WO2006098415A1 - Vecteur de medicament - Google Patents

Vecteur de medicament Download PDF

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Publication number
WO2006098415A1
WO2006098415A1 PCT/JP2006/305304 JP2006305304W WO2006098415A1 WO 2006098415 A1 WO2006098415 A1 WO 2006098415A1 JP 2006305304 W JP2006305304 W JP 2006305304W WO 2006098415 A1 WO2006098415 A1 WO 2006098415A1
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WIPO (PCT)
Prior art keywords
hydrophilic
binding site
site
binding
drug
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PCT/JP2006/305304
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English (en)
Japanese (ja)
Inventor
Shinji Takeoka
Yosuke Okamura
Hideo Kanazawa
Shuji Hisamoto
Kohei Kubota
Yosuke Obata
Original Assignee
Oxygenix Co., Ltd.
Waseda University
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Application filed by Oxygenix Co., Ltd., Waseda University filed Critical Oxygenix Co., Ltd.
Priority to US11/886,319 priority Critical patent/US8647613B2/en
Priority to JP2007508214A priority patent/JP5038128B2/ja
Publication of WO2006098415A1 publication Critical patent/WO2006098415A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent

Definitions

  • the present invention relates to a drug carrier preparation that controls a pharmacokinetic by utilizing a phenomenon in which a drug is supported on a molecular assembly and an amphiphilic molecule that is a part of a component of the molecular assembly is released from the molecular assembly. .
  • a drug delivery system incorporating a drug in a molecular assembly is called a drug delivery system.
  • hydrophilic drugs there are bilayer vesicles constructed by assembling phospholipid molecules, so-called drug carriers encapsulated in the inner aqueous phase of ribosomes.
  • drug carriers encapsulated in the inner aqueous phase of ribosomes.
  • hydrophobic drugs it is physically dissolved or chemically dissolved in the hydrophobic part of molecular aggregates such as lipid microspheres that are o / w emulsions, micelles composed of surfactants or amphiphilic polymers.
  • lipid microspheres that are o / w emulsions, micelles composed of surfactants or amphiphilic polymers.
  • a hydrophobic drug may be embedded in the hydrophobic part of the bimolecular membrane of the ribosome.
  • these drug carriers When these drug carriers are administered into blood, for example, they are taken up by phagocytic cells mainly developed in the organs of the reticuloendothelial system (for example, spleen, liver, etc.), so the residence time in blood is extremely short. Limited to drug carriers that target multiple organs. For this reason, measures have been taken to extend the residence time in the blood. For example, ribosomes coated with polyethylene dallicol chains, which are water-soluble and highly biocompatible polymers, are often used as so-called stealth ribosomes.
  • solid cancer tissue is characterized by abnormally developed new blood vessels with many branches and thin and discontinuous blood vessel walls. If the size of PEG-ribosome with high blood retention is reduced to 300 nm or less, it becomes possible to leak from the blood into the stroma via the highly permeable blood vessel wall of cancer tissue. Accumulate with difficulty in returning to the lumen side. Therefore, PEG-ribosome has an effect of higher accumulation in solid cancer tissue than a low molecular weight drug, that is, has an EPR effect, and is one of the important factors in targeting a tumor tissue. is there.
  • the liposome which is a molecular assembly, disperses the amphiphilic molecules constituting it into water by some energy irradiation, and self-assembles by hydrophobic interaction. It is a metastable state in terms of physico-chemistry because it is prepared using the phenomenon. Therefore, aggregation or fusion may occur during storage, and precipitation may occur over time. Therefore, in consideration of the stability in blood, the solution has been achieved by mixing negatively charged lipids or cholesterol with the constituent lipids of the ribosome and modifying the surface of the ribosome with a polyethylene glycol chain or sugar chain.
  • lipids in which polyethylene glycol chains are bound to diacylphosphatidylethanolamine or cholesterol are widely used.
  • This polyethylene glycol-linked diacyl-type lipid has been reported to be released from phospholipid bilayer vesicles (JRSilvius and MJZuckermann, Biochemistry, 32, 3153, 1993, K. Sou, et al, Bioconjugate, 11, 372, 2000).
  • the release rate of this lipid depends on the molecular weight of the polyethylene glycol chain and the size of the hydrophobic part (the number of carbons constituting the acyl chain), that is, the hydrophilic-hydrophobic balance, and the hydrophilic part is relatively large. Easy to release lipids. Therefore, we have developed a lipid that combines a single polyethylene glycol chain and a number of alkyl chains using the monodendron structure, and a series that is difficult to desorb even when a polyethylene molecular chain with a large molecular weight is bound. Of amphipathic molecules (Patent No. 3181276). Disclosure of the invention
  • the present inventors have used a series of polyethylene glycol-binding lipids synthesized so far, and have used hydrophilic recognition sites such as proteins at the end of the polyethylene glycol chain of the polyethylene glycol-binding lipid. It has been clarified that liberation occurs because the hydrophilic-hydrophobic balance tends to become more hydrophilic. For this reason, it has been studied to increase the hydrophobic part of the binding lipid in order to stably carry the recognition part on the surface of the ribosome so that the hydrophilic recognition part is not released. However, as a result, liberation still occurred and it was difficult to completely control the liberation.
  • the present inventors have the drug carried on a molecular assembly composed of amphiphilic molecules, and controlled the pharmacokinetics of the drug carrier by utilizing the release phenomenon of the lipid (for example, polyethylene glycol-binding lipid) constituting the molecular assembly.
  • An object of the present invention is to provide a drug carrier preparation to be used. If it is further expanded and released by various external stimuli such as dilution effect (concentration change), temperature change, pH change, chemical reaction, etc., it can be used as an active control method for the pharmacokinetics of drug carriers. This is another issue It was. We also examined expanding this issue to include not only ribosomes, which are phospholipid molecular assemblies, but also the entire drug delivery system based on molecular assemblies such as emulsions or micelles. ⁇
  • amphiphilic molecules of amphiphilic molecules by changing the external environment of the molecular aggregates to a predetermined molecular structure.
  • the inventors have found that release can be adjusted arbitrarily, and have completed the present invention.
  • the present invention is a drug carrier including a molecular assembly carrying a drug, and a part of the amphiphilic molecules constituting the molecular assembly is released from the molecular assembly by a change in the external environment. By doing so, it is a drug carrier whose pharmacokinetics in the living body are controlled. More specifically, the present invention provides the following drug carriers and the like.
  • a drug carrier including a molecular assembly on which a drug is supported, and a part of the amphiphilic molecules constituting the molecular assembly is released from the molecular assembly due to a change in the external environment.
  • a drug carrier whose pharmacokinetics are controlled in vivo.
  • binding site A is the site that binds the hydrophilic part and drug
  • binding site B is the site that binds the hydrophilic part and hydrophobic part
  • binding site C is the part that binds the hydrophobic part and drug.
  • binding site B is the site that binds the hydrophilic part and hydrophobic part
  • binding site D is the site that binds the hydrophilic polymer and the hydrophilic part
  • binding site E is the recognition site and highly hydrophilic. The pharmacokinetics in the living body is controlled by the release of the amphipathic molecule.
  • binding site B is the site that binds the hydrophilic part and the hydrophobic part
  • binding site E is the part that binds the recognition site and the hydrophilic part.
  • the binding site B is the part that binds the hydrophilic part and the hydrophobic part
  • the binding site D is a site that binds the hydrophilic polymer and the hydrophilic part.
