JP2013053103A - Liver-accumulative nanoparticle having drug encapsulated therein - Google Patents

Liver-accumulative nanoparticle having drug encapsulated therein Download PDF

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JP2013053103A
JP2013053103A JP2011192838A JP2011192838A JP2013053103A JP 2013053103 A JP2013053103 A JP 2013053103A JP 2011192838 A JP2011192838 A JP 2011192838A JP 2011192838 A JP2011192838 A JP 2011192838A JP 2013053103 A JP2013053103 A JP 2013053103A
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drug
liver
nanoparticles
ion
ribavirin
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Tsutomu Ishihara
務 石原
Toru Mizushima
徹 水島
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Ltt Bio-Pharma Co Ltd
株式会社Lttバイオファーマ
Nihon Univ
学校法人日本大学
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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00Nanotechnology for materials or surface science, e.g. nanocomposites
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51Nanocapsules; Nanoparticles
    • A61K9/5107Excipients; Inactive ingredients
    • A61K9/5115Inorganic compounds
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51Nanocapsules; Nanoparticles
    • A61K9/5107Excipients; Inactive ingredients
    • A61K9/5123Organic compounds, e.g. fats, sugars
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/48Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51Nanocapsules; Nanoparticles
    • A61K9/5107Excipients; Inactive ingredients
    • A61K9/513Organic macromolecular compounds; Dendrimers
    • A61K9/5146Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153Polyesters, e.g. poly(lactide-co-glycolide)
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Abstract

PROBLEM TO BE SOLVED: To provide a liver-integrated nanoparticle in which a drug is encapsulated, which efficiently encapsulates the drug, and stably traps the drug in the particle and easily accumulates in the liver.
SOLUTION: Drugs include poly DL or L-lactic acid or poly (DL or L-lactic acid / glycolic acid) copolymer, conjugate of arabinogalactan and biodegradable cationic polymer, metal ion and basic Drug-encapsulated nanoparticles obtained by encapsulating in nanoparticles composed of low molecular weight compounds. Examples of drugs include anti-hepatitis virus drugs, liver cancer therapeutic drugs or liver function improving drugs, and excellent liver accumulation and sustainability. It is a drug-encapsulated nanoparticle exhibiting sex.
[Selection] Figure 7

Description

  The present invention relates to a nanoparticle encapsulating a drug, and more particularly to a liver-integrated nanoparticle encapsulating a drug that can further reduce the pharmacological effects and side effects of the drug by accumulating in the liver.

When infected with hepatitis C virus, it progresses to chronic hepatitis at a high rate, and may lead to cirrhosis and liver cancer. In Japan, it is estimated that there are about 2 million people infected with hepatitis C virus, and 20,000 people die each year due to liver cancer that progressed from hepatitis C caused by this infection.
As the treatment method, interferon or a combination therapy of peginterferon obtained by chemically modifying polyethylene glycol with interferon and an anti-hepatitis virus drug such as ribavirin is mainly used.

  However, the cure rate of combination therapy with peginterferon and ribavirin, an anti-hepatitis virus drug, is only about 50% after 48 weeks of administration, and the treatment involves side effects. In particular, the side effect of hemolytic anemia caused by ribavirin is a major barrier to complete healing.

  In recent years, a drug delivery system, that is, a drug delivery system (DDS: Drug Delivery System) based formulation (DDS formulation) has been actively developed, targeting the affected area, sustained action, sustained drug release, side effects It is widely applied to various drugs for the purpose of mitigation.

The inventors of the present invention aim at sustained-release targeting in which a water-soluble non-peptide low-molecular-weight drug is hydrophobized with a metal ion and encapsulated in polylactic acid (PLA) or polylactic acid-glycol copolymer (PLGA) microparticles. Proposed a nanoparticle preparation (Patent Document 1).
Furthermore, the present inventors hydrophobized a water-soluble non-peptide low molecular weight drug with a metal ion, added a basic low molecular weight compound, and added this to polylactic acid (PEG-PLA) or polylactic acid-glycol to which polyethylene glycol was bound. We have proposed nanoparticles encapsulated in a copolymer (PEG-PLGA). These nanoparticles are referred to as stealth-type nanoparticles and have reduced retention and accumulation in the liver (Patent Document 2).

