art Kidney diseases are generally classified according to their disease site. That is, glomerular diseases (glomerulonephritis), diseases of the renal tubules, diseases of blood vessels in the kidney, diseases of the renal pelvis, and the like. The most common case is glomerulonephritis.

[0003] For example, in glomerulonephritis, various pathogenic mechanisms such as streptococcus and IgG are deposited on glomerular mesangial cells and capillary loops, which triggers proliferation and hardening of mesangial cells. Alternatively, it is said that permeabilization of plasma proteins in the glomerular basement membrane occurs, and it appears as a systemic disease state, but as a whole it is very complicated and it has not yet been grasped clearly. Is the current situation. The reason is that kidneys and glomeruli have unique properties. That is, 1) the blood flow in the kidney reaches 1.2 liters / minute in adults, which is 1/3 to 1/4 of the cardiac output, and the immune complex easily flows in. 2) thread Mesangium, which binds the capillaries of the spherical capillaries, is not just stromal tissue, but has receptors for various hormones and autacoid drugs on its surface, and plays an important role in regulating glomerular filtration rate.

Various drug therapies have been implemented to date for such various renal diseases. For example, for glomerular disorder factors, administration of corticosteroid drugs to suppress inflammatory response, immunosuppressive therapy for antibody production, and the like. However, in reality, these drug therapies have not been able to realize an efficient drug treatment even at present due to the strength of side effects on organs other than the kidney.

On the other hand, in recent years, research on a drug delivery system (DDS) for efficiently distributing a drug to a target organ has been actively conducted. For example, there is a method of using closed vesicles such as liposomes, emulsions, lipid microspheres and nanoparticles as a drug carrier, and a method of enhancing the targeting property by forming a polymer between a specific sugar chain and a drug.

However, there are various problems in putting these to practical use. For example, the biggest problem in using closed vesicles in the DDS field is that it is extremely difficult to control the pharmacokinetics. In particular, closed vesicles are likely to be captured in the liver and spleen, which makes it difficult to apply them to other organs. In particular, targeting to the kidney was hardly considered.

That is, the kidney passes a large amount of drug due to its large blood flow volume, but targeting for accumulating the drug in the kidney, more specifically, in organelles in the kidney such as glomerulus is considered at all. Was not done. Although some drugs were distributed to the kidneys, they were excreted immediately and had little retention in the kidneys, and their drug effects were not sufficiently exerted. Further, a drug carrier that assists retention is not realized.

[0008]

SUMMARY OF THE INVENTION In view of the above problems, the object of the present invention is to have a high drug encapsulation rate and excellent in-body stability, and to recognize kidneys and renal organelles such as glomeruli. The purpose of this is to provide a drug carrier capable of actively accumulating the drug.

[0009]

Means for Solving the Problems In the course of research on factors that control the pharmacokinetics of drug carriers, the present inventors have surprisingly surprisingly found that the surface of drug carriers is exposed to physiological pH in the physiological pH range. It was found that the retention of the charged residue dramatically improves the accumulation property in the kidney, particularly in the glomerulus which is mainly distributed in the renal cortex.

Even more surprisingly, when a hydrophilic polymer is bound to the surface of a carrier to which the positively charged residue is bound, even if such a polymer binds to the surface, Accumulation in the kidney (glomerulus) is not impeded at all, while on the other hand, stability of the drug carrier in the blood is prevented by preventing aggregation in the blood, and retention in the blood is prevented by avoiding capture in the liver. The present invention has been completed by finding that improvement is possible.

The above-mentioned present invention is as follows.

(1) A drug characterized in that it has a residue having a positive charge in the physiological pH range on the surface, and a drug for diagnosing / treating the kidney or organs within the kidney is enclosed therein. Carrier.

(2) The surface has a residue which is positively charged in a physiological pH range, and further has a hydrophilic polymer bonded thereto, and a drug for diagnosing / treating the kidney or organs within the kidney is enclosed therein. The drug carrier according to (1) above, characterized in that

(3) The residue having a positive charge in a physiological pH range has one or more aliphatic primary, secondary, tertiary and quaternary amino groups, amidino group, aromatic primary, secondary, tertiary and aromatic groups. The drug carrier according to (1) or (2) above, which is a quaternary amino group and a residue thereof having one or more hydroxyl groups bonded thereto.

(4) The drug carrier according to the above (2) to (3), wherein the hydrophilic polymer is polyethylene glycol.