  • the amphiphilic molecule 3 represented by The drug carrier according to (1), wherein pharmacokinetics in the living body is controlled by expressing the recognition ability of the amphipathic molecule 5 by release.
  • binding site A is a site that binds the drug to the hydrophilic portion
  • binding site B is a site that binds the hydrophilic portion to the hydrophobic portion
  • binding site B is the site that binds the hydrophilic part and hydrophobic part
  • binding site D is the site that binds the hydrophilic polymer and the hydrophilic part
  • binding site E is the recognition site and highly hydrophilic.
  • the release of the amphiphilic molecule from the molecular assembly is due to a concentration change of at least one selected from the group consisting of protons, alkali metal ions and alkaline earth metal ions ( The drug carrier according to any one of 1) to (1 1).
  • a disulfide bond is contained at the site where the hydrophilic part and hydrophobic part of the liberated amphiphilic molecule or hydrophobic part are bonded, and the release of the amphiphilic molecule from the molecular assembly reduces the disulfide bond.
  • the drug carrier according to any one of (1) to (1 3). is
  • the drug retained in the molecular assembly is released by partial or total destruction of the molecular assembly structure due to the release of the amphiphilic molecule (1) to (1 4)
  • the drug carrier according to any of the above.
  • a drug carrier capable of controlling the pharmacokinetics of a drug to be transported according to the purpose.
  • FIG. 1A ′ is a diagram showing a model structure of an amphiphilic molecule used in the drug carrier of the present invention.
  • FIG. 1B is a diagram showing a model structure of an amphiphilic molecule used in the drug carrier of the present invention.
  • Figure 2 is a graph comparing the release behavior of PEG lipids from the endoplasmic reticulum.
  • Figure 3 is a graph comparing the desorption behavior of protein-bound transmembrane lipids from protein-loaded ribosomes.
  • Figure 4 is a graph comparing the detachment behavior of proteins from protein-bound ribosomes with the addition of a reducing agent.
  • Fig. 5 is a graph showing the results of CMC measurement of glycolipids due to differences in the number of alkyl chains of glycolipids.
  • Fig. 6 is a graph comparing the desorption rates of protein-bound membrane lipids from protein-bound ribosomes due to protein differences.
  • Figure 7 is a graph comparing the desorption rates of protein-bound glycolipids from protein-bound ribosomes due to differences in protein molecular weight. .
  • Fig. 8 shows the results of detection of thin-layer chromatography after hydrolysis of pH-responsive ribosomes at low pH.
  • Fig. 9 is a graph showing the release behavior of fluorescent molecules encapsulated in pH-responsive ribosomes due to pH changes.
  • FIG. 10 is a graph showing the temporal release behavior of fluorescent molecules encapsulated in pH-responsive ribosomes at each pH.
  • Fig. 11 is a graph showing the release behavior of fluorescent molecules encapsulated in pH-responsive ribosomes due to pH changes.
  • Fig. 12 is a graph showing the release behavior of lucanin encapsulated in ribosomes containing disulfide lipids when a reducing agent is added.
  • Fig. 16 is a graph showing that H12-vesicle specifically binds to activated platelets and that binding is suppressed by introducing PEG into H12-vesicle.
  • FIG. 17 is a graph comparing the binding rate of PEG (H12) -vesicle to activated platelets accompanying PEG elimination.
  • the flats of amphiphilic molecules that are desired to be released are inclined to the aggregates.
  • the equilibrium state is inclined to the free state into the aqueous phase, and the amphiphilic molecules to be released are released to reach a new equilibrium state.
  • the equilibrium state is a function of temperature, when the temperature is raised, the amphiphilic molecules are released and reach a new equilibrium state. Release can be controlled by such physical factors.
  • the hydrophilic part of hydrophilic / hydrophobic water is made hydrophilic by chemically cutting the hydrophobic part of the amphiphilic molecule or the binding part between the hydrophobic part and the hydrophilic part to make the hydrophobic part small. Tilt to the sexual side and promote release.
  • a hydrolyzable ester bond, an amide bond, a urethane bond, a Schiff base or the like, or a disulfide bond cleaved by a reducing agent can be used as the chemical bond to be cleaved.
  • amphiphilic molecule capable of adjusting the hydrophilic-hydrophobic balance is polyethylene glycol having a large hydrophilic portion, recall type lipid, such as polyethylene glycol on the amino group of diacylphosphatidylethanolamine.
  • Lipids covalently bonded to polyethylene lipids obtained by covalently bonding polyethylene glycol to hydroxyl groups of diacyl derivatives of glycerol, lipids obtained by covalently bonding polyethylene glycol to carboxyl groups of dialkyl derivatives of trifunctional amino acids (glutamic acid lysine)
  • the molecular design is based on the necessity of having a large hydrophobic part to stabilize the bimolecular membrane for the so-called large hydrophilic part, and the inventors have led to the invention of multi-acyl lipids (Patent No. 3181276). issue).
  • the present inventors analyzed the effect of hydrophilic monohydrophobic balance on the release rate of the amphiphilic molecule using these amphiphilic molecules, and the ribosome of the two amphiphilic molecules having a large hydrophobic part.
  • the immobilization and release to the body a lot of knowledge that is the basis of the present invention was accumulated, and further, physical factors and chemical factors that promote the release were arranged.
  • the present inventor has come up with the idea that the release of this lipid can effectively control the pharmacokinetics of a drug in response to an external stimulus, contrary to the fixation of polyethylene glycol-type lipids.
  • the molecular assembly used in the drug carrier of the present invention is not particularly limited as long as it can carry a drug.
  • examples of such molecular assemblies include polymer assemblies, polymer micelles, emulsions, lipid microspheres, bilayer vesicles (liposomes), and other molecular assemblies (tubes, fibers, ribbons, sheets) Etc.) can be used.
  • the components of this molecular assembly include amphiphilic molecules that can be released from the molecular assembly due to changes in the external environment and polymers that have the property of forming molecular assemblies with this free amphiphilic molecule. It is.
  • the polymer that forms a molecular assembly has the property of forming a molecular assembly with a free amphiphilic molecule, and is not particularly limited as long as it uses a synthetic or natural polymer. Can do.
  • the synthetic polymer include an amphiphilic block copolymer or a comb polymer having a hydrophobic substituent in a hydrophilic main chain such as a polysaccharide, or an amphiphilic membrane protein.
  • the shape of the molecular aggregate formed by these polymers is generally polymeric micelles, but they may be in the shape of endoplasmic reticulums, fibers, tubes, or sheets.
  • aggregates formed by temperature-responsive vinyl polymers such as poly [N-2 (hydroxypropyl) methacrylamide] (PHPMA) or poly [-(isopropyl) acrylamide] (PNIPAM) above the transition temperature
  • PPMA poly [N-2 (hydroxypropyl) methacrylamide]
  • PNIPAM poly [-(isopropyl) acrylamide]
  • O / w emulsion gel lipid microspheres which are oil droplets such as triglycerides that do not mix with water and stabilized with phospholipids and other surfactants, are frequently used for the stable dispersion of fat-soluble drugs.
  • the drug is not particularly limited, and for example, doxorubicin derivative, paclitaxel or methotrexate can be used.
  • Phospholipid vesicles or ribosomes which are widely used as drug carriers, are intermolecular interactions (hydrophobic interactions, It is a molecular assembly of a vesicle structure that has a membrane that is constructed without a covalent bond by electrostatic interaction, hydrogen bonding, etc., and the membrane is a monolayer (a single bilayer membrane) Or, a multi-layer (lamellar) film is formed.