  By the way, a preparation in which a synthetic polymer chain bonded with galactose is coated on the surface of polylactic acid (PLA) nanoparticles is known (Non-patent document 1, Non-patent document 2). The therapeutic effect has been verified (Non-patent Document 3). In addition, it has been confirmed that a preparation in which a conjugate obtained by binding arabinogalactan and polylysine and DNA (polylactic acid is not used) reaches the liver (Non-patent Document 4). Furthermore, nanoparticles composed of a conjugate of dextran and polylysine and polylactic acid (PLA) are disclosed (Non-patent Document 5).

  However, the drug is applied to nanoparticles composed of polylactic acid or poly (lactic acid / glycolic acid) copolymer, a conjugate of arabinogalactan and a biodegradable cationic polymer, a metal ion and a basic low molecular weight compound. The encapsulated liver-accumulating nanoparticles are not known.

International publication WO 2004/084871 International Publication WO 2007/076044

Biomaterials, 15.13. 1035-1042 (1994) Biomaterials, 22. 45-51 (2001) Hepatology. 32 (6), 1300-8, (2000) Prep. Biochem. Biotech., 29, 353-370 (1999) Bioconjugate Chemistry, 8, 735-742 (1997)

  Therefore, an object of the present invention is to provide liver-integrated nanoparticles encapsulating a drug that can encapsulate the drug efficiently, stably confine the drug in the particle, and easily accumulate in the liver.

  In order to solve this problem, the present inventors have intensively studied. As a result, the drug was converted to poly DL or L-lactic acid or poly (DL or L-lactic acid / glycolic acid) copolymer, arabinogalactan and biodegradable cation. Confirming that drug-encapsulated nanoparticles obtained by encapsulating in a nanoparticle composed of a conjugate with a functional polymer, a metal ion, and a basic low molecular weight compound exhibit excellent liver accumulation and persistence, The present invention has been completed.

  In particular, the present invention is characterized in that it exhibits excellent liver accumulation and durability by incorporating a conjugate of arabinogalactan and a biodegradable cationic polymer into the particle surface.

Therefore, the present invention provides nanoparticles that are particularly advantageous for drugs for treating liver diseases because they efficiently encapsulate drugs, stably confine the drugs in the particles, and accumulate in the liver.
For example, ribavirin, an anti-hepatitis virus drug, has strong side effects due to hemolytic anemia, so it was difficult to administer until the patient was completely cured, but it was stabilized by encapsulating the nanoparticles in the present invention. The side effect of hemolytic anemia can be avoided by eliminating the interaction between red blood cells and ribavirin in the blood.
Accordingly, the present invention provides liver-integrating nanoparticles encapsulating in particular anti-hepatitis virus drugs, liver cancer therapeutic drugs or liver function improving drugs.
Furthermore, this invention provides the pharmaceutical which uses the said nanoparticle as an active ingredient.