(5) The drug carrier according to the above (1) to (4), which has at least one form of macromolecules, microaggregates, fine particles, microspheres, nanospheres, liposomes and emulsions.

(6) The drug carrier as described in (1) to (5) above, which has a particle size of 0.02 to 250 μm.

(7) Diagnosis of the kidney or organs within the kidney
The encapsulated drug for treatment is at least one of an anti-inflammatory agent, an aldose reductase inhibitor, a mesangial cell growth inhibitor, a lipoxygenase inhibitor, an antioxidant, and a radical scavenger (1) to ( 6)
The drug carrier according to 1.

The drug carrier of the present invention has a residue having a positively charged residue in the physiological pH range on the surface thereof, and a kidney in which a drug for diagnosing and treating the kidney, particularly the glomerulus, is encapsulated therein, particularly the glomerulus. It is a drug carrier that recognizes and, if necessary, a hydrophilic polymer bound to the surface of the drug carrier.

Various structures are conceivable as the structure of the drug carrier of the present invention. In particular, macromolecules, microaggregates, microparticles, microspheres having a potential function capable of encapsulating a drug therein at a high concentration, Examples include nanospheres, liposomes, and emulsions, and a plurality of these may be combined. Of these, liposomes and emulsions are most desirable.

The drug carrier of the present invention has a particle size of 0.02 to 25.
A size of 0 μm, especially 0.05 to 0.4 μm is preferable.

The drug carrier of the present invention can be blended with other constituents, and in its form, it can bind to the surface a residue having a positive charge in the physiological pH range and further bind a hydrophilic polymer. The formulation is not particularly limited as long as it can form a carrier, but from the viewpoint of its safety and the provision of a stable drug carrier in vivo, phospholipid or its derivative, other than phospholipid It is desirable to add a lipid or its derivative, a stabilizer, an antioxidant and the like.

In the present invention, the residue 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 as the residue having a positive charge, 1 or Multiple aliphatic first, second,
Most preferred are tertiary and quaternary amino groups, amidino groups, aromatic primary, secondary, tertiary and quaternary amino groups, and combinations of these with residues having one or more hydroxyl groups.

Various methods are conceivable for binding these residues to a drug carrier. As a method for stably binding to a phospholipid or its derivative, a drug carrier composed of a lipid other than phospholipid or its derivative, , A residue is bonded directly or through a residue having one or more hydroxyl groups to a hydrophobic compound such as long-chain aliphatic alcohol, sterol, polyoxypropylene alkyl, or glycerin fatty acid ester, and these hydrophobic moieties A method of stably inserting the drug carrier into the surface of the drug carrier is desirable.

As the substance having a residue having a positive charge in the above physiological pH range and capable of being inserted into the surface of the drug carrier, derivatives of amino sugars such as galactosamine, derivatives of basic amino acids such as lysine and arginine, and bases Examples include derivatives of sex peptides.

In the present invention, the physiological pH range refers to the pH range in the living body, particularly in the blood, the cytoplasm, and the inside of the organelle present therein, and specifically, the pH range of 4
-10, pH 6-8.

In the present invention, the hydrophilic polymer bound to 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 acids, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan and the like. Among them, polyethylene glycol is most desirable because it has a remarkable effect of improving blood retention.

Various methods are conceivable for binding the hydrophilic polymer to the surface of the drug carrier. Stable binding to the drug carrier consisting of phospholipid or its derivative, or lipid other than phospholipid or its derivative. As a method of making the polymer, long-chain aliphatic alcohol, sterol,
A method in which a hydrophobic compound such as polyoxypropylene alkyl or glycerin fatty acid ester is bound and the hydrophobic moiety is used to stably insert into the drug carrier surface is desirable.

The drug to be encapsulated inside the drug carrier is
A pharmaceutically acceptable pharmacologically active substance, a physiologically active substance or a diagnostic substance can be used according to the purpose of diagnosis / treatment of the kidney, and an anti-inflammatory agent, an aldose reductase inhibitor, a mesangial cell growth inhibitor, Examples include lipoxygenase inhibitors, and specific examples include dexamethasone as an anti-inflammatory agent, esculetin, baicalein, BW-A4C as a lipoxygenase inhibitor, and heparin as a mesangial cell growth inhibitor.

As a drug for the substance to be encapsulated, any substance is basically acceptable, but a substance that is electrically neutral or anionic is highly encapsulated because the surface of the drug carrier has a positive charge. You can expect a rate.

