JP2000319200A - Amidine derivative and drug carrier containing the same as constituent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new amidine derivative useful as a constituent of drug carrier capable of efficiently and safely carrying out the transfection of nucleic acids, polypeptides, genes and their analogues to cells. SOLUTION: The compound of the formula [A is an aromatic ring; X is -OCOO- or -(CH2)i-O-; (m) and (n) are each 0 or 1; (i) is 0-6; Y is a steroid], e.g. α-(5-cholesten-3β-oxy)-4-amidino-toluene hydrochloride, is obtained by condensing a compound of the formula: Z-(CH2)m-A-X [X is -OH or -(CH2)ii-Y (Y is a halogen; Z is a halogen, CN or a protected amidino)] to a steroid and then introducing thiourea into the part of Z when Z is a halogen, transforming Z to an amidino group when Z is CN or deprotecting Z when Z is a protected amidino group.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel amidine derivative and a drug carrier containing the same as a constituent.

[0002]

2. Description of the Related Art In recent years, studies have been actively conducted to apply closed packages such as liposomes, emulsions, and lipid microspheres as drug carriers to drug delivery systems (hereinafter, referred to as DDS). These closed parcels are generally prepared using phospholipids or derivatives thereof, or lipids other than sterols and phospholipids as basic membrane constituents. However, these basic constituents alone cause aggregation of closed parcels and retention in the body. It was not possible to overcome difficulties in various aspects of practical application such as a decrease in sex. Furthermore, it was very difficult to actually reach a target site as a DDS preparation.

[0003] Therefore, in order to improve the drug encapsulation rate and the cell adhesion of the closed package, a small amount of a cationized lipid such as stearylamine is blended to cationize the surface of the closed package within a physiological pH range. Attempts have been made to do so. In particular, cationic liposomes containing DNA are DNA
Is known to promote the transfer into cells, that is, transfection. However, it is desired to further enhance the transfection efficiency, expression rate, and safety. However, lipids that can be selected as cationized lipids are limited, and there is a strong demand for the development of cationized lipids for drug carriers that are highly safe and exhibit high functions. At present, as such cationized lipids, U.S. Pat.No. 4,897,355, U.S. Pat.No. 5,337,461, Japanese Patent Publication No. 2-292246, and Japanese Patent Publication No. 4-108391 have been reported. Sufficient effects have not been obtained.

[0004]

Therefore, DNA can be efficiently and safely introduced into cells, and it can be used for purposes other than transfer of DNA into cells, such as sites of vascular endothelial damage, nephritis, renal cancer, pneumonia, and lungs. There is a strong demand for the development of drug carriers that can reliably, efficiently and safely target drugs to affected sites such as cancer, hepatitis, liver cancer, pancreatic cancer, and lymphoma and are effective for DDS therapy.

Accordingly, it is an object of the present invention to provide a drug carrier capable of efficiently and safely transfecting nucleic acids, polynucleotides, genes and their analogs into cells, and a cationized lipid capable of forming the same. . It is also an object of the present invention to provide a drug carrier that effectively delivers drugs, peptides and proteins to a target site, and a cationized lipid and a drug carrier that can form the same.

[0006]

The above-mentioned objects are attained by the following invention.

(1) An amidine derivative represented by the following general formula (1) and a salt thereof.

[0008]

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(In the formula 1, A is an aromatic ring. X is —OCOO— or — (CH ₂ ) _i —O—, m is 0 or 1, n is 0 or 1, i
Represents a natural number from 0 or 1 to 6. Y is a steroid. (2) A drug carrier comprising the amidine derivative according to (1) as a constituent.

(3) The drug carrier according to (2), wherein a drug for diagnosis and / or treatment is enclosed in the carrier.

(4) The drug carrier according to (2) or (3), wherein the carrier comprises fine particles having a diameter of 0.02 to 250 μm.

(5) The above-mentioned (2) to (2) to wherein said carrier is composed of at least one of macromolecules, microaggregates, microparticles, microspheres, nanospheres, liposomes and emulsions.
The drug carrier according to any one of (4).

(6) The carrier comprises a phospholipid or a derivative thereof, and / or a lipid other than the 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 (2) to (5) above.

(7) The drug carrier according to any one of the above (2) to (6), wherein the drug for diagnosis and / or treatment is a nucleic acid, a polynucleotide, a gene or an analog thereof.