More specifically, the present invention has the following configuration. That is,
(1) A drug comprising a poly DL or L-lactic acid or a poly (DL or L-lactic acid / glycolic acid) copolymer, a conjugate of an arabinogalactan and a biodegradable cationic polymer, a metal ion and a basic low Drug-encapsulated liver-integrating nanoparticles, characterized in that they are encapsulated in nanoparticles composed of molecular compounds;
(2) Liver-accumulating nanoparticles encapsulating the drug according to 1 above, wherein the particle diameter is 20 to 300 nm;
(3) Liver-accumulating nanoparticles encapsulating the drug according to 1 above, wherein the metal ion is one or more of zinc ion, iron ion, copper ion, nickel ion, beryllium ion, manganese ion or cobalt ion ;
(4) Basic low molecular weight compounds are (dimethylamino) pyridine, pyridine, piperidine, pyrimidine, pyrazine, pyridazine, quinoline, quinuclidine, isoquinoline, bis (dimethylamino) naphthalene, naphthylamine, morpholine, amantadine, aniline, spermine, spermidine , Hexamethylenediamine, putrescine, cadaverine, phenethylamine, histamine, diazabicyclooctane, diisopropylethylamine, monoethanolamine, diethanolamine, triethanolamine, ethylamine, diethylamine, triethylamine, methylamine, dimethylamine, trimethylamine, triethylenediamine, diethylenetriamine, One selected from the group consisting of ethylenediamine and trimethylenediamine, or Liver Accumulation nanoparticles encapsulating drugs according to the above 1 is more than a species;
(5) The drug is a drug having a phosphate group, a sulfate group or a carboxyl group to be hydrophobized by the metal ion in the molecule, or a group to which these groups are bonded. Liver-accumulated nanoparticles encapsulating the drug according to 1 above;
(6) Liver-integrated nanoparticles encapsulating the drug according to 1 or 5 above, wherein the drug is an anti-hepatitis virus drug, a liver cancer therapeutic drug or a liver function improving drug;
(7) Liver-accumulating nanoparticles encapsulating the drug according to 6 above, wherein the anti-hepatitis virus drug is ribavirin or ribavirin-phosphate;
(8) A pharmaceutical comprising as an active ingredient liver-accumulating nanoparticles encapsulating the drug according to 1 to 7 above;
It is.

  That is, the present invention relates to a drug comprising poly DL or L-lactic acid or poly (DL or L-lactic acid / glycolic acid) copolymer, a conjugate of arabinogalactan and a biodegradable cationic polymer, a metal ion and a base. It is characterized by producing liver-accumulating nanoparticles encapsulated in nanoparticles composed of a low molecular weight compound.

  The liver-accumulating nanoparticles encapsulating a drug provided by the present invention are particularly advantageous for drugs for treating liver diseases because they efficiently encapsulate the drug, stably entrap the drug in the particle, and accumulate in the liver. Demonstrate sex. Further, since the constituent component of the nanoparticles is a biodegradable polymer, the encapsulated drug is released little by little by being gradually degraded in vivo, and as a result, the pharmacological effect can be sustained over a long period of time.

The greatest feature of the present invention is that liver targeting is possible because of its high accumulation in the liver. It is known that a molecule having galactose binds to a liver parenchymal cell receptor and is taken up into the cell via endocytosis. Therefore, the drug can be transported to the liver by arranging a sugar chain mainly composed of galactose on the surface of the nanoparticle of the present invention.
As a result, the liver-accumulating nanoparticles encapsulating the drug provided by the present invention have the feature that they can be easily prepared into any dosage form of oral and parenteral dosage forms.

  Therefore, it is particularly excellent in terms of providing nanoparticles that improve targeting to the liver and sustained sustained release, which have not been sufficiently achieved so far, and efficiently exert pharmacological effects and reduce side effects in the affected area. Is.

It is the figure which showed the result of Example 1, and showed the production | generation of the conjugate | bonded_body of arabinogalactan and polylysine. It is the figure which showed the result of Example 2, and showed the result of the iron ion and diethanolamine, and the particle | grain encapsulation rate. It is the figure which showed the result of Example 2, and showed the result of the conjugate | bonded_body of arabinogalactan and polylysine, diethanolamine, and particle | grain encapsulation rate. It is the figure which showed the result of Example 2, and showed the result of addition conditions and particle | grain enclosure rate. It is the figure which showed the result of Example 3, and showed the result of the aggregation test of a nanoparticle. It is the figure which showed the result of Example 4. A microscopic image of the cells was shown. It is the figure which showed the result of Example 4, and showed the result of the intracellular uptake | capture of ribavirin. It is the figure which showed the result of Example 5, and showed the result of the liver accumulation property test of ribavirin.