  • the size ranges from several tens of nanometers to several tens of micrometers, but is preferably 10 nanometers to 1 micrometer, and more preferably 30 nanometers to 300 nanometers.
  • there are aggregate forms such as a bilayer sheet, ribbon, tube or fiber, and these are also included in the molecular assembly referred to in the present invention.
  • Molecular assemblies with an endoplasmic reticulum structure can retain water-soluble or lipophilic drugs in the inner aqueous phase or bilayer membrane of the endoplasmic reticulum, thereby extending the residence time in the blood. Therefore, it is preferable.
  • phospholipid endoplasmic reticulum or ribosome is particularly preferable.
  • Phospholipid vesicles or ribosomes are under development in the area of drug carriers and are composed of phospholipids alone or a mixture of phospholipids and cholesterol and / or fatty acids.
  • phospholipids examples include egg yolk lecithin, soybean lecithin, hydrogenated egg yolk lecithin, hydrogenated soybean lecithin, diacylphosphatidylcholine, diacylphosphatidylethanolamine, sfungomyelin, and various glycolipids. These phospholipids may contain unsaturated parts such as phen (double bond), in (triple bond), gen, diyne, and triene, and may contain a polymerizable group such as a vinyl group, such as a styryl group. May be included.
  • polymerizable phospholipids include 1,2-di (octadeca-trans-2, trans-4-dienoyl) phosphatidylcholine, 1,2-di (octadeca-2,4-dienoyl) phosphatidic acid, 1,2-biseleostaroylphosphatidylcholine and the like.
  • the phospholipid content is preferably 5 to 70 mol%, more preferably 20 to 50 mol%, based on the total number of moles of the constituent lipids of the molecular assembly.
  • fatty acid constituting the acyl chain a saturated or unsaturated fatty acid having 12 to 20 carbon atoms is used.
  • trifunctional amino acids such as amphoteric lipids such as glutamic acid and lysine skeleton can be used.
  • Fatty acid content Yuryou is the total number of moles of constituent lipids of the molecular assembly, lay preferred that 1 to 70 mol%, more preferably 5 to 30 mol 0/0.
  • a negatively charged lipid can also be added as a component to the molecular assembly. This is because mixing of negatively charged lipids in the membrane component suppresses aggregation of the endoplasmic reticulum, reduces the number of coating layers, and increases the encapsulation efficiency.
  • the negatively charged lipid include diacyl phosphatidyl glycerol, diacyl phosphatidic acid, diacyl phosphatidyl inositol, diacyl phosphatidyl serine, and anionic amino acid type fats.
  • the content of the negatively charged lipid is preferably 1 to 70 mol%, particularly preferably 5 to 30 mol%, based on the total number of moles of the constituent lipids of the molecular assembly.
  • sterols may be added to the molecular assembly as stabilizers as membrane components of the lipid vesicles.
  • examples of such sterols include all steroloids having perhydrocyclopentanophenanthrene such as ergosterol and cholesterol, and preferably cholesterol.
  • Taire the total number of moles of constituent lipids of the molecular assembly, 5 to 50 mol 0 /. It is preferably, more preferably 15 to 40 mole 0/0.
  • the molecular assembly includes amphiphilic molecules that can be released from the molecular assembly due to a change in external environment.
  • the content of the amphipathic molecule is appropriately determined according to the target organ / tissue, the type of drug, the drug loading site, the loading method, the form of the molecular assembly, etc. no. for example, based on the total molarity of the constituent lipid of the molecular assembly, it forces S preferably 0.01 to 100 mol%, more preferably from 0.05 to 50 mol 0/0, 0.1 to 30 moles 0 / 0 is particularly preferred.
  • ribosome production methods include single or mixed lipid powders or thin films that are hydrated and dispersed, followed by high pressure extrusion (extrusion) method, ultrasonic irradiation method, stirring (portex mixing, homogenizer) method , Freeze-thaw method, microfluidizer method, etc., or a solution of a single or 'mixed fat dissolved in an organic solvent is injected into the aqueous phase, followed by ethanol, ether, etc.
  • a method for loading a drug on such a molecular assembly may be appropriately selected according to the type of drug.
  • a water-soluble drug for example, it can be prepared by dissolving the drug in an aqueous phase at the time of ribosome production.
  • a water-soluble drug can be added to the outer aqueous phase, and the drug can be introduced into the inner aqueous phase using the permeability of the ribosome membrane.
  • the water-soluble drug not encapsulated can be separated from the encapsulated endoplasmic reticulum by gel filtration, ultracentrifugation, or ultrafiltration membrane treatment.
  • the drug is introduced into the hydrophobic part of the bilayer by mixing the drug in a state where a single or mixed lipid is dissolved in an organic solvent and forming a ribosome by the method described above. can do. It is also possible to form a liposome containing an amphiphilic molecule having a functional group and then carry the drug on the functional group exposed on the surface of the ribosome using a chemical reaction in the aqueous phase. it can. Alternatively, after the ribosome is produced in advance, the drug can be dissolved in an organic solvent mixed with water and added to the outer aqueous phase to introduce the drug into the hydrophobic part of the bilayer membrane.
  • a phenomenon in which some amphiphilic molecules constituting the molecular assembly are released from the molecular assembly due to a change in the external environment of the molecular assembly is used.
  • the drug carrier of the present invention controls pharmacokinetics in a living body by the release phenomenon of the amphiphilic molecule.
  • “External environment” means the environment surrounding the molecular assembly, and includes temperature, pH, dilution of molecular assembly, ionic environment, reducing atmosphere, and the like.
  • Amphiphilic molecules released from a molecular assembly can control the release rate from the molecular assembly by the hydrophilic part-hydrophobic part balance in the molecule.
  • the release rate can be appropriately adjusted according to the target organ / tissue, type of drug, drug loading site, loading method, morphology of the molecular assembly, and the like.
  • the release rate can be reduced by designing the ratio of the hydrophobic part to the hydrophilic part to be large.
  • the release rate can be increased by increasing the ratio of the hydrophilic part to the hydrophobic part.
  • the drug carrier of the present invention is not particularly limited as long as pharmacokinetics can be controlled by releasing some amphiphilic molecules from the molecular assembly.
  • the drug carrier of the present invention includes, for example, the following four modes depending on the hydrophilic part, hydrophobic part, combination of drug and hydrophilic polymer and control method of the amphiphilic molecule to be released.
  • the first aspect of the present invention is an aspect in which the pharmacokinetics is controlled by loading a drug in the hydrophilic or hydrophobic part of the liberated amphiphilic molecule (FIG. 1A (a)).
  • the pharmacokinetics is controlled by supporting a hydrophilic polymer or a hydrophilic polymer having a recognition site bonded to the hydrophilic part of the liberated amphiphilic molecule.
  • Figure l A (b) the pharmacokinetics is controlled by releasing the amphiphilic molecule from the molecular assembly and expressing the recognition site on the surface of the carrier such as a liposome (active targeting, Fig. L A ( c)).
  • the fourth aspect of the present invention is an aspect in which the pharmacokinetics is controlled by the liberated amphiphilic molecule having a transmembrane structure (FIG. 1B (d)). Each aspect will be described below.
  • the amphiphilic molecule to be separated is composed of a drug, a hydrophilic part and a hydrophobic part, and the drug is carried by supporting the drug on the liberated amphiphilic molecule. It controls the dynamics (Fig. L A (a)).
