JP2007204469A - Drug delivery material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a drug delivery material which can deliver a drug to a desired site with good efficiency, can prevent the influence of the drug on any site other than the desired site during the delivery of the drug (therefore, is less likely to cause any adverse effect), and can release the drug only at the desired site so that the drug can exert its effect at the desired site, without the need of any external means. <P>SOLUTION: The drug delivery material comprises a conjugate of (1) a drug-carried molecule aggregate, (2) a linker and (3) a substance capable of recognizing an activated platelet, a damaged part in a blood vessel and/or an inflammatory tissue. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a drug carrier that is a conjugate of 1) a drug-carrying molecular assembly, 2) a linker, and 3) a substance that recognizes activated platelets, vascular injury sites and / or inflammatory tissues. More specifically, the present invention relates to a drug carrier useful as a prophylactic / therapeutic agent for diseases, a diagnostic agent and a reagent, particularly as a platelet substitute or antiplatelet agent.

  Various glycoproteins (glycoproteins: GP) exist on the platelet membrane surface and are involved in the expression of platelet functions. As such platelet glycoproteins, GPIB, GPIIb, GPIIIa, GPIIIb, GPIV, GPIX and the like are known. Of these, GPIb functions as a receptor for von Willebrand factor (vW factor). GPIb is a heterodimer with a molecular weight of 160,000, in which α and β chains are disulfide bonded.

  Recently, a platelet substitute is known in which a functional polymer such as GPIb, a recombinant body (rGPIbα) of a hydrophilic part of αkDa of 45 kDa, GPIIb / IIIa is bound to the surface of some microparticles. Among these, those having rGPIbα are expected to act as platelet substitutes by having an adhesive action based on the interaction of rGPIbα and vW factor, while those having GPIIb / IIIa are expected to act as GPIIb / IIIa and fibrinogen. It is expected to act as a platelet substitute by having an aggregating action based on the interaction of and / or vW factors.

  Further, as fine particles for binding these functional polymers, lipid membranes such as endoplasmic reticulum, human albumin or a polymer thereof, or human erythrocytes are known. Patent Document 1 discloses that rGPIbα is contained in the endoplasmic reticulum. What has been combined is described.

  On the other hand, there is also known one that induces blood aggregation by assisting the activity of platelets remaining in patient blood, rather than acting as a platelet substitute by having a functional polymer on platelets. For example, one that induces platelet aggregation by interacting with GPIIb / IIIa on platelets is known. Specifically, Non-Patent Document 1 discloses that the endoplasmic reticulum has a GPIIb / IIIa recognition site in fibrinogen. The thing which combined the peptide containing the contained dodecapeptide (H12) is described.

  Patent Documents 2 and 3 describe a conjugate in which a linker is inserted between GPIb or dodecapeptide and the endoplasmic reticulum, and a physiologically or pharmacologically effective drug by accumulating at the site of vascular injury. It is described that the conjugates described in these documents can be used as drug carriers.

  Non-Patent Document 2 describes an endoplasmic reticulum carrying adenosine diphosphate (ADP). ADP is a substance known to promote platelet aggregation and thrombus formation. The endoplasmic reticulum releases ADP by laser irradiation and causes platelet aggregation.

However, none of the above-described documents discloses a drug carrier that achieves a pharmacological effect by spontaneously releasing the drug only at a desired location. In Patent Documents 2 and 3, it is described that the conjugates described in these documents are used as a drug carrier, but specific embodiments thereof are not described. Non-Patent Document 2 describes an ADP-supporting vesicle, but the vesicle requires an external means of laser irradiation in order to release ADP.
Japanese Patent Laid-Open No. 9-208599 International Publication No. 01/64743 Pamphlet International Publication No. 2004/069862 Pamphlet T. T. et al. Nishiya et al., Thromb Haemost, 91, 1158-67 (2004). B. Khobehi et al., Lasers Sur Med. , 12 (6), 609-14, 1992

  The present inventors can efficiently transport a drug to a desired place, and the drug being transported does not affect a place other than the desired place (thus, it is less likely to cause a side effect). An object of the present invention is to provide a drug carrier that releases a drug only at a desired place without requiring an external means and exerts an effect on the drug.