  The liver-accumulating nanoparticles encapsulating a drug, which is a basic aspect of the present invention, contains a drug, polyDL or L-lactic acid or poly (DL or L-lactic acid / glycolic acid) copolymer, arabinogalactan and in vivo degradation. It is obtained by encapsulating in a nanoparticle composed of a conjugate with a cationic cationic polymer, a metal ion, and a basic low molecular weight compound.

  The liver-accumulating nanoparticles encapsulating a drug provided by the present invention has one feature in that a conjugate of arabinogalactan and a biodegradable cationic polymer is used as one of the components forming the nanoparticles. Have. That is, by using arabinogalactan and a biodegradable cationic polymer, nanoparticles can be easily accumulated in the liver.

  Liver-accumulating nanoparticles encapsulating the drug of the present invention provided by the above are tablets, capsules, orally administered drugs such as powders, intravenous preparations, topical injection preparations, nasal drops, eye drops, inhalation It can be administered by preparing a preparation for parenteral administration such as an agent or a spray.

The liver-accumulating nanoparticles encapsulating the drug provided by the present invention can be prepared, for example, by the following means.
That is, a drug, a poly DL or L-lactic acid or poly (DL or L-lactic acid / glycolic acid) copolymer, a conjugate of arabinogalactan and a biodegradable cationic polymer, a metal ion, a base Nanoparticles can be produced by dissolving or suspending a low molecular weight compound in an organic solvent and gradually adding it to a large amount of water (Oil-in-Water type solvent diffusion method).
In this case, nanoparticles having various physical properties can be produced by adjusting the amount and the addition rate of the drug.

  Examples of biodegradable cationic polymers used in the conjugates of arabinogalactan and biodegradable cationic polymers include polylysine, polyarginine, polyhistidine, polypeptides rich in basic amino acids, chitin, chitosan, etc. Polylysine is preferably used.

  Conjugates of arabinogalactan and biodegradable cationic polymer are prepared by dissolving dialyzed and purified arabinogalactan together with sodium cyanoborohydride and biodegradable cationic polymer in an aqueous solvent such as sodium hydroxide. It can produce | generate by making it basic of pH 7.5-9.0 with this base, and heating and stirring at 40-100 degreeC.

  The metal ions are any of zinc ions, iron ions, copper ions, nickel ions, beryllium ions, manganese ions, and cobalt ions, and one or more of these water-soluble metal salts are used. Of these, zinc ions and iron ions are preferable, and zinc chloride, iron chloride and the like can be preferably used.

  Solvents used in the above reaction include organic solvents such as acetone, acetonitrile, ethanol, methanol, propanol, dimethylformamide, dimethyl sulfoxide, dioxane, tetrahydrofuran, and water-containing solvents thereof, acetone, dimethylformamide, dimethyl sulfoxide. , Dioxane and tetrahydrofuran are preferred.

The drug used in the present invention has a phosphate group, a sulfate group or a carboxyl group in the molecule so that it can be easily hydrophobized by binding to the above metal ions, or these groups are bonded to the drug molecule. It is preferable that the drug has been used.
The molecular weight of the drug is preferably 1,000 or less.

Examples of such drugs include various drugs, and among them, drug-encapsulated nanoparticles accumulate in the liver, which is particularly advantageous for drugs for treating liver diseases, such as anti-hepatitis virus drugs, liver cancer therapeutic drugs, A liver function improving drug or the like is preferable.
More specifically, anti-hepatitis virus drugs such as ribavirin, lamivudine, adefovir, entecavir, vidarabine; drugs for treating liver cancer such as cyclophosphamide, fluorouracil, doxyfluridine, tegafur, cytarabine, doxorubicin, epirubicin, mitoxantrone, mitomycin; Examples include, but are not limited to, liver function improving drugs such as ursodeoxycholic acid, furopropion, glutathione, and glycyrrhizic acid. Of these, ribavirin and ribavirin-phosphate having ribavirin and phosphoric acid bonded thereto are preferred.