  • the structure of the amphiphilic molecule (hereinafter sometimes referred to as “drug-binding amphiphilic molecule”) to which the drug released from the molecular assembly is bound is the amphiphilic molecule 1 and 2: ) — (Binding site A) — (Hydrophilic part) One (Binding part B) — (Hydrophobic part) 1
  • binding site A is a site that binds a hydrophilic part and a drug
  • binding site B is a part that binds a hydrophilic part and a hydrophobic part
  • binding site C is a part that binds a hydrophobic part and a drug. Is].
  • the (hydrophilic part) -one (binding site B)-(hydrophobic part) can target all known amphiphilic molecules known as phospholipids, sphingolipids, amino acid-type lipids, and the like.
  • the hydrophilic part is made of hydrophilic polymers such as polyethylene glycol, polysaccharides, polyvinyl alcohol, polyvinyl pyrrolidone, polyglutamic acid, and polypeptides. May be included. '
  • the binding site B is a site that binds the hydrophilic part and the hydrophobic part, and is not particularly limited as long as it is generally known to have such a function in an amphiphilic molecule.
  • the binding site B is defined as glycerol, oligosaccharide, oligopeptide, polyester, bull oligomer, amino acid such as glutamic acid or lysine, or a multi-branched structure composed of these, for example, a dendron structure. Two hydrophilic portions and one or more hydrophobic portions are bonded.
  • the binding site B is preferably at least one selected from the group consisting of an oligosaccharide chain, an oligopeptide chain, a polyester chain, and a vinyl-based oligomeric dendron structure.
  • an oligopeptide chain consisting of repeating glutamic acid with glycine unit as a spacer has an arbitrary number of carboxylic acid groups in the side chain
  • the hydrophobic part can be arbitrarily defined by an amide bond or an ester bond.
  • the hydrophilic part can be bound to the N-terminus of the oligopeptide chain.
  • the hydrophobic part can be bonded to the carboxylic acid group or the hydroxyl group of oligoacrylic acid or vinyl alcohol by a polymer reaction.
  • molecular faeces can be controlled and a functional group can be introduced at the terminal, so that a hydrophilic part can be bonded to this terminal.
  • the number of terminal functional groups can be controlled by the number of generations. 2 in the 1st generation, 4 in the 2nd generation, 8 in the 3rd generation.
  • the number of hydrophobic parts can be controlled by bonding hydrophobic parts here.
  • the hydrophilic part can be bonded to one functional group on the opposite side of the monodendron.
  • the hydrophobic part is not particularly limited as long as it can impart sufficient hydrophobicity to the amphiphilic molecule.
  • saturated fatty acids, unsaturated fatty acids, saturated higher alcohols, unsaturated higher alcohols, and sterolides are used as the hydrophobic part. These may be linear or branched.
  • the hydrophobic part of the liberated amphiphilic molecule preferably has two or more hydrocarbon chains.
  • “the hydrophobic part has two or more hydrocarbon chains” means that two or more hydrocarbon chains are bonded to the binding site B.
  • the hydrocarbon chain may be a branched chain, and examples of the branched chain include those having a long chain having an isoprenoid structure.
  • the number of hydrocarbon chains is not particularly limited, but is preferably 2 or more. There is no particular upper limit to the number of hydrocarbon chains, but it is usually preferably 18 or less in view of easy synthesis.
  • the hydrocarbon chain introduced into the hydrophobic part is preferably an alkyl chain, an alkenyl chain or an alkynyl chain, and an alkyl chain is particularly preferred because steric hindrance can be minimized and it can be easily introduced into a molecular assembly.
  • These hydrocarbon chains may have a substituent as long as they are sufficiently hydrophobic as the hydrophobic part of the amphiphilic molecule and do not inhibit introduction into the molecular assembly.
  • the hydrocarbon chain is interrupted by an oxygen atom, a sulfur atom, one NR— (wherein R represents an alkyl group, an alkenyl group, or an alkynyl group which may have a substituent). May be.
  • the number of carbon atoms in the hydrocarbon chain is not particularly limited, but is preferably 4 to 24, more preferably 10 to 20 and particularly preferably 12 to 18.
  • glutamic acid or lysine is included as the binding site B, the above-mentioned hydrophobic part can be bonded to two carboxylic acid groups or two amino groups possessed by these.
  • amphiphilic molecules represented by (hydrophilic part) (binding site B)-(hydrophobic part) include polyethylene glycol lipids, glycolipids, peptide-binding lipids, protein (antibodies, enzymes, etc.) binding lipids
  • Drug-binding amphiphilic molecules such as nucleic acids, multi-acyl chain lipids, transmembrane lipids, etc. can be employed.
  • polyethylene glycol lipids are those having a molecular weight of about 200 Da to 12500 Da, preferably about 1000 Da to 5000 Da, or a substituent such as an amino group, a carboxyl group, a hydroxyl group, or a maleimide group at both ends.
  • the glycolipid has a reducing end group, and may be an oligosaccharide polysaccharide having a branched or almost linear sugar polymerization degree of 2 to 400, and may be either natural sugar or synthetic sugar.
  • An oligosaccharide is a sugar in which one or more of, for example, gnolecose, funolectose, xylose, galactose, mannose, darcosamine, etc. are ⁇ -bonded or iS-bonded.
  • Gentio-oligosaccharides nigg mouth oligosaccharides, lacto-oligosaccharides, merigo-oligosaccharides, inuro-oligosaccharides and the like.
  • polysaccharides starch, cellulose, mucopolysaccharide (hyaluronic acid, chondroitin, chondroitin sulfate, dermantan sulfate, ketalan sulfate, beparin, etc.), chitin, chitosan, other polysaccharide degradation products, cells, and complex sugars derived from bacteria Quality and so on.
  • the drug is an amphiphilic molecule 1 represented by Formula 1 bound to the hydrophilic part by a binding site A, or an amphiphile represented by Formula 2 bound to the end of a hydrophobic part by a binding site C. It is introduced by co-assembly into the molecular assembly by sex molecule 2.
  • Binding sites A and C are sites that bind the drug to the hydrophilic or hydrophobic part, respectively. Examples of the binding sites A and C include an amide bond, an ester bond, an ether bond, a urethane bond, a disulfide bond, and a bond in which a mercapto group and a maleimide group are added. '
  • the drug that can be carried on the drug carrier of the present invention is not particularly limited as long as it acts on at least one target organ or tissue.
  • examples of such drugs include enzymes, peptides or proteins, various antibiotics, various peptide hormones, DNA, RNA, siRNA, plasmids, various anticancer agents, drugs for central nervous system, Peripheral nerve agent, Sensory organ agent, Cardiovascular agent, Respiratory agent agent, Gastrointestinal agent, Hormonal agent, Urogenital and anal drug, Skin agent, Oral dental agent, Vitamin agent, Nutrition Tonics, blood and body fluids, artificial dialysis drugs, other metabolic drugs, cell rejuvenation drugs, oncology drugs, radiopharmaceuticals allergy drugs, herbal medicines and herbal medicines, antibiotics, chemotherapy Drugs, biologics, diagnostics.
  • Examples of peptides or proteins include various sites such as interleukins, cell transmission factors, fibrinogen, collagen, keratin, pro
  • Examples include polypeptides as extracellular matrix agents such as theoglucan or oligo bodies as a part of the structure, or functional polypeptides such as oxytocin, bradykinin, thyrotropin releasing factor, enkephalin and the like.