(8) The drug for diagnosis and / or treatment is an anti-inflammatory agent, a steroid agent, an anticancer agent, an enzyme agent, an enzyme inhibitor, an antibiotic, an antioxidant, a lipid uptake inhibitor, a hormone agent, an angiotensin. Converting enzyme inhibitor,
Angiotensin receptor antagonist, smooth muscle cell growth / migration inhibitor, platelet aggregation inhibitor, chemical mediator release inhibitor, vascular endothelial cell growth or inhibitor, aldose reductase inhibitor, mesangial cell growth inhibitor,
The drug carrier according to any one of the above (2) to (6), which is a lipoxygenase inhibitor, an immunosuppressant, an immunostimulant, an antiviral agent or a radical scavenger.

(9) The drug carrier according to any one of the above (2) to (6), wherein the drug for diagnosis and / or treatment is glycosaminoglycan and a derivative thereof.

(10) The drug carrier according to the above (2) to (6), wherein the drug for diagnosis and / or treatment is an oligo and / or polysaccharide and a derivative thereof.

(11) The drug carrier according to any one of the above (2) to (6), wherein the drug for diagnosis and / or treatment is a protein or a peptide.

(12) The drug for diagnosis and / or treatment is an in-vivo diagnostic agent such as an X-ray contrast agent, a radioisotope-labeled nuclear medicine diagnostic agent, or a diagnostic agent for nuclear magnetic resonance diagnosis. ) The drug carrier according to any one of (1) to (6).

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The compounds of the present invention are all novel compounds, and examples of their synthetic routes are shown below. However,
The present invention is not limited to these.

After the compound represented by the following general formula (2) is bonded to the steroid, the thiourea is introduced into the Z site when Z is a halogen, and the thiourea is introduced into the amidino when Z is CN. By converting to a group, or
When Z is a protected amidino group, it can be produced by deprotection.

Examples of steroids used as raw materials for synthesis include general compounds having a steroid skeleton such as cholesterol, cholesterol chloroformate, estrone, pregnenolone, estradiol and estriol.

[0023]

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(In the formula 2, A is an aromatic ring. X is —OH or — (CH ₂ ) _i —Y. M is 0 or 1, and i is 0 or a natural number from 1 to 6. Y represents a halogen, Z represents a halogen, CN or a protected amidino group.) The amidine derivative (1) thus produced can be obtained by a known separation and purification means (for example, chromatography, recrystallization). Can be isolated and collected.

The carrier of the present invention preferably has a particle size of 0.02 to 250 μm, particularly preferably 0.05 to 0.4 μm.

The structure may take various forms, and it is not necessary to limit the structure. In particular, macromolecules, microaggregates, fine particles, and the like having a potential function of encapsulating a drug at a high concentration therein can be considered. Most desirably, it consists of at least one of microspheres, nanospheres, liposomes and emulsions.

In the present invention, the components of the drug carrier need not be particularly limited as long as they can form the above-mentioned form, but in consideration of their safety and stability in vivo, It is desirable to incorporate a phospholipid or a derivative thereof, a lipid other than the phospholipid or a derivative thereof, or a stabilizer, an antioxidant, or another surface modifier.

Examples of the phospholipid include natural or synthetic phospholipids such as phosphatidylcholine (= lecithin), phosphatidylglycerol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, and cardioribine. Examples thereof include those hydrogenated according to the method.

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

Examples of the antioxidant include tocopherol homologs, that is, vitamin E and the like. Tocopherol has four isomers of α, β, γ, and δ, and any of them can be used in the present invention.

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

Examples of the hydrophilic polymer include 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 has a remarkable effect of improving blood retention.

The hydrophilic polymer may be a long-chain aliphatic alcohol, sterol, polyoxypropylene alkyl,
Alternatively, by using a derivative bonded to a hydrophobic compound such as glycerin fatty acid ester, the hydrophobic compound site can be stably inserted into the membrane of a drug carrier (for example, a liposome). This allows the hydrophilic polymer to be present on the drug carrier surface. Examples of the hydrophilic polymer derivative that can be specifically used in the present invention include polyethylene glycol-phosphatidylethanolamine and the like.

In the present invention, a pharmaceutically acceptable pharmacologically active substance, physiologically active substance or diagnostic substance can be used as the drug encapsulated in the drug carrier, depending on the purpose of diagnosis and / or treatment. .

There is basically no problem with the properties of the drug to be encapsulated in any substance, but the electrically neutral or anionic substance is more highly encapsulated due to the characteristic of the carrier surface having a positive charge. Rate can be expected.