  We combine specific molecular assemblies carrying a drug with a linker and a substance that recognizes activated platelets, sites of vascular injury and / or inflammatory tissue, for example, to activate desired cells (eg, activated Platelets) and / or biological tissues (for example, vascular injury sites, inflamed tissues), and in these desired locations, cells and / or biological tissues and drug-carrying molecular aggregates are drugs. It interacts via a substance that recognizes activated platelets, vascular injury sites and / or inflamed tissue bound to the carrier molecule assembly, which allows the drug-carrying molecule assembly to physically stimulate from cells and / or living tissue Thus, the drug is released from the drug-carrying molecular assembly, and it is found that the drug can exert a desired effect only at a desired location, and the present invention is completed. Was Tsu.

The gist of the present invention is as follows.
[1] A drug carrier which is a conjugate of 1) a drug-carrying molecular assembly, 2) a linker, and 3) a substance that recognizes activated platelets, vascular injury sites and / or inflammatory tissues.
[2] The drug carrier according to [1], wherein the drug-carrying molecular assembly is a lipid bilayer vesicle in which a drug is encapsulated in an inner aqueous phase.
[3] Drug delivery according to [1] or [2], represented by (drug-carrying molecular assembly)-(linker)-(substance recognizing activated platelet, vascular injury site and / or inflammatory tissue) body.
[4] The linker includes an amphipathic molecule that becomes a part of the components of the drug-carrying molecular assembly when bound to the drug-carrying molecular assembly, and the linker and the drug-carrying molecular assembly are interposed via the amphiphilic molecule. The drug carrier according to [3], which is bound.
[5] The drug carrier according to [3], wherein the linker includes a hydrophobic molecule, and the linker and the drug-carrying molecular assembly are bonded to the drug-carrying molecular assembly through the hydrophobic molecule.
[6] The drug carrier according to any one of [1] to [5], wherein the linker includes a spacer portion.
[7] The drug carrier according to [6], wherein the spacer portion is polyoxyethylene.
[8] The drug carrier according to any one of [1] to [7], wherein the drug is selected from the group consisting of a platelet aggregation inducer, a platelet aggregation inhibitor, a vasoconstrictor, a vasodilator, and an anti-inflammatory agent. .
[9] The drug according to any one of [1] to [7], wherein the drug is selected from the group consisting of adenosine diphosphate, collagen, collagen-derived peptide, convulxin, serotonin, aspirin, dipyridamole, ticlopidine, cilostazol, and beraprost. Drug carrier.
[10] A substance that recognizes activated platelets, vascular injury sites and / or inflamed tissues is integrin or selectin exposed to activated platelets, collagen exposed to vascular injury sites, exposed to vascular injury sites A substance that recognizes von Willebrand factor bound to collagen, selectin exposed to inflamed tissue, and / or selectin ligand exposed to leukocytes, and is taken into activated platelets and / or leukocyte aggregates, And / or the drug carrier according to any one of [1] to [9], which is a substance that accumulates in a vascular injury site and / or an inflamed tissue.
[11] The substance that recognizes activated platelets, vascular injury sites and / or inflammatory tissues is selected from the group consisting of H12, GPIbα, GPIa / IIa, GPVI, MAC-1, fibrinogen, P-selectin and PSGL-1. The drug carrier according to [10].
[12] The lipid bilayer vesicle is composed of a mixed lipid containing 20 to 100% of cholesterol in a molar ratio with respect to phosphatidylcholine which is hydrogenated egg yolk lecithin, hydrogenated soybean lecithin, distearoyl phosphatidylcholine or dipalmitoylphosphatidylcholine. The drug carrier according to [2], wherein a conjugate of a linker and a substance that recognizes activated platelets, vascular injury sites and / or inflammatory tissues is contained in an amount of 0.001 to 20% relative to the phosphatidylcholine.
[13] The drug carrier according to [12], wherein the lipid bilayer vesicle has a particle size of 50 to 300 nm and the lipid bilayer has 1 to 4 layers.
[14] A drug carrier that is a conjugate of 1) a drug-carrying molecular assembly, 2) a linker, and 3) a substance that recognizes activated platelets, vascular injury sites and / or inflamed tissues, A drug carrier in which a drug is released from a drug-carrying molecular assembly upon receiving physical stimulation from a cell or a biological tissue when reaching the biological tissue.
[15] The cell according to [14], wherein the cell is activated platelet or leukocyte, and the drug is selected from the group consisting of a platelet aggregation inducer, a platelet aggregation inhibitor, a vasoconstrictor, a vasodilator, and an anti-inflammatory agent. Drug carrier.
[16] The biological tissue is a vascular injury site or an inflammatory tissue, and the drug is selected from the group consisting of a platelet aggregation inducer, a platelet aggregation inhibitor, a vasoconstrictor, a vasodilator, and an anti-inflammatory agent. [14] The drug carrier according to 1.
[17] A diagnostic agent comprising the drug carrier according to any one of [1] to [16].
[18] A reagent comprising the drug carrier according to any one of [1] to [16].
[19] A platelet replacement agent comprising the drug carrier according to any one of [1] to [16].
[20] An antiplatelet agent comprising the drug carrier according to any one of [1] to [16].