  • Examples of the enzyme include force talase, chymotrypsin, cytochrome, and amylase, but are not limited thereto. These drugs may be used alone or in combination of two or more.
  • the hydrophilic-hydrophobic balance of the amphiphilic molecule 1 or 2 may be appropriately designed according to the type of drug, target organ or tissue, pathology, kinetics of the amphiphilic molecule 1 or 2 itself, etc. .
  • Such an amphiphilic molecule 1 or 2 can be introduced into a bilayer membrane of a liposome composed of a molecular assembly, for example, a phospholipid, by the method described above.
  • the content of amphiphilic molecules 1 and 2 is appropriately determined according to the type of drug, target organ or tissue, pathology, etc. It is preferably 0.01 to 30 mol%, particularly preferably 0.1 to 10 mol%, based on the total number of moles of the constituent lipid.
  • the drug carrier according to the second aspect of the present invention controls pharmacokinetics by supporting a hydrophilic polymer or a hydrophilic polymer having a recognition site bonded to a hydrophilic part of a liberated amphiphilic molecule.
  • the “drug” part of the first aspect is a “hydrophilic polymer”, and basically the hydrophilic polymer, the hydrophilic part and the hydrophobic part. Is included.
  • this embodiment includes those in which a recognition site is further bound to a hydrophilic polymer.
  • binding site B is the site that binds the hydrophilic part and hydrophobic part
  • binding site D is the site that binds the hydrophilic polymer and the hydrophilic part
  • binding site E is the recognition site and highly hydrophilic. It is a site that binds to a molecule.
  • the amphiphilic molecule represented by the formula 3 or 4 can modify the surface of the liposome with a hydrophilic polymer, for example, by co-assembling with a phospholipid.
  • a hydrophilic polymer the same polymer as described in the description of the hydrophilic portion of the first embodiment can be used.
  • the molecular weight of the hydrophilic polymer is preferably about 200 Da to 20000 Da, and particularly preferably about 2000 Da to 12500 Da. If the hydrophilic polymer is a polyethylene glycol chain, it has the effect of greatly extending the residence time of the ribosome in the blood compared to the unmodified ribosome.
  • the hydrophilic polymer itself a polysaccharide that is easily recognized by cells constituting the organ or tissue, the specificity to the organ or tissue can be increased.
  • the amphiphilic molecule 4 has a recognition site such as an oligopeptide chain or sugar chain at the end of a hydrophilic polymer such as a surface-modified polyethylene glycol chain, and controls the pharmacokinetics of the drug carrier. Can do.
  • the pharmacokinetics of the drug carrier can be more accurately controlled by releasing the amphiphilic molecule 3 or 4 due to a change in the external environment.
  • polyethylene glycol-binding lipids that modify liposomes increase the residence time of ribosomes in the blood, but release from ribosomes can shorten the residence time in blood.
  • the release rate can be adjusted by the molecular weight of polyethylene glycol or the size of the hydrophobic part.
  • amphiphilic molecule 4 • Ribosomes can increase specific accumulation in cells that make up organs or tissues that recognize amphipathic molecules 4, but the release of amphipathic molecules 4 reduces the specificity, and The liberated amphiphilic molecule 4 binds to the site to be recognized and inhibits the recognition of liposomes in which both amphiphilic molecules 4 are modified, resulting in lower specificity. Furthermore, the internal kinetics of the drug carrier can be controlled with higher accuracy by mixing the amphiphilic molecules 3 and 4 and introducing them onto the ribosome surface and further controlling their release rates.
  • the binding site D or E is not particularly limited as long as it is a bond between a hydrophilic polymer and a hydrophilic moiety, or a bond between a hydrophilic polymer and a recognition site.
  • Such binding sites D or E include, for example, amide bonds, ester bonds, ether bonds, urethane bonds, disulfide bonds, condensation bonds of mercapto groups and maleimide groups, and Schiffs of aldehyde groups and amino groups. Examples include bases.
  • the recognition site is not particularly limited as long as it specifically recognizes a target organ or tissue.
  • Examples of recognition sites include, but are not limited to, the oligosaccharides, antibodies, peptide hormones, lectins, .glycoproteins described above.
  • the content of the amphiphilic molecules 3 and 4 is appropriately determined according to the desired blood convection time and recognition ability, but each of the constituent lipids of the molecular assembly.
  • the amount is preferably 0.01 to 50 mol%, particularly preferably 0.1 to 20 mol%, based on the total number of moles.
  • the recognition site on the surface of the molecular assembly is expressed by the release of a part of the amphiphilic molecules constituting the molecular assembly, and thereby the pharmacokinetics is improved. It is to control (Fig. L A (c)).
  • the drug carrier of this embodiment is, for example, a molecular assembly in which an amphiphilic molecule 5 is supported, and the amphiphilic molecule 3 introduced into the molecular assembly is recognized by the amphiphilic molecule 5. And a drug carrier whose pharmacokinetics is controlled by the release of the amphiphilic molecule 3 and the recognition ability of the amphiphilic molecule 5 being expressed by the release of the amphiphilic molecule 3.
  • amphiphilic molecules 5 and 3 (Recognition site) One (Binding site E) One (Hydrophilic part) One (Binding part B) One (Hydrophobic part) 5
  • the binding site B and the binding site D have the same meaning as described above, and the binding site E is a site that binds the recognition site and the hydrophilic portion.
  • the hydrophilic polymer of the amphiphilic molecule 3 is preferably a hydrophilic polymer having a molecular weight larger than the recognition site of the amphiphilic molecule 5.
  • the amphiphilic molecule 3 When the molecular assembly is administered into the blood in this state, the amphiphilic molecule 3 is released due to a change in the external environment such as a dilution effect.
  • the recognition site of the amphipathic molecule 5 when the recognition site of the amphipathic molecule 5 is exposed on the surface of the liposome ⁇ , the recognition site increases the specific accumulation in the cells constituting the organ or tissue. The pharmacokinetics can be controlled. Furthermore, the release of the amphiphilic molecule 5 reduces the specific accumulation property, which can also control the pharmacokinetics of the drug carrier.
  • the recognition site possessed by the amphipathic molecule 5 the same recognition site as described in the embodiment of Clay 2 can be shown.
  • examples of the amphiphilic molecule 5 include those having a recognition site such as an oligopeptide chain or a sugar chain at the end of a hydrophilic portion such as glutamic acid or lysine.
  • a recognition site such as an oligopeptide chain or a sugar chain at the end of a hydrophilic portion such as glutamic acid or lysine.
  • sugar chains are recognized in a structure-specific manner by lectins, which are sugar-binding proteins, molecular aggregates containing amphipathic molecules having sugar chains as recognition sites are used for proteins and sugars on the cell membrane surface. It can be used as a carrier that specifically recognizes a ligand such as a chain and is highly useful.
  • amphiphilic molecule 3 can be the same as those described in the second embodiment.
  • the recognition ability of the amphiphilic molecule 5 is inhibited by the hydrophilic polymer such as the polyethylene glycol chain of the amphiphilic molecule 3.
  • the hydrophilic polymer in the amphiphilic molecule 3 is not particularly limited as long as it can inhibit the recognition ability of the amphiphilic molecule 5, but preferably has a molecular weight of about 200 Da to 20000 Da. It is particularly preferable that it is about 2000 Da to 12500 Da.
  • the content of the amphiphilic molecule 5 is appropriately determined according to the desired recognition ability, but is 0.01 to 50 mol% with respect to the total number of moles of the constituent lipids of the molecular assembly. It is preferable that it is 0.1-30 mol%.