[0031]

EXAMPLES Next, the present invention will be described more specifically by showing examples and test examples.

Example 1 Glucamine-palmitic acid derivative (the structure of which is shown in the following formula 1 )
Is shown as a membrane constituent component.
Preparation of some

[0033]

Embedded image

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 100 mM): 840 μl Cholesterol (concentration 100 mM): 240 μl Glucamine-palmitic acid derivative (10 mM): 1200 μl 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 μg of heparin as a mesangial cell growth inhibitor was dissolved 3
00 mM sorbitol / 10 mM Tris-HCl buffer 4
By adding ml to the flask and vigorously stirring and stirring, a liposome (MLV) dispersion was obtained.

The 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 /
By dispersing in 10 mM Tris-hydrochloric acid, the title liposome dispersion liquid retaining heparin was obtained.

(Example 2) Glucamine-palmitic acid derivative and polyethylene glycol
Recall-phosphatidyl ethanolamine derivative
Containing (the structure is shown in the following formula 2) as a film constituent component
Of liposomes containing heparin

[0038]

Embedded image

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 100 mM): 840 μl Cholesterol (concentration 100 mM): 240 μl Glucamine-palmitic acid derivative (10 mM): 1200 μl Polyethylene glycol-phosphatidylethanolamine derivative (concentration 0.4 W / V%): 1000 μl Below, the same procedure as in Example 1 was carried out to obtain the title liposome dispersion retaining heparin.

(Test Example 1) Measurement of drug encapsulation ratio The encapsulation ratio of heparin, which is the mesangium cell growth inhibitor 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, polyethylene glycol-
A liposome dispersion liquid was prepared in the same manner as in the above example without using the phosphatidylethanolamine derivative and the glucamine-palmitic acid derivative. Then, the encapsulation rate of heparin was determined by the same method as described above.

The results are shown in FIG. FIG.
The numbers in the liposome dispersion column in the table are as follows. Indicates a liposome containing a glucamine-palmitic acid derivative as a membrane constituent, and a liposome containing a glucamine-palmitic acid derivative and a polyethylene glycol-phosphatidylethanolamine derivative as a membrane constituent represents a neutral liposome.

As is clear from the results, the drug carrier containing the cationic compound or the cationic compound and the hydrophilic polymer derivative showed a higher encapsulation rate of heparin than the neutral drug carrier containing no cationic compound.

Test Example 2 Measurement of Blood Kinetics A liposome dispersion 500 encapsulating 100 mM carboxyfluorescein prepared according to Examples 1 and 2 from the femoral vein of SD male rats anesthetized with urethane.
μl was injected intravenously. Blood was collected in Eppendorf tubes over time. Blood kinetics was measured by measuring the fluorescence intensity of the collected blood.

For comparison, a liposome dispersion prepared according to the above Example without using a glucamine-palmitic acid derivative or a polyethylene glycol-phosphatidylethanolamine derivative was prepared in the same manner as above. Blood kinetics was measured.

The results are shown in FIG. The numbers in the column of liposome dispersion in FIG. 2 are as follows. Indicates a liposome containing a glucamine-palmitic acid derivative as a membrane constituent, and a liposome containing a glucamine-palmitic acid derivative and a polyethylene glycol-phosphatidylethanolamine derivative as a membrane constituent represents a neutral liposome.

As is clear from the results, it was confirmed that the drug carrier of the present invention containing a cationic compound as a membrane constituent has higher blood retention than a drug carrier not containing it. Further, it was confirmed that the drug carrier further surface-modified with the hydrophilic polymer has higher retention in blood than the case where only the cationic compound is used.

Test Example 3 Measurement of Accumulation in Kidney (1) 100 mM carboxyfluorescein prepared according to Examples 1 and 2 was encapsulated from the femoral vein of SD male rats anesthetized with urethane. Liposome dispersion 500
μl was injected intravenously. After 3 hours, the kidney of the rat was removed.
Carboxyfluorescein was extracted from the excised kidney, and the distribution of liposomes in the kidney was measured by measuring the fluorescence intensity.

For comparison, polyethylene glycol-
With respect to the liposome dispersion prepared according to the above example without using the phosphatidylethanolamine derivative and the glucamine-palmitic acid derivative, the distribution of liposomes to the kidney was measured by the same method as above.