  The drug carrier of the present invention induces unnecessary thrombus formation and intravascular coagulation by causing aggregation with inactive platelets in blood vessels before reaching desired cells and / or living tissues. Without further using the external means such as a laser, the drug-carrying molecular assembly is disintegrated and released only at a desired location without using an external means such as a laser. Can be delivered to desired cells and / or living tissue. For this reason, the drug carrier of the present invention has high drug absorption efficiency and can significantly reduce side effects.

  The present invention is a conjugate of 1) a drug-carrying molecular assembly, 2) a linker, and 3) a substance that recognizes activated platelets, vascular injury sites and / or inflammatory tissues (sometimes abbreviated as a recognition substance). A drug carrier (also referred to as a drug carrier of the present invention) is provided. The drug carrier of the present invention is represented by (drug-carrying molecular assembly)-(linker)-(substance recognizing activated platelet, vascular injury site and / or inflammatory tissue), that is, each component in this order. Those in which the elements are arranged are preferred.

  In the present specification, the “drug-carrying molecular assembly” is a molecular assembly carrying a drug, and a desired cell and / or biological tissue (for example, when it is combined with a linker and a recognition substance) , Activated platelets, vascular injury sites, and inflamed tissues) means a molecular assembly that releases the carried drug but does not release the carried drug in other places and hardly exerts the effect of the drug. Such molecular assemblies include biocompatible carriers capable of parenteral administration for medical use. Preferable materials for the molecular assembly include vesicles, micelles, polymer micelles, microspheres and the like. Of these, endoplasmic reticulum is particularly preferred.

The endoplasmic reticulum is a particle composed of a lipid artificial membrane, and is produced as a lipid bilayer from phospholipid, glyceroglycolipid, cholesterol and the like. In the present invention, phospholipid bilayer vesicles are more preferred. As for the number of carbon atoms of the hydrocarbon chain which comprises a lipid bilayer, 12-18 are preferable. It is also possible to adjust the strength of the membrane by introducing 1 to 3 unsaturated groups into the hydrocarbon chain. If the number of carbon atoms in the hydrocarbon chain is too large, the strength of the film will be too high, and it will be difficult to release the drug when it is loaded.
In the present invention, a mixed lipid containing (preferably consisting essentially of) phosphatidylcholine, which is hydrogenated egg yolk lecithin, hydrogenated soybean lecithin, distearoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine, and cholesterol, more specifically, hydrogenated Particularly preferred is a phospholipid bilayer vesicle composed of egg yolk lecithin, hydrogenated soybean lecithin, mixed lipid containing 20 to 100% of cholesterol in a molar ratio with respect to phosphatidylcholine which is distearoylphosphatidylcholine or dipalmitoylphosphatidylcholine.