The type of the drug to be encapsulated for treatment is not particularly limited as long as the formation of the drug carrier is not impaired. Specifically, nucleic acids, polynucleotides, genes and their analogs, anti-inflammatory agents, steroids, anticancer agents, enzyme agents, antibiotics, antioxidants, lipid uptake inhibitors, hormonal agents, angiotensin converting enzyme inhibitors, angiotensin Receptor antagonist, smooth muscle cell proliferation / migration inhibitor, platelet aggregation inhibitor, chemical mediator release inhibitor, vascular endothelial cell growth promoter or inhibitor, aldose reductase inhibitor, mesangial cell growth inhibitor, Examples include lipoxygenase inhibitors, immunosuppressants, immunostimulants, antiviral agents, radical scavengers, proteins, peptides and the like.

The type of the diagnostic drug to be enclosed is not particularly limited as long as the formation of the drug carrier is not impaired. Specifically, the drug carrier of the present invention, which includes an X-ray contrast agent, a radioisotope-labeled diagnostic agent for nuclear medicine, a diagnostic agent for nuclear magnetic resonance diagnosis, and the like, can be easily obtained by an ordinary method. It is shown below. The amidine derivative represented by the general formula (1) and other carrier components such as phospholipids are mixed in an organic solvent such as chloroform in the flask, and the organic solvent is distilled off, followed by vacuum drying to form a thin film on the inner wall of the flask. Let it form. Next, a drug is added to the flask and stirred vigorously to obtain a liposome dispersion. The drug carrier can be obtained as a dispersion by centrifuging the obtained liposome dispersion and decanting the supernatant to remove the unencapsulated drug. Further, it can also be obtained by mixing the above-mentioned respective components and discharging the mixture under high pressure by a high-pressure discharge type emulsifier.

[0038]

Next, the present invention will be described in more detail with reference to examples and test examples, but the present invention is not limited to these examples and test examples.

Example 1 α- (5-Cholesten-3β-oxy) -4-amidinotoluene Hydrochl
Synthesis of oride 0.020 g of 60% sodium hydride was dispersed in 5 mL of tetrahydrofuran, 5 mL of a tetrahydrofuran solution containing 0.986 g of cholesterol was added dropwise, and 10 mL of a 0.50 g solution of α-bromo-p-tolunitrile was further added dropwise.
After stirring overnight, the reaction mixture was poured onto ice, extracted with chloroform and dried over sodium sulfate. The solvent was distilled off under reduced pressure to give 4- (5-Cholesten-3β-oxy) as pale yellow crystals.
0.4308 g of methyl) benzonitrile was obtained. This compound 0.200
0 g was dissolved in a mixed solvent of 2 mL of methanol and 5 mL of chloroform, hydrogen chloride gas was introduced under ice-cooling for 10 minutes, and the mixture was stirred at room temperature overnight, and the solvent was distilled off under reduced pressure. Recrystallize with Methoxyiminomethyl-4
-(5-Cholesten-3β-oxymethl) benzene Hydrochloride
0.1226 g was obtained. 0.1220 g of this compound in 5 mL of chloroform
And 2 mL of a 2 mol / L ammonia methanol solution was added thereto. The mixture was heated and refluxed for 24 hours, and then the solvent was distilled off under reduced pressure. The obtained solid was recrystallized from chloroform and n-hexane to obtain α
-(5-Cholesten-3β-oxy) -4-amidinotoluene Hydrochlor
16 mg of ide were obtained.

The instrumental analysis data supports the structure of the following formula (3).

¹ H-nmr (CDCl3) δ (ppm): 8.80 (d, 8H,),
7.80 (d, 2H), 7.40 (d, 2H), 5.35 (t, 1H), 4.44 (s, 2H),
3.27-3.19 (m, 1H), 2.40-0.66 (m, 43H)

[0042]

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Example 2 Synthesis of 5-Cholesten-3β-yl 4-amidinophenoxyformate 4- (N-benzyloxycarbonylamidino) phenol
200 g, 0.225 g of triethylamine, 10 mL of methylene chloride and 5 mL of dimethylformamide were mixed, and a solution of 0.332 g of cholesterol chloroformate in 10 mL of methylene chloride was added dropwise under ice cooling. After stirring at room temperature all day and night, water was added and extracted with chloroform. After drying over sodium sulfate and evaporating the solvent under reduced pressure, the residue was purified by silica gel column chromatography and purified by 5-Cholesten-3β-yl 4- (N-benzyloxycarbonyl) amidin
0.2937 g of ophenoxyformate was obtained. This was dissolved in a mixed solvent of 10 mL of methylene chloride and 10 mL of ethanol, 0.1 g of 10% palladium on carbon was added, and the mixture was stirred under a hydrogen atmosphere for 1 hour.
After filtering off the palladium carbon, the solid obtained by evaporating the solvent under reduced pressure was purified by silica gel column chromatography to give 5-Cholesten-3β-yl 4-amidinophenoxyformate
0.0456 g was obtained.