The results are shown in FIG. The numbers in the column of liposome dispersion in FIG. 3 are as follows. Indicates a liposome containing a glucamine-palmitic acid derivative as a membrane constituent, and a liposome containing a glucamine-palmitic acid derivative and a polyethylene glycol-phosphatidylethanolamine derivative as a membrane constituent represents a neutral liposome.

As is clear from the results, the drug carrier of the present invention containing a cationic compound, or a cationic compound and a hydrophilic polymer derivative as a membrane constituent,
It has a higher degree of accumulation in the kidney than drug carriers that do not contain them. Therefore, the drug carrier of the present invention has excellent renal targeting properties.

Test Example 4 Measurement of Accumulation in Kidney (2) Encapsulation of 100 mM carboxyfluorescein prepared according to Examples 1 and 2 from the femoral vein of SD male rats anesthetized with urethane. Liposome dispersion 500
μl was injected intravenously. After 3 hours, the kidney of the rat was removed.
The excised kidney was carefully excised with scissors to obtain a renal cortex and further subdivided. The obtained piece of renal cortex was placed on a 60-mesh sieve and crushed with a spatula to give a paste.

After washing with physiological saline from above the sieve, it was passed through a sieve of 83 mesh, 120 mesh and 200 mesh to finally obtain a glomerular fraction remaining on the sieve of 200 mesh. Carboxyfluorescein was extracted from the glomerular fraction, and the fluorescence intensity was measured to measure the distribution of liposomes in the glomerulus. Furthermore, carboxyfluorescein was extracted from the fractions other than the glomerulus, and the fluorescence intensity was measured.

For comparison, polyethylene glycol-
The distribution of liposomes in the glomerulus was measured in the same manner as above for the liposome dispersion prepared according to the above example without using the phosphatidylethanolamine derivative and the glucamine-palmitic acid derivative.

The results are shown in FIG. The numbers in the column of liposome dispersion in FIG. 4 are as follows. Indicates a liposome containing a glucamine-palmitic acid derivative as a membrane constituent, and a liposome containing a glucamine-palmitic acid derivative and a polyethylene glycol-phosphatidylethanolamine derivative as a membrane constituent represents a neutral liposome.

As is clear from the results, the drug carrier of the present invention containing a cationic compound, or a cationic compound and a hydrophilic polymer derivative as a membrane constituent,
Accumulation in the glomerulus is higher than that of drug carriers that do not contain them. Therefore, the drug carrier of the present invention is excellent in the targeting property to the glomerulus.

Test Example 5 Acute Toxicity Test 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 the conventional drug carrier. 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. In addition, since renal function decline was reported due to basic drugs (such as the antibiotic puromycin), a separate renal function test was performed.

A test solution was prepared as follows.

(1) Glucamine-palmitic acid derivative
A liposome dispersion containing the glucamine-palmitic acid derivative as a membrane constituent was obtained in the same manner as in Example 1, except that the heparin of the liposome dispersion of the present invention contained in the membrane constituent was not added. This was concentrated using an ultrafiltration membrane and further diluted with sterile distilled water for injection to obtain a test solution.

(2) Glucamine-palmitic acid derivative and
And polyethylene glycol-phosphatidyl ethano
Of the present invention containing a ruamine derivative as a membrane constituent
In the same manner as in Example 2, except that heparin was not added, a liposome dispersion containing a glucamine-palmitic acid derivative and a polyethylene glycol-phosphatidylethanolamine derivative as membrane constituents was obtained. . This was concentrated using an ultrafiltration membrane and further diluted with sterile distilled water for injection to obtain a test solution.

(3) For comparison of conventional liposome dispersion liquids , neutral liposome dispersion liquids were obtained in the same manner as in the examples without using glucamine-palmitic acid derivative and polyethylene glycol-phosphatidylethanolamine derivative. It was This was concentrated using an ultrafiltration membrane and further diluted with sterile distilled water for injection to obtain a test solution.

The test was conducted by the following method.

(1) Lethal Toxicity Test Quarantine 5-week-old SD male rats were divided into 3 groups per group,
The above test solution (2.0 ml) was intraperitoneally administered once. On the other hand, as a solvent control group, 2.0 ml of physiological saline was administered. After the administration of the test solution, the general condition was carefully observed at least once a day for 16 days, and toxicity signs and mortality were recorded.