  The endoplasmic reticulum can be produced by a known method such as a surfactant removal method, a hydration method, an ultrasonic method, a reverse phase distillation method, a freeze-thaw method, an ethanol injection method, an extrusion method, and a high-pressure emulsification method. Details of the preparation of the endoplasmic reticulum are described in JP-A-9-208599 and T.W. Nishiya et al., Biochim. Biophys. Res. Commun. , 224, 242-245, 1996.

  The particle size of the endoplasmic reticulum after introduction of the linker and the recognition substance is preferably 50 to 300 nm, more preferably 100 to 270 nm, and more preferably 150 to 250 nm, from the viewpoints of the introduction amount of the linker and the recognition substance and their functional expression and pharmacokinetics. Most preferred. Here, the particle diameter is a particle diameter that is controlled by using a filter after introducing the linker and the recognition substance. When the particle diameter is smaller than 50 nm, it becomes difficult to release the drug from the molecular assembly when the drug is supported.

The number of lipid bilayer membranes of the endoplasmic reticulum is preferably 1 to 4 and more preferably 1 to 2 when the bilayer is considered as one. When the number of layers exceeds 4, it becomes difficult to release the drug from the molecular assembly when the drug is supported.
The number of layers can be controlled by the pore size of the filter and the dispersion medium (pH, temperature, ionic strength) of the endoplasmic reticulum. As a method for measuring the number of layers, there are a freeze cleaving method, a small angle X-ray scattering method, an electron spin resonance (ESR) using a spin-labeled lipid, a measuring method using ³¹ P-NMR, and 6-p-toluidino-2-naphthalene. Examples include a measurement method using sulfonic acid (TNS).

  As the drug-carrying molecular assembly, a lipid bilayer vesicle in which a drug is encapsulated in the inner aqueous phase is most preferable.

In the present specification, the “linker” is capable of cross-linking a drug-carrying molecular assembly and a substance that recognizes activated platelets, vascular injury sites and / or inflammatory tissues, and is biocompatible. If there is no particular limitation. The linker is preferably a saturated or unsaturated acyclic hydrocarbon having 2 to 10 carbon atoms or an aliphatic or aromatic cyclic hydrocarbon having a functional group that becomes a binding site with a drug-carrying molecular assembly or a recognition substance, The chain or ring may contain a hetero atom (for example, an oxygen atom or a nitrogen atom). Two or more different linkers may be used in combination. As the linker, a compound having a functional group that reacts with any of the SH group, OH group, COOH group, and NH ₂ group is more preferable. Examples of the linker include those synthesized from dicarboxylic acid, aminocarboxylic acid, bismaleimide compound, bishalocarbonyl compound, halocarbonylmaleimide compound, dithiomaleimide, dithiocarboxylic acid, maleimide carboxylic acid, and the like. As a linker having a functional group that reacts with any of SH group, OH group, COOH group, and NH ₂ group, for example, N- (α-maleimidoacetoxy) succinimide ester, N- [4- (p-azidosalisyl) Amido) butyl] -3 ′-(2′-pyridyldithio) propionate, N-β-maleimidopropionic acid, N- (β-maleimidopropionic acid) hydrazide, N- (β-maleimidopropoxy) succinimide ester, N- ε-maleimidocaproic acid, N- (ε-male Midocaproic acid) hydrazide, N- (ε-maleimidocaproyloxy) succinimide ester, N- (γ-maleimidobutyryloxy) succinimide ester, N-κ-maleimidoundecanoic acid, N- (κ-maleimidoundecanoic acid) hydrazide, Succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxy- (6-amidecaprate), succinimidyl-6- [3- (2-pyridyldithio) -propionamide] hexanate, m-maleimidobenzoyl-N- Examples include linkers synthesized using hydroxysuccinimide ester, 4- (4-N-maleimidophenyl) butyric acid hydrazide and the like as raw materials.