  • the content of the amphiphilic molecule 3 is not limited as long as the recognition ability of the amphiphilic molecule 5 can be inhibited, but is 0.01 to 30 mol% with respect to the total number of moles of the constituent lipids of the molecular assembly. It is preferable that it is 0.05 to 10 mol%.
  • the release rate of the amphiphilic molecule 3 can be controlled by the molecular weight or charge amount of the hydrophilic polymer or the size of the hydrophobic part.
  • the release rate of the amphiphilic molecule 5 can be controlled by the same method. Therefore, the relative release rate of these amphiphilic molecules can be appropriately designed according to the purpose.
  • the fourth aspect of the present invention is to control the pharmacokinetics of the drug transporter by releasing transmembrane amphipathic molecules that penetrate the membrane of the molecular assembly (FIG. 1B (d )).
  • the structures of the amphiphilic molecules exemplified in the first to third embodiments described above are those in which the liberated amphiphilic molecules are simply classified into structures represented by a hydrophilic part and a hydrophobic part.
  • the amphiphilic molecules used in the invention may be classified according to the structure represented by hydrophilic part-hydrophobic part-hydrophilic part. In the present embodiment, an amphiphilic molecule having such a structure is preferable.
  • amphiphilic molecule used in this embodiment, specifically,
  • Amphiphilic molecules represented by -These amphiphilic molecules are introduced into the assembly in a structure that penetrates the bilayer in the ribosome bilayer.
  • the hydrophobic part may be single-stranded or double-stranded, but this depends on the binding site B.
  • the length of the hydrophobic portion may be any length that can penetrate the bimolecular membrane, but is 20 to 5.0, preferably 28 to 36 in terms of carbon number.
  • Symmetrical structures such as amphipathic molecules 7 or 9 are forces that are easy to synthesize.
  • a hydrophilic polymer or drug is oriented on the inner aqueous phase side of the ribosome. This may be convenient for controlling the release rate, that is, controlling pharmacokinetics, but because the hydrophilic polymer or drug is not exposed to the ribosome surface, these cannot function effectively and are wasted. There is. In the latter case, after introducing an amphiphilic molecule into the ribosome, the drug or hydrophilic polymer is bound only to the end of the amphiphilic molecule exposed on the surface via binding site A or binding site D. It can also be made.
  • the control of the release rate is not only a hydrophilic / hydrophobic balance, but also the size and charge amount of the hydrophilic part facing the inner aqueous phase. That is, even if the transmembrane amphiphilic molecule is low in hydrophobicity, the transmembrane amphiphilic molecule is difficult to be released from the ribosome if the hydrophilicity facing the inner aqueous phase is strong.
  • the content of the amphiphilic molecule 6-1 1 is not particularly limited, respectively, based on the total molarity of the constituent lipids of the molecular assembly is preferably a Dearuko 0.01 to 100 mole 0/0 0.1 to 25 mol% is particularly preferable.
  • the release of amphiphilic molecules from the molecular assembly is caused by changes in the external environment.
  • “External environment” means the environment surrounding the molecular assembly as described above.
  • Examples of the external environment include a diluting solution for diluting the molecular assembly and an ambient temperature for changing the temperature of the molecular assembly. , Ambient hydrogen ion concentration to change the pH of the molecular assembly.
  • the change in the external environment is not limited to the shift from the equilibrium state to the non-equilibrium state due to dilution when administered in blood.
  • the equilibrium state also shifts with temperature. For example, when the temperature rises, the equilibrium state shifts to the free side, which can promote the release. It is also affected by the mobility of the molecular assembly.
  • the gel-liquid crystal phase transition temperature is at 42 ° C, but it is 40 ° C near the phase transition temperature or higher than the phase transition temperature than 35 ° C below the phase transition temperature.
  • the molecular packing state of the membrane is more disturbed and is more likely to be released. Therefore, amphipathic molecules can be locally released from the molecular assembly by local heating with a warmer, infrared, microwave, or catheter.
  • the temperature of the ribosome can be locally controlled by encapsulating iron oxide fine particles in a ribosome and heating it with a microwave.
  • the phase transition temperature can be changed by changing the combination of lipids having different phase transition temperatures and their composition, for example, dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine.
  • a temperature-responsive polymer such as poly- (N-isopropyl acrylamide) (PNIPAM) is a hydrophilic polymer of a free amphiphilic molecule, the hydrophilicity increases and is released below the phase transition temperature. It becomes easy.
  • the release rate of the amphiphilic molecules from the molecular assembly can be adjusted, for example, by utilizing changes in the concentrations of protons, alkali metal ions, and alkaline earth metal ions. For example, if an amino group is provided in the hydrophilic part, the hydrophilicity is improved by the protonation of the amino group at a low pH, and the release rate is increased. Also, if a hydrophilic rubonic acid group is added to the hydrophilic part, the hydrophilicity is improved by dissociation of the carboxylic acid group at high pH The release rate increases.
  • the binding site B is lysine
  • a long chain alcohol is ester-bonded to the strong lpoxyl group to introduce a hydrophobic part
  • a long-chain fatty acid is amide-bonded to the amino group to introduce a hydrophobic part.
  • This release greatly disturbs the molecular packing state of the bilayer membrane of the ribosome and promotes the release of the encapsulated drug, so that the pharmacokinetics of the drug carrier can be controlled.
  • This pharmacokinetics can include not only blood flow, organs, and tissues but also intracellular cells, and a system that releases amphipathic molecules in response to pH changes is also used to control intracellular kinetics.
  • amphiphilic molecules having a phosphate group in the hydrophilic part bind to divalent cations such as calcium ions, and the hydrophilic monohydrophobic balance tilts toward the hydrophobic side, reducing the release rate.
  • This phenomenon can also be used in the present invention.
  • the suppressed release rate can be promoted by capturing this calcium ion with a chelating agent such as taenoic acid.
  • charge shielding occurs as the ionic strength of the aqueous phase increases. Movement to the hydrophobic side decreases the release rate.
  • the binding site B of the liberated amphiphilic molecule contains a disulfide bond at the hydrophobic part, and that the release of the amphiphilic molecule from the molecular assembly is a cleavage by disulfide bond reduction.
  • an amphiphilic molecule for example, a fatty acid is introduced into the amino group of cystine through an amide bond, and the other hydrophobic part is an alkane- Examples include those introduced into the mercapto group of cysteine via a disulfide bond of Honore, and having a hydrophilic moiety introduced into the carboxylic acid group.
  • a method of introducing a disulfide group into the hydrophobic alkyl chain is also effective. This includes the case of introduction into the hydrophobic part of the transmembrane amphiphilic molecule from 6 to 11 described above. Multiple disulfide groups can be introduced. When introduced into the binding site, it is preferably introduced into binding site B.
  • lysine was used as a spacer (corresponding to the binding site B), and a compound having polyethylene glycol (PEG) in the hydrophilic part and two alkyl groups in the hydrophobic part was synthesized.
  • PEG polyethylene glycol
  • PEG lipid the introduction stability of the lysine-type PEG lipid obtained by the method of Example 1 (hereinafter referred to as “PEG lipid”) into the endoplasmic reticulum and the inhibitory effect on the endoplasmic reticulum aggregation were correlated with the PEG lipid structure. Revealed.