In the liver-accumulating nanoparticles encapsulating the drug of the present invention, the incorporation rate of the drug in the nanoparticles is increased by further mixing a basic low molecular weight compound.
Such basic low molecular weight compounds include (dimethylamino) pyridine, pyridine, piperidine, pyrimidine, pyrazine, pyridazine, quinoline, quinuclidine, isoquinoline, bis (dimethylamino) naphthalene, naphthylamine, morpholine, amantadine, aniline, spermine, spermidine , Hexamethylenediamine, putrescine, cadaverine, phenethylamine, histamine, diazabicyclooctane, diisopropylethylamine, monoethanolamine, diethanolamine, triethanolamine, ethylamine, diethylamine, triethylamine, methylamine, dimethylamine, trimethylamine, triethylenediamine, diethylenetriamine, Examples include ethylenediamine and trimethylenediamine. Details, a secondary or tertiary amine, diethanolamine being particularly preferred.

In the liver-accumulating nanoparticles encapsulating drugs provided by the present invention, the diameter of the particles is in the range of 20 to 300 nm, preferably 50 to 200 nm, depending on the therapeutic purpose of each drug. Its particle size can be determined.
For example, when the drug is an anti-hepatitis virus drug, nanoparticles having a particle size of 50 to 200 nm are preferably administered orally or intravenously.

  In the liver-accumulating nanoparticles encapsulating the drug provided by the present invention, the encapsulation rate varies depending on the amount of each drug, the rate of addition to water in the solvent diffusion method, and the amount of addition of metal ions and basic low molecular weight compounds. The diameter also changes.

  The liver-accumulating nanoparticles encapsulating the drug of the present invention thus prepared are appropriately purified by operations such as centrifugation, ultrafiltration, gel filtration, filter filtration, and fiber dialysis of the nanoparticle solution or suspension. It is preferably obtained and stored by lyophilization.

  At that time, it is preferable to add a stabilizer and / or a dispersing agent in order to resuspend the lyophilized preparation so that it can be administered, and then lyophilized. Sucrose, trehalose, sodium carboxymethyl cellulose and the like are preferably used.

  The liver-accumulating nanoparticles encapsulating the drug provided by the present invention are used as pharmaceuticals for preparations for parenteral administration such as intravenous injection preparations, topical injection preparations, nasal drops, eye drops, inhalants, and sprays. In particular, the characteristics and effects of the nanoparticles can be better exhibited by preparing a preparation for intravenous injection. In addition, it is used as a pharmaceutical for oral administration such as tablets, capsules and powders using ordinary excipients and additives.

  Examples of bases and other additive components used in the preparation of these parenteral preparations and oral administration preparations include various bases and ingredients that are pharmaceutically acceptable and used. . Specifically, saccharides such as physiological saline, monosaccharides, disaccharides, sugar alcohols, polysaccharides; polymer additives such as hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, crystalline cellulose; ionic or nonionic interfaces Activators; etc. can be appropriately selected and used depending on the dosage form.

  EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Example 1: Synthesis of a conjugate of arabinogalactan (AG) and polylysine (PLL) (AG-PLL) Arabinogalactan (AG, manufactured by Tokyo Chemical Industry Co., Ltd.) was purified by dialysis and freeze-dried for 3 days, and then recovered. . 600 mg of this purified arabinogalactan, 300 mg of sodium cyanoborohydride, and 100 mg of polylysine (PLL, manufactured by Sigma) were dissolved in 8 mL of water, and 4N sodium hydroxide aqueous solution was added to adjust the pH to 8.5. The solution was stirred at 60 ° C. for 18 hours. The reprecipitation purification was repeated three times using 5 times the amount of ethanol, and then lyophilized. The resulting reaction product was analyzed by size exclusion chromatography (SEC). The columns used were TSKgel α3000 and TSKgel α2500 (manufactured by Tosoh Corporation), and were detected by a differential refractometer and an ultraviolet / visible spectrophotometer. Further, trinitrobenzenesulfonic acid (TNBS), which is an amino group detection reagent, was added to the obtained reaction product, and analysis was similarly performed by size exclusion chromatography.