  The linker may include a spacer part between the drug-carrying molecular assembly and the recognition substance and capable of adjusting the length between the drug-carrying molecular assembly and the recognition substance. The arrangement position of the spacer part may be a drug-carrying molecular assembly-linker-spacer-recognizing substance or a drug-carrying molecular assembly-spacer-linker-recognizing substance. In addition, a spacer part may be inserted between the same or different linkers. That is, it may be a drug-carrying molecular assembly-linker-spacer-linker-recognizing substance. The spacer is not particularly limited as long as it has biocompatibility, and a substance selected from the group consisting of polyoxyethylene, polypeptide, polysaccharide, albumin, and antibody can be used. Recombinants may be used for albumin and antibodies. As the spacer in the present invention, polyoxyethylene or a derivative thereof is particularly preferable.

  As a preferred drug carrier of the present invention, the linker includes an amphipathic molecule that becomes a part of a component of the drug-carrying molecule assembly when bound to the drug-carrying molecule assembly, and through the amphiphilic molecule The linker or spacer and the drug-carrying molecular assembly are bonded, and the linker contains a hydrophobic molecule, and the linker or spacer and the drug-carrying molecular assembly are converted into the drug-carrying molecular assembly via the hydrophobic molecule. The thing which has couple | bonded is mentioned.

  Examples of amphiphilic molecules and hydrophobic molecules include dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dioleoylphosphatidylethanolamine, and the like. In the present invention, Glu2C18 represented by the following formula is particularly preferred as an amphiphilic molecule.

  In the case of a lipid bilayer vesicle composed of a mixed lipid containing 20 to 100% of cholesterol in a molar ratio with respect to phosphatidylcholine, a linker and a substance that recognizes activated platelets, vascular injury sites and / or inflamed tissues The conjugate is preferably contained in an amount of 0.001 to 20% with respect to the phosphatidylcholine.

In the present specification, the “substance recognizing activated platelets, vascular injury site and / or inflammatory tissue” means that the drug carrier of the present invention is recognized by recognizing activated platelets, vascular injury site and / or inflammatory tissue. It refers to a substance that is directed to activated platelets, sites of vascular injury and / or inflamed tissue and serves to accumulate the drug carrier of the present invention at these locations. Recognizing substances include integrins or selectins exposed to activated platelets, collagen exposed to vascular injury sites, von Willebrand factor bound to collagen exposed to vascular injury sites, exposed to inflammatory tissues Preferred is a substance that recognizes the selectin and / or the selectin ligand that is exposed to leukocytes, and that is incorporated into activated platelets and / or leukocyte aggregates and / or that accumulates at sites of vascular injury and / or inflamed tissue Used. More specifically, as a recognition substance, H12 (HHLGGAKQAGDV, SEQ ID NO: 1), GPIbα, GPIa / IIa (integrin α2β1), GPVI, MAC-1, fibrinogen, P-selectin, PSGL-1, and the like are preferable.
There are no particular restrictions on the method for preparing H12, GPIbα, GPIa / IIa, GPVI, MAC-1, fibrinogen, P-selectin, and PSGL-1, methods for extraction and isolation from platelet membranes, cell culture methods, genes It can be prepared by an engineering production method or the like.
As long as the object of the present invention is achieved, the recognition substance used in the present invention is one obtained by subjecting one or more amino acids in the amino acid sequence to any mutation such as deletion, substitution, addition or modification. For example, substitutions, analogs, mutants, modifications, derivatives, sugar chain adducts and the like of natural recognition substances are also included.
Examples of GPIbα include GPIbα [His (1) to Leu (610)], GPIbα fragments such as a fragment of the vWF binding region of the α chain, and GPIbα fragments lacking a transmembrane site. More preferred in the present invention is a GPIbα fragment lacking a transmembrane site.
More specific GPIbα chain fragments include His (1) to Cys (485), His (1) to Pro (340), His (1) to Thr (304), His (1) to Ala (302), His. (1) to Arg (293) [JP-A-1-221394, EP0317278], Ala (165) to Leu (184), Gln (180) to Phe (199), His (195) to Leu (214), Asn (210) to Val (229), Glu (225) to Ala (244) and Thr (240) to Tyr (259) [Japanese Patent Laid-Open No. 1100196], Asn (61) to Thr (75), Gln (71) to Ser (85), Thr (81) to Leu (95), Gln (97) to Arg (111), Leu (136) to Leu (150), A n (210) ~Ala (224), Gln (221) ~Asp (235) and Ser (241) ~Asp (255) [Kohyo 5-503708 discloses, WO91 / 09,614], etc. are exemplified. In addition, examples of the substitute include GPIbα chain fragments consisting of His (1) to Ala (302), in which Gly (233) or Met (239) are each substituted with Val [WO93 / 16712]. All of these GPIbα chain fragments lack the transmembrane site. The transmembrane site corresponds to Leu (486) to Gly (514) in the GPIbα chain (Proc. Natl. Acad. Sci. USA, 84, 5615-5619, 1987).