The instrumental analysis data supports the structure of the following formula (4).

¹ H-nmr (CDCl 3) δ (ppm): 8.90 (d, 3H),
7.96 (d, 2H), 7.21 (d, 2H), 5.40 (t, 1H), 4.48-4.42 (m,
1H), 2.42-0.72 (m, 43H) Melting point 186-190 ℃

[0046]

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Example 3 Liposomes containing the amidine derivative of the present invention as a membrane component were prepared as follows.

3 μmol of the amidine derivative, 21 μmol of dipalmitoylphosphatidylcholine (DPPC) and 6 μmol of cholesterol are weighed in a 10 mL eggplant-shaped flask, and completely dissolved in 1 mL of chloroform. Chloroform is distilled off under reduced pressure by an evaporator to form a lipid thin film on the inner wall of the flask. Then contains 0.5 μmol calcein
1 mL of 10 mmol / L Tris-HCl buffer was added to the flask, and the mixture was vigorously shaken to obtain a calcein-encapsulated liposome (SUV) dispersion.

(Test Example 1) Measurement of Calcein Encapsulation Rate The encapsulation rate of calcein in liposomes was determined as follows. Add 40 mL of the 50-fold diluted liposome suspension to 2.0 mL of 10
Add to a mmol / L Tris-HCl buffer and measure the fluorescence intensity (excitation wavelength 490 nm, fluorescence wavelength 530 nm). The fluorescence intensity at this time is defined as Ft. Next, calcein not encapsulated in the liposome is quenched by adding 20 μL of a 10 mmol / L CoCl ₂ aqueous solution, and the fluorescence of calcein encapsulated in the liposome is measured. The fluorescence intensity at this time is defined as Fin. 20% Tr
By adding 20 μL of the itonX-100 solution, the liposomes are broken, and all calcein binds to Co ²⁺ and is quenched. The fluorescence intensity at this time is defined as Fq. The liposome retention efficiency is calculated by the following equation.

Retention efficiency (%) = (Fin−Fq × r) / (Ft
-Fq × r) × 100 r in the formula is a value corrected for the volume change of the reaction solution due to the addition of the liposome suspension and the drug, and is r = (2.0
+ 0.04 + 0.02 + 0.02) /2.0=1.04. Table 1 shows the results.

[0051]

[Table 1]

Example 4 A liposome for gene transfer containing an amidine derivative as a compound of the present invention as a membrane component was prepared as follows.

10 μmol of amidine derivative, 20 μmol of dilauroylphosphatidylcholine (DLPC), 20 μmol of dioleoylphosphatidylethanolamine (DOPE)
Is weighed to obtain 1 mL of chloroform solution A.

20 μL of chloroform solution A was placed in a 10 mL eggplant-shaped flask, and 1 mL of chloroform was further added. Chloroform was distilled off under reduced pressure by an evaporator to form a lipid thin film on the inner wall of the flask. Next, 1 mL of 40 ° C. distilled water was added, and the mixture was vigorously shaken and stirred to prepare liposomes.

After mixing 20 μg of the plasmid pcDNA / Amp (Invitrogen) incorporating the β-Galactosidase gene and the liposome, the mixture was stirred with a vortex mixer to prepare a lipid DNA complex.

(Test Example 2) Cells Cos-1 cells (ATCC N) purchased from Dainippon Pharmaceutical Co., Ltd.
o. CRL-1650) with Iscove's containing 10% fetal calf serum (FCS)
5% CO ₂ , 37 using modifided Dalbecco's medium (IMDM)
The cells were subcultured in an incubator at ° C.

Transfection Method 2 × 10 ⁴ Cos-1 cells were seeded on a 6-well multiwell plate to which 10% FCS IMDM medium (2 mL) was added, and cell culture was performed.
After culturing for about 24 hours, a lipid-DNA complex containing 0.2 μg of DNA was added, and the cells were cultured at 37 ° C. for 1 hour. After the culture, the medium used for gene transfer was replaced with a new one, and the culture was continued for another 24 hours.

Method for Detecting β-Galactosidase 24 hours after transfection, cells were fixed in a phosphate buffer (PBS) containing 2% formaldehyde for 5 minutes. After washing the cells with PBS, 2 mL of 5 mmol / L potassium ferricyanide.5 mmol / L potassium ferrocyanide.2
mmol / L magnesium chloride ・ 1mg 5-bromo-4-chloro-3-in
4 with PBS containing dolyl-β-galactopyranoside (X-gal)
Color developed for hours. The ratio of cells into which the gene was introduced was determined by counting the cells stained blue or image-processing the area of the blue-stained portion in five randomly extracted visual fields using a microscope.