  As a result of analysis with a differential refractometer, the reaction product was eluted slightly earlier than arabinogalactan. Furthermore, when TNBS was added and detected at 365 nm as its absorption wavelength, no peak was observed with arabinogalactan, whereas the reaction product absorbed at the same position as that observed with a differential refractometer. (See FIG. 1). Therefore, it was revealed that this reaction product was a conjugate (AG-PLL) in which arabinogalactan and polylysine were combined. The yield was 94%.

Example 2: Preparation of nanoparticles (1) Production method of ribavirin-phosphate (RMP) Ribavirin-phosphate was prepared according to the method described in J. Med. Chem., 21 (8), 742-746 (1978). Manufactured.
That is, ribavirin, trimethyl phosphate, phosphoryl chloride and water were mixed and stirred for 5 hours while cooling with ice. 160 g of ice was added and dissolved, and then 4N aqueous sodium hydroxide solution was added to adjust the pH to 3. This solution was washed with chloroform, adsorbed on activated carbon, and eluted with a mixed solvent of water / ethanol / aqueous ammonia (10: 10: 1). The obtained ribavirin-phosphoric acid was confirmed by NMR and mass spectrometry.

(2) Preparation of nanoparticles containing ribavirin-phosphate (solvent diffusion method)
Poly DL-lactic acid (manufactured by Taki Chemical), a conjugate of arabinogalactan and polylysine, ribavirin-phosphoric acid, ferric chloride and diethanolamine are dissolved in dimethyl sulfoxide, and this solution is stirred in a large amount of water with a stir bar. While adding gradually, nanoparticles were obtained. At that time, the amount of each compound used, the addition rate, and the like were changed, and the influence on the particle size of the nanoparticles and the encapsulation rate of ribavirin-phosphoric acid in the particles was evaluated. The respective results are shown in FIG. 2, FIG. 3, and FIG.

It can be seen from FIG. 2 that the ribavirin-phosphate (RMP) encapsulation rate has a great influence by changing the amounts of ferric chloride and diethanolamine (DEA). That is, 7.5, 11.25 and 15.0 μmol of iron were used, and at the same time, the amount of ribavirin-phosphate encapsulated in nanoparticles adjusted by changing the amount of diethanolamine (DEA) was also measured by high performance liquid chromatography. Was quantified.
As a result, there was an optimal amount of diethanolamine to obtain the maximum encapsulation efficiency. It was also found that the amount of diethanolamine required increased as the amount of iron increased.

In FIG. 3, in the nanoparticles prepared by using 0, 2.5, 5.0 mg as the conjugate of arabinogalactan and polylysine (AG-PLL), and at the same time changing the amount of diethanolamine (DEA) The result of having quantified the amount of ribavirin-phosphate (RMP) encapsulated in is determined by high performance liquid chromatography.
As a result, there was an optimal amount of diethanolamine to obtain the maximum encapsulation efficiency. In addition, it was found that the optimum amount and maximum encapsulation efficiency did not depend much on the amounts of arabinogalactan and polylysine.

FIG. 4 shows the amount of ribavirin-phosphoric acid (RMP) encapsulated in nanoparticles prepared by changing the addition rate of dimethyl sulfoxide solution and the amount of diethanolamine (DEA). The results quantified by chromatography were shown.
As a result, it was found that stirring was effective and there was an optimum amount of diethanolamine to obtain the maximum encapsulation efficiency. It was also found that the maximum encapsulation efficiency depends on the addition rate of the dimethyl sulfoxide solution.