When the drug carrier of the present invention reaches a cell or biological tissue, the drug-carrying molecular assembly is physically stimulated from the cell or biological tissue, whereby the drug is released from the drug-carrying molecular assembly. That is, the recognition substance constituting the drug carrier directs the drug carrier of the present invention to the target cell or the target biological tissue. When the drug carrier of the present invention reaches the target cell or the target biological tissue, the drug carrier of the present invention interacts with the target cell or the target biological tissue through the linker and the recognition substance, thereby causing physical stimulation. The received and loaded drug is released from the molecular assembly. More specifically, the recognition substance is attracted to the target cell or the target biological tissue, and a physical load is applied to the drug-carrying molecular assembly due to the morphological change of these cells or the biological tissue. Collapses and drug is released.
Here, when the target is a cell, the target is preferably activated platelet or leukocyte. In this case, the drug carrier is engulfed in an aggregate of activated platelets and leukocytes, and the molecular aggregates are broken down and released by physical stimulation due to morphological changes of the platelets and leukocytes. When the target is a living tissue, the target is preferably a vascular injury site or an inflamed tissue.

  It is preferable that the number of molecules of the recognition substance bound on the drug-carrying molecular assembly is high because the possibility of binding to cells or biological tissue is increased and the formation of aggregates proceeds rapidly. The number can be appropriately adjusted by those skilled in the art according to the desired degree of aggregation and aggregation rate.

Preparation of a drug-carrying molecular assembly, a linker, and a recognition substance conjugate may be prepared by binding a linker to the molecular assembly after the preparation of the molecular assembly, and then reacting the recognition substance. Alternatively, a reaction product of the linker and the recognition substance may be prepared in advance, and then the reaction product may be combined with the molecular assembly.
A drug-carrying molecule in which a linker and a drug-carrying molecular assembly are bound to the drug-carrying molecular assembly via an amphiphilic molecule that is a part of a component of the drug-carrying molecular assembly or via a hydrophobic molecule When preparing an aggregate, a linker, and a recognition substance combination, a reaction product of a linker, a recognition substance, and an amphiphilic molecule or a hydrophobic molecule is prepared in advance, and then the reaction product is converted into a molecule. You may combine with an aggregate.
The drug may be supported on the molecular assembly from the beginning, or may be finally supported on the molecular assembly after preparing a conjugate of the molecular assembly, the linker, and the recognition substance. As the reaction conditions, conditions known per se can be used according to the raw material of the molecular assembly. The mixing ratio between the drug-carrying molecular assembly and the recognition substance is adjusted according to the desired value of the density of the recognition substance in the final conjugate.