  • a PEG lipid aqueous solution (17.0 ⁇ M, 22.6 mL) was mixed with the endoplasmic reticulum dispersion (17.0 mM, 15.0 mL) and stirred at 37 ° C. to obtain a PEG lipid-introduced endoplasmic reticulum dispersion.
  • This PEG lipid-introduced endoplasmic reticulum dispersion was diluted 6-fold with phosphate buffer (PBS, pH 7.0), and the amount introduced was measured over time until 12 hours later. The amount introduced can be calculated as follows (Yoshioka, H. Biomaterials 1991, 12, 861). ,
  • [PEG-lipid] AX (Hp EG + / H PEG- ) (mol%)
  • Fig. 2 shows the observation results (1H-NMR) of PEG lipid desorption from the endoplasmic reticulum after 6-fold dilution with PBS (pH 7.0).
  • the symbols in Figure 2 are ( ⁇ ) P50-2C14, ( ⁇ ) P125-2C14, ( ⁇ ) P50-2C16, ( ⁇ ) P125-2C16, ( ⁇ ) P125-2C18, ( ⁇ ) P125-4C16, ( ⁇ ) P50-DPPE, (PE) P125-DSPE.
  • P125-2C18 PEG molecular weight (12500), hydrophobic group (-(CH 2 ) 16 CH 3 X 2)
  • P125-4C16 PEG molecular weight (12500), hydrophobic group (-( CH 2 ) 14 CH 3 X 4)
  • P50-2C16 PEG molecular weight (5000) and hydrophobic groups (-(CH 2 ) 14 CH 3 X 2)) were not desorbed, but P125- DSPE (PEG lipid with PEG molecular weight (12500) bound to distearoylphosphatidylethanolamine DSPE) or P125-2C16 (PEG molecular weight (12500), hydrophobic group (-(CH 2 ) 14 CH 3 X 2)) for 3 hours Later, about 8% and about 10% were detached from the endoplasmic reticulum, respectively.
  • P50-2C14 PEG molecular weight (5000), hydrophobic group (-(CH 2 ) 12 CH 3 X 2)
  • P125-2C14 PEG molecular weight (12500), hydrophobic group (-(C3 ⁇ 4) 12 CH 3 X 2 In)
  • about 20 and 30% were detached from the endoplasmic reticulum after 12 hours, respectively. Therefore, by increasing the molecular weight of the PEG chain or shortening the alkyl chain length, the PEG chain on the surface of the ribosome can be easily detached, and the directivity can be increased while controlling the blood retention.
  • P50-2C16 showed no detachment
  • P50-DPPE PEG molecular weight (5000) hydrophobic group (-(CH 2 ) 16 CH 3 X 2)
  • P50-DPPE is a PEG lipid with a PEG molecular weight (5000) bound to dipalmitoylphosphatidylethanolamine.
  • the hydrophilicity of the whole lipid can be affected by the hydrophilicity of the phospholipid head, while the lysine-type PEG lipid does not have that effect, but also has an amide binding site and endoplasmic reticulum in the lysine backbone. It is thought that hydrogen bonds in the phospholipid ester part contribute to stable introduction.
  • the disulfide in the hydrophobic part may be cleaved by (catalytic reduction or strong acid)
  • the Boc group that can be deprotected with a weak acid was selected. In order to prevent cleavage of the Boc group, acid mixing and heating were avoided as much as possible.
  • 2,2'-dipyridyl disulfide (2-PD, 4.3 g, 19.2 mmol) was stirred in 20 mL distilled THF for 1 hour in a nitrogen atmosphere and deoxygenated, then 1,10-decanedithiol (1.0 g, 4.8 mmol) was added.
  • PD groups were introduced at both ends of 1,10-decanedithiol by the conversion reaction (0.82 g, yield 40%).
  • the amount introduced was fluorescently labeled with fluorescamine on both terminal amino groups of the transmembrane lipid and quantified by fluorescence measurement.
  • fluorescamine on both terminal amino groups of the transmembrane lipid and quantified by fluorescence measurement.
  • Thvrogrobulin 670 4.5 [Preparation of protein].
  • the ⁇ -Lactalbumin-bound ribosome with the least detachment from the above detachment evaluation was evaluated by allowing L-cysteine (Cys), a membrane-permeable reducing agent, to coexist in the outer aqueous phase.
  • Cys L-cysteine
  • Fig. 4 The symbols in Fig. 4 represent ( ⁇ ) ⁇ ; -Lactalbumin (Cys-free system) and ( ⁇ ) a -Lactalbumin + Cys solution (Cys-added system), respectively. .
  • a benzene solution (100 mL) of p_toluesulfonic acid monohydrate (4.56 g, 24 mmol) was refluxed at 85 ° C., and water was removed using Ginster before the reaction.
  • Glutamic acid (2.96 g, 20 mmol) and hexadecyl alcohol (10.7 g, 44 mmol), or octadecyl alcohol (10.7 g, 44 mmol) were added to the reaction solution, and the generated water was removed for 10 hours. The boiling point was refluxed. As the reaction progressed, the suspension gradually dissolved and turned yellow and transparent.
  • the CMC of compound 21 was 20 ⁇ ⁇ ⁇ , whereas that of compound 22 was 18 ⁇ , and the CMC decreased slightly by changing the alkyl chain length from C16 to C18.
  • the CMC of Compound 23 was 8.0 ⁇ and that of Compound 24 was 7.0 ⁇ , and a significant decrease in CMC was observed when the number of alkyl chains was increased from 2 to 4. From this, it was confirmed that increasing the number of alkyl chains is effective in increasing hydrophobic interaction.
  • the results are shown in FIG.
  • the symbols in FIG. 5 represent (b) 21 (2C16), (concealed) 22 (2C18), (0) 23 (4C16), ( ⁇ ) 24 (4C18), respectively.
  • granulation (000 ⁇ 8QO ⁇ 650 ⁇ 450 ⁇ 300 ⁇ 220 nm X 2) was performed using an extractor, and the particle size was controlled.
  • the outer aqueous phase was removed by centrifugation (10,000 rpm, 5 min ⁇ 3) and redispersed in PBS to prepare a glycolipid-containing ribosome dispersion.
  • Catalase (from bovie liver) 232 5.5
  • Thyroglobulin ffrom bovie thvroid 670 4.5
  • SPDP-introduced protein solution 0.4 g / dL
  • Figure 7 shows the relationship between protein molecular weight and desorption rate.
  • the symbols in Fig. 7 represent (D) 21 (2C16), (Re) 22 (2C18), (0) 23 (4C16), (Reference) 24 (4C18), respectively.
  • the desorption rate was accelerated by 10.7 times (1.0% / hr to 10.7% / hr) with respect to an increase in protein molecular weight of 31 times (14 kDa to 440 kDa).
  • the desorption rate was 5.0 times (0.7% / hr to 3.9% / hr), and the desorption rate was hardly accelerated even when the molecular weight increased to a protein molecular weight of 440 kDa.
  • Nt-Boc-L-glutamic acid-g-butyl ester (764 mg, 2.52 mmol) and DCC (513 mg, 2.52 mmol) were dissolved in dichloromethane and stirred at 4 ° C for 30 minutes, then Glu2C16 (lg, 1.68 mmol) was added dropwise to the dissolved dichloromethane solution. The reaction solution was stirred at room temperature for 5 hours, and then the reaction solution was filtered and the solvent was removed under reduced pressure.
  • Compound 29 was prepared in the same manner as Compound 27, except that myristic acid was used instead of lauric acid.