Nanoparticles were prepared using ribavirin without a phosphate group in place of ribavirin-phosphate.
Poly DL-lactic acid (manufactured by Taki Chemical), a conjugate of arabinogalactan and polylysine, ribavirin, ferric chloride and diethanolamine are dissolved in dimethyl sulfoxide, and this solution is gradually stirred in a large amount of water with a stirrer. By adding to, nanoparticles were obtained.
The encapsulation efficiency of ribavirin was 0.53% by weight, which was low compared to 1.58% by weight in the case of ribavirin-phosphate, but it was found that nanoparticles encapsulating ribavirin could be prepared. From this point, it is understood that the phosphate group in ribavirin-phosphate plays an important role in the encapsulation in the nanoparticles.
When prepared without using ferric chloride, ribavirin was not encapsulated in the nanoparticles at all.

  Drug-unencapsulated nanoparticles that do not contain drugs such as ribavirin-phosphate and ribavirin are dissolved in dimethyl sulfoxide using only a conjugate of polylactic acid, arabinogalactan, and polylysine. Adjustment was possible by law.

Example 3 Evaluation of Physical Properties of Nanoparticles By carefully examining the results of Example 2, conditions for adjusting nanoparticles having a relatively small particle size and high ribavirin-phosphate encapsulation efficiency were determined. That is, the particle diameter of the obtained nanoparticles was 105 nm, the ribavirin-phosphoric acid encapsulation rate was 1.7% by weight, and the arabinogalactan content in the nanoparticles was 5.7% by weight. The value of the zeta potential was -43.4 mV for the nanoparticles prepared without adding arabinogalactan, whereas it was -7.7 mV for the nanoparticles of the present invention. Further, when a heanut lectin (PNA) that specifically recognizes a galactose residue was added to the suspension of nanoparticles, aggregation of the nanoparticles was observed (see FIG. 5, left: nanoparticle suspension). , Right: Nanoparticle suspension with PNA added).

  From the above results, it was found that the arabinogalactan molecule was coordinated and exposed on the surface of the nanoparticle, and that galactose residues could be recognized by lectins.

Example 4: Evaluation of interaction with cells HepG cells (human hepatoma-derived cell line) were seeded on 50,000 Cells / 8 well chamber slides and incubated overnight. The medium was replaced with Opti-MEM, and nanoparticles encapsulating the labeled fluorescent substance rhodamine 123 were added and incubated for 3 hours. The cells were washed three times with physiological saline, fixed with 4% formaldehyde solution, and then observed with a fluorescence microscope.

  FIG. 6 shows a microscopic image of the cells. Nanoparticles prepared using a conjugate of arabinogalactan and polylysine (AG-PLL) are significantly incorporated into cells compared to nanoparticles prepared without using them (unmodified sugar nanoparticles). Turned out to be.

  Further, ribavirin incorporated into the cells was quantified. HepG cells were seeded at 2 million Cells / plate and incubated overnight. The medium was changed to Opti-MEM medium, and particles or ribavirin was added and incubated at 37 ° C. for 3 hours. The cells were trypsinized and the cell pellet collected by centrifugation was sonicated. Acetonitrile was added to dissolve the particles, and after acid phosphatase treatment, dephosphorylation was performed. After purification with a cartridge column, the amount of ribavirin was quantified by high performance liquid chromatography.

  The results are shown in FIG. It was found that by encapsulating ribavirin-phosphate (RMP) in the nanoparticles rather than ribavirin alone, a large amount of ribavirin was taken up into the cells.

Example 5: Liver-accumulating animal experimental mice (C57BL6 mice, female, 6 weeks old, body weight about 20 g) were injected with ribavirin aqueous solution or ribavirin-phosphate encapsulated nanoparticle suspension via tail vein, and after a predetermined time the liver Collected and homogenized. Acetonitrile was added to the homogenized solution to dissolve the particles, treated with acid phosphatase, dephosphorylated, purified with a cartridge column, and then ribavirin was quantified by high performance liquid chromatography.

The results are shown in FIG. It was revealed that ribavirin was significantly accumulated in the liver by administering nanoparticles encapsulating ribavirin, compared with the case of administering an aqueous solution of ribavirin. In addition, it was found that ribavirin accumulated in the liver gradually decreased after administration and remained after 7 days, and it was found that ribavirin was gradually released from the nanoparticles in the liver and had sustained drug efficacy.
In the figure, RBV means a ribavirin aqueous solution, and NP means a babylin-phosphate encapsulated nanoparticle.