(2) Renal Function Test Five-week-old SD male rats quarantined were divided into three groups per group,
1.0 ml of the above test solution was administered once a day for 10 days. On the other hand, a puromycin aqueous solution was similarly administered as a renal function decline positive control, and physiological saline was similarly administered as a solvent control. Urine was collected during 10 days of the test period and its protein content was measured.

The results of the lethal toxicity test are shown in Table 1. Also,
The results of the renal function test are shown in FIG. The symbols in the test substance column in the table and in FIG. 5 are as follows.
Is a liposome containing a glucamine-palmitic acid derivative as a membrane constituent, a liposome containing a glucamine-palmitic acid derivative and a polyethylene glycol-phosphatidylethanolamine derivative as a membrane constituent, a neutral liposome, Solvent control (saline), puromycin solution (only renal function test)
Indicates.

[0067]

[Table 1]

As shown in the above test results, the drug carrier of the present invention did not die during the observation period. 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 because from the subsequent organ findings, conventional drug carriers cause the formation of aggregates in the blood due to the association with blood cell components or the like in the blood or the association between liposomes and the formation of embolisms in the lungs and the like. It was confirmed that there is.

In the renal function test, puromycin, which is a positive control, increased the urinary protein content 4 days after the start of administration, whereas the various liposome administration groups and the solvent control group showed urinary protein content. No increase in quantity was observed.

From these results, the drug carrier of the present invention has an effect on renal function as compared with conventionally known drug carriers.
It can be said that it has extremely low toxicity and is highly safe.

[0071]

INDUSTRIAL APPLICABILITY As described above, the drug carrier of the present invention has a higher encapsulation rate of a neutral or anionic substance than conventional drug carriers, and is a thread that is often present in the kidney, particularly in the renal cortex. Very good targeting ability to the sphere. It is also very safe.

From these characteristics, the novel drug carrier of the present invention in which a pharmaceutically acceptable pharmacologically active substance, physiologically active substance or diagnostic substance is encapsulated is used for the purpose of diagnosis and treatment in the kidney, particularly in the glomerulus. Very effective against. For example, encapsulation of heparin or other sulfated polysaccharides is considered to be useful as a therapeutic agent for glomerulonephritis that suppresses the proliferation of glomerular mesangial cells in the kidney.

[Brief description of drawings]

FIG. 1 shows the measurement results of the drug (heparin) encapsulation rate of neutral liposomes as a comparison with the drug carrier (liposomes) of the present invention.

FIG. 2 shows the measurement results of blood kinetics of neutral liposomes as a comparison with the drug carrier (liposomes) of the present invention.

FIG. 3 shows the results of measuring the accumulation of neutral liposomes in the kidney as a comparison with the drug carrier (liposomes) of the present invention.

FIG. 4 shows the measurement results of the accumulation of neutral liposomes in the glomerular fraction and non-glomerular fraction of the kidney as a comparison with the drug carrier (liposome) of the present invention.

FIG. 5 shows the measurement results of urinary protein after administration of the drug carrier (liposome) of the present invention and each comparative product.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display area A61K 49/00 Z

Claims (7)

[Claims] 1. A drug carrier characterized in that it has a residue having a positive charge in a physiological pH range on the surface thereof, and a drug for diagnosing / treating the kidney or organs within the kidney is enclosed therein. . 2. A surface has a residue having a positive charge in a physiological pH range, and further has a hydrophilic polymer bonded thereto, and a drug for diagnosing / treating a kidney or an organ within the kidney is enclosed therein. The drug carrier according to claim 1, wherein 3. A residue having a positive charge in a physiological pH range comprises:
One or more aliphatic primary, secondary, tertiary and quaternary amino groups, amidino group, aromatic primary, secondary, tertiary and quaternary amino groups, and residues having one or more hydroxyl groups The drug carrier according to claim 1, which is bound to the drug carrier. 4. The drug carrier according to claim 2, wherein the hydrophilic polymer is polyethylene glycol. 5. The drug carrier according to claim 1, which has a form of at least one of a macromolecule, a microaggregate, a microparticle, a microsphere, a nanosphere, a liposome, and an emulsion. 6. The drug carrier according to claim 1, which has a particle size of 0.02 to 250 μm. 7. The encapsulated drug for diagnosing / treating the kidney or organs within the kidney is at least one of an anti-inflammatory agent, an aldose reductase inhibitor, a mesangial cell growth inhibitor, and a lipoxygenase inhibitor. Claim 1
7. The drug carrier according to items 6 to 6.