Table 2 shows the results.

[0060]

[Table 2]

(Acute toxicity) As a result of an acute toxicity test by oral and intravenous administration using an ICR male mouse (5 weeks old), the LD50 of the compound of the present invention was 320 mg / kg or more, and the efficacy was Higher safety was confirmed compared to.

[0062]

As described above, the present invention provides a novel amidine derivative. As shown in the test examples, it was clarified that the drug carrier containing the amidine derivative of the present invention as a constituent had a higher transfection efficiency into cells than a conventional drug carrier.

From the above characteristics, the drug carrier of the present invention, in which a pharmaceutically acceptable pharmacologically active substance, physiologically active substance or diagnostic substance is encapsulated, is very effective for therapeutic and diagnostic purposes. It is a target.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 31/726 A61K 31/715 609 45/00 45/00 48/00 48/00 49/00 49/00 C 51/00 49/04 A 49/04 C07J 1/00 C07J 1/00 A61K 49/02 A F-term (reference) 4C076 AA17 AA19 AA95 CC05 CC07 CC11 CC15 CC16 CC17 CC27 CC30 CC32 CC35 CC50 DD38 DD49 DD63 DD70 FF31 FF70 4C084 AA02 AA03 AA13 AA19 AA27 BA44 DB01 DC40 MA24 NA12 NA13 ZB261 ZC031 4C085 HH03 HH05 HH07 JJ05 KA29 KB65 KB79 KB82 KB92 KB95 4C086 AA01 EA02 EA01 EA25 EA26 MA01 MA01 MA01 NA01 NA01 NA01 NA01 NA01 NA01 NA01 NA01 NA01 NA01 CC PA02 PA05 PB05 QQ01 RR08

Claims (12)

[Claims] An amidine derivative represented by the following general formula (1) and a salt thereof: (In the formula 1, A is an aromatic ring. X is -OCOO- or-(CH ₂ ) _i -O
-, Where m is 0 or 1, n is 0 or 1, i is 0 or 1.
Indicates a natural number up to 6. Y is a steroid. ) 2. A drug carrier comprising the amidine derivative according to claim 1 as a constituent. 3. The drug carrier according to claim 2, wherein a drug for diagnosis and / or treatment is encapsulated inside the carrier. 4. The drug carrier according to claim 2, wherein the carrier comprises fine particles having a diameter of 0.02 to 250 μm. 5. The method according to claim 1, wherein the carrier is a macromolecule, a fine aggregate, a fine particle,
The drug carrier according to any one of claims 2 to 4, comprising at least one of a microsphere, a nanosphere, a liposome, and an emulsion. 6. The carrier according to claim 1, wherein the carrier is a phospholipid or a derivative thereof.
The lipid according to any one of claims 2 to 5, comprising a lipid other than a phospholipid or a derivative thereof, and / or a stabilizer, and / or an antioxidant, and / or another surface modifier. Drug carrier. 7. The drug carrier according to any one of claims 2 to 6, wherein the drug for diagnosis and / or treatment is a nucleic acid, a polynucleotide, a gene, or an analog thereof. 8. The drug for diagnosis and / or treatment may be an anti-inflammatory agent, a steroid, an anticancer agent, an enzyme, an enzyme inhibitor, an antibiotic, an antioxidant, a lipid uptake inhibitor, a hormone, an angiotensin conversion. Enzyme inhibitor, angiotensin receptor antagonist, smooth muscle cell proliferation / migration inhibitor, platelet aggregation inhibitor, chemical mediator release inhibitor, vascular endothelial cell growth or inhibitor, aldose reductase inhibitor, mesangial cell proliferation An inhibitor, a lipoxygenase inhibitor, an immunosuppressant, an immunostimulant, an antiviral agent or a radical scavenger.
The drug carrier according to any one of the above. 9. The drug carrier according to any one of claims 2 to 6, wherein the drug for diagnosis and / or treatment is glycosaminoglycan and a derivative thereof. 10. The drug carrier according to claim 2, wherein the drug for diagnosis and / or treatment is an oligo and / or polysaccharide and a derivative thereof. 11. The drug carrier according to claim 2, wherein the drug for diagnosis and / or treatment is a protein or a peptide. 12. The drug for diagnosis and / or treatment is an in-vivo diagnostic agent such as an X-ray contrast agent, a radioisotope-labeled nuclear medicine diagnostic agent, or a diagnostic agent for nuclear magnetic resonance diagnosis. 7. The drug carrier according to any one of 6.