  • the stability of the prepared ribosome was determined by particle size measurement using light scattering. As a result, the particle size of ribosome did not change with the membrane content of compound 28 (Table 7),
  • the dried mixed lipid powder was dissolved in black mouth form and detected by thin layer chromatography (silica gel plate, black mouth form / methanol (8/1) (volume / volume), detection: iodine). The detection results are as shown in Fig. 8.
  • FIG. Fig. 10 shows the results of measuring the release of encapsulated molecules over time at each pH.
  • the symbols in Fig. 10 indicate the measurement results at (country) ⁇ 3.5, ( ⁇ ) pH 4.5, ( ⁇ ) pH 5.5, ( ⁇ ) pH 6.5, (mouth) pH 7.3, ( ⁇ ) pH 11, respectively. .
  • Approximately 20% of the encapsulated fluorescent molecules were released at pH 3.5, but were hardly released at pH 6 or higher.
  • the ribosome with Compound 28 as a membrane component hydrolyzes Compound 28 at low pH, and the hydrolyzed lyso form of Compound 28 is more hydrophilic and is released from the endoplasmic reticulum. It is considered that the molecular packing state of the molecular film was disturbed and the release rate of the encapsulated fluorescent molecules was increased.
  • ribosomes containing 10 mol% of each of compounds 28 and 29 as membrane components were prepared and their pH responsiveness was verified.
  • the release of the encapsulated molecule was calculated from the above formula 1.1 based on the measurement result by measuring the fluorescence intensity in the same manner as described above.
  • the release rate after 10 minutes at each pH is shown in FIG.
  • Ribosomes containing Compound 28 or 29 as membrane components (10 mol%) released encapsulated molecules rapidly at pH 4.
  • Degradability due to increased release rate due to low pH The hydrolysis of lipid (compound 28 or 29) liberates the resulting lyso form, which disrupts the molecular packing state of the liposomal membrane and promotes the release of the encapsulated molecules.
  • a disulfide lipid having a disulfide group in the hydrophobic part was synthesized.
  • F 100 fluorescence intensity after complete solubility of liposomes
  • Figure 12 shows the results.
  • Fig. 12 - ⁇ -indicates a cysteine-added system, and -mouth- indicates a gluta-thione-added system.
  • the release of force lucein was promoted in the ribosome mixed with Glu-Cys-SC 16 as a membrane component due to the presence of the reducing agent.
  • the cysteine-added system showed a high release rate at each concentration, while the glutathione-added system increased the release rate in a concentration-dependent manner. This is because cystine is opposite to the endoplasmic reticulum membrane.
  • ⁇ the MP-Glu2C18 introduction rate 0 mol%
  • ⁇ is MP-Glu2C18 introduction rate 1 mole 0 /.
  • ⁇ indicates change over time at MP-Glu2C18 introduction rate of 2 mol%
  • mouth indicates MP-Glu2C18 introduction rate of 10 mol%.
  • An increase in solution turbidity was observed immediately after the addition of the Con A aqueous solution. This is thought to be because the added Con.
  • ⁇ indicates turbidity change after 0.5 minutes
  • ⁇ indicates turbidity change after 5 minutes.
  • An increase in turbidity was observed depending on the amount of MP-Glu2C18 introduced on the ribosome surface, and a sharp increase in turbidity was observed at an introduced amount of 2 mol% or more.
  • MAL-vesicle Removal of maleimide-introduced phospholipid endoplasmic reticulum (MAL-vesicle) was obtained (4 wt%, 6 mL).
  • Dodecapeptide (H12, 100 mM, lOO L) into which cysteine was introduced in advance at the N-terminal was added to MAL-vesicle and shaken (r ⁇ , 12 hours). Unbound H12 was removed by centrifugation (100,000 g, 30 min) to obtain H12-vesicle. ,
  • the amount of H12 bound on the surface of the endoplasmic reticulum was determined using HPLC (TSK-GEL G3000PW XL column, 7.8 mm od x 300 mm h, 1 mL / min, 36% (v / v) acetonito linole, 0.1% ( v / v) Quantified with trifluoroacetic acid) and back-calculated.
  • the H12-vesicle dispersion (17 mM, 5 mL) was mixed with an aqueous PEG lipid (P125-2C14) solution (17.0 ⁇ ,, 15.8 mL) and stirred at 37 ° C for 2 hours. Ultracentrifugation (33,000 rpm, 30 After removal of unintroduced PEG lipid at min), PEG lipid-introduced vesicles (PEG (H12) vesicle) were obtained.
  • the binding rate of PEG (H12) -vesicle (PEG (H12) -vesicle in FIG. 17) to activated platelets was 11.0%.
  • the binding rate increased with increasing dilution ratio of PEG (H12) -vesicle, and was almost equivalent to that of H12-vesicle after 300-fold dilution. This suggests that the detachment of PEG lipids by dilution was promoted and the function of H12 was expressed.
  • the drug carrier of the present invention can control the pharmacokinetics according to changes in the external environment, it is extremely useful as a preparation for the prevention / treatment of various diseases.

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Abstract

L’un des objectifs de l’invention est de fournir un vecteur de médicament pouvant réguler la pharmacocinétique in vivo. L’invention concerne un vecteur de médicament comprenant un assemblage moléculaire servant de vecteur à un médicament et l’objectif peut être atteint en détachant une partie des molécules amphiphiles constituant l’assemblage moléculaire du reste de l’assemblage moléculaire sous l’effet de modifications de l’environnement extérieur. L’invention exploite une particularité des molécules amphiphiles de se détacher de l’assemblage moléculaire à la suite d’un basculement de l’équilibre hydrophiles-hydrophobes vers l’hydrophilicité sous l’effet de modifications de l’environnement extérieur.
PCT/JP2006/305304 2005-03-16 2006-03-10 Vecteur de medicament WO2006098415A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
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JP2007210953A (ja) * 2006-02-09 2007-08-23 Oxygenix:Kk ｐＨ応答性分子集合体
WO2008096904A1 (fr) * 2007-02-08 2008-08-14 Tokyo Metropolitan Organization For Medical Research Conjugué de mannose 6-phosphate-polyéthylène glycol
JP5241711B2 (ja) * 2007-05-17 2013-07-17 学校法人早稲田大学 両親媒性分子、それを含む分子集合体及びその用途
CN113456587A (zh) * 2021-06-08 2021-10-01 中国农业大学 一种靶向乳腺癌干细胞的谷胱甘肽响应型纳米药物载体制备及应用
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JP2007210953A (ja) * 2006-02-09 2007-08-23 Oxygenix:Kk ｐＨ応答性分子集合体
WO2008096904A1 (fr) * 2007-02-08 2008-08-14 Tokyo Metropolitan Organization For Medical Research Conjugué de mannose 6-phosphate-polyéthylène glycol
JP2008195757A (ja) * 2007-02-08 2008-08-28 Tokyoto Igaku Kenkyu Kiko マンノース６−リン酸−ポリエチレングリコール結合体
JP5241711B2 (ja) * 2007-05-17 2013-07-17 学校法人早稲田大学 両親媒性分子、それを含む分子集合体及びその用途
CN113456587A (zh) * 2021-06-08 2021-10-01 中国农业大学 一种靶向乳腺癌干细胞的谷胱甘肽响应型纳米药物载体制备及应用
WO2023095874A1 (fr) * 2021-11-25 2023-06-01 国立大学法人長崎大学 Composé lipidique, liposome, exosome, nanoparticule lipidique et système d'administration de médicament

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