  The lipid and bilayer vesicles are linked to the lipid bilayer vesicles via amphipathic molecules that are part of the components of the lipid bilayer or via hydrophobic molecules. When preparing a conjugate of a molecular membrane endoplasmic reticulum, a linker, and a recognition substance, the above-mentioned parents that can be a constituent component of a lipid bilayer membrane in which a linker, a recognition substance, and an amphipathic molecule or a hydrophobic molecule are combined. The surface of the endoplasmic reticulum can be modified with a recognition substance by mixing the medicinal molecule with the lipid constituting the endoplasmic reticulum in an organic solvent and preparing the endoplasmic reticulum by a conventional method.

  Then, if necessary, the conjugate prepared above is washed with a physiologically acceptable aqueous solution, subjected to sterilization filtration, dispensing, etc., and the drug carrier of the present invention is used as a solution, pellet, or suspension. It can be. Formulation can be carried out according to a known method in the field of pharmaceutical production. Moreover, after freezing a liquid agent, it can also be dried under reduced pressure and it can also be set as a freeze-dried formulation. In addition, when freeze-drying, you may mix | blend monosaccharides (for example, glucose etc.), disaccharides (for example, sucrose) etc. as a protective agent. In the preparation, polymers such as albumin, dextran, vinyl polymer, gelatin and hydroxylethyl starch may be added as a stabilizer. The added amount of the stabilizer is preferably 0.5 to 10 parts by weight, more preferably 1 to 5 parts by weight with respect to 1 part by weight of the lipid.

The drug carried on the drug-carrying molecular assembly is not particularly limited as long as it has a desired physiological activity in cells such as activated platelets and leukocytes, or in biological tissues such as vascular injury sites and inflamed tissues, Platelet aggregation inducers, platelet aggregation inhibitors, vasoconstrictors, vasodilators and anti-inflammatory agents are preferred. When used as a diagnostic agent, a fluorescent reagent or a contrast agent having no physiological activity can be carried.
Platelet aggregation inducers include adenosine diphosphate (ADP), collagen, collagen-derived peptides, convulxin, serotonin, epinephrine, vasopressin, carbazochrome, blood coagulation factors (FVIII, FIX), thrombin, antiplasmin agents (for example, epsilon- Aminocaproic acid, tranexamic acid), protamine sulfate, ethanesylate, phytonadione, conjugated estrogens (for example, sodium estrone sulfate, sodium equilin sulfate) and the like.
Platelet aggregation inhibitors include mucopolysaccharides such as aspirin, dipyridamole, ticlopidine, cilostazol, beraprost, heparin, natural extracts such as coumarin anticoagulants, hirudin and their derivatives, physiological activities such as thrombomodulin and active protein C Examples include substances.
Examples of the vasoconstrictor include noradrenaline, norphenephrine, phenylephrine, metallaminol, methoxamine, prostaglandin F ₁ α, prostaglandin F ₂ α, thromboxane A _{2 and the} like.
The vasodilator prostaglandins E, such as prostaglandin I ₂ and the like.
Anti-inflammatory agents include steroidal anti-inflammatory agents (dexamethasone, hydrocortisone, prednisolone, betamethasone, triamcinolone, methylprednisolone, etc.), non-steroidal anti-inflammatory agents (indomethacin, acemetacin, flurbiprofen, aspirin, ibuprofen, flufenamic acid, Ketoprofen, etc.).
Particularly preferred examples of the drug carried on the drug-carrying molecular assembly include adenosine diphosphate (ADP), collagen, collagen-derived peptide, convulxin, serotonin, aspirin, dipyridamole, ticlopidine, cilostazol and beraprost.

  Since the amount of the drug to be carried on the drug-carrying molecular assembly varies depending on the kind of drug to be carried and the purpose of use, it is difficult to define it generally. For example, by encapsulating ADP in a phospholipid bilayer, When using the drug carrier of the present invention to activate platelets at a desired location, it is preferable to encapsulate 0.1 to 25 mM, more preferably 0.5 to 10 mM in 10 mg / mL of lipid. It is even more preferable to include 1 to 6 mM.

  Since the dosage of the preparation containing the drug carrier of the present invention is appropriately determined according to the amount of drug carried, the sex, age, symptoms, etc. of the patient, it cannot be generally determined. About 0.