As described above, the liver-accumulating nanoparticles encapsulating the drug provided by the present invention improve targeting and sustainability to the liver, which has not been sufficiently achieved so far, and efficiently exert pharmacological effects in the affected area. Nanoparticles to be made.
Therefore, it can be used for the treatment of liver diseases such as anti-hepatitis virus drugs, liver cancer therapeutic drugs, liver function improving drugs, etc., and it can achieve the sustainability in the liver. It is a thing.

Claims (8)

  1.   Drug, poly DL or L-lactic acid or poly (DL or L-lactic acid / glycolic acid) copolymer, conjugate of arabinogalactan and biodegradable cationic polymer, metal ion, and basic low molecular weight compound A liver-accumulating nanoparticle encapsulating a drug, which is encapsulated in a nanoparticle comprising:
  2.   The liver-accumulating nanoparticles encapsulating the drug according to claim 1, wherein the particle has a diameter of 20 to 300 nm.
  3.   The liver-accumulating nanoparticles encapsulating the drug according to claim 1, wherein the metal ion is one or more of zinc ion, iron ion, copper ion, nickel ion, beryllium ion, manganese ion or cobalt ion.
  4.   Basic low molecular weight compounds are (dimethylamino) pyridine, pyridine, piperidine, pyrimidine, pyrazine, pyridazine, quinoline, quinuclidine, isoquinoline, bis (dimethylamino) naphthalene, naphthylamine, morpholine, amantadine, aniline, spermine, spermidine, hexamethylene Diamine, putrescine, cadaverine, phenethylamine, histamine, diazabicyclooctane, diisopropylethylamine, monoethanolamine, diethanolamine, triethanolamine, ethylamine, diethylamine, triethylamine, methylamine, dimethylamine, trimethylamine, triethylenediamine, diethylenetriamine, ethylenediamine, triethylene One or more selected from the group consisting of methylenediamine Liver Accumulation nanoparticles encapsulating drugs according to claim 1 is of.
  5.   The drug has a phosphate group, a sulfate group, or a carboxyl group to be hydrophobized by the metal ion in the molecule, or is a drug to which these groups are bonded. Liver-accumulated nanoparticles encapsulating the drug described in 1.
  6.   The liver-accumulating nanoparticles encapsulating the drug according to claim 1 or 5, wherein the drug is an anti-hepatitis virus drug, a liver cancer therapeutic drug or a liver function improving drug.
  7.   The liver-accumulating nanoparticles encapsulating the drug according to claim 6, wherein the anti-hepatitis virus drug is ribavirin or ribavirin-phosphate.
  8.   The pharmaceutical which uses the liver accumulation | aggregation nanoparticle which enclosed the medicine of Claims 1-7 as an active ingredient.
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Cited By (1)

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AT151991T (en) * 1990-12-19 1997-05-15 Advanced Magnetics Inc Targeting of therapeutic agents by use of polysaccharides
CA2518223A1 (en) * 2003-03-26 2004-10-07 Ltt Bio-Pharma Co., Ltd. Intravenous nanoparticles for targeting drug delivery and sustained drug release
CA2520475C (en) * 2003-04-03 2012-10-09 Jessie L.-S. Au Tumor-targeting drug-loaded particles
US8916206B2 (en) * 2005-12-26 2014-12-23 Ltt Bio-Pharma Co., Ltd. Nanoparticles containing water-soluble non-peptide low-molecular weight drug
US20100129456A1 (en) * 2007-05-14 2010-05-27 Ltt Bio-Pharma Co., Ltd. Sustained-release nanoparticle containing low-molecular-weight drug with negatively charged group

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016527308A (en) * 2013-08-06 2016-09-08 ドン クック ファーマシューティカル カンパニー リミテッド Entecavir microspheres and pharmaceutical composition for parenteral administration containing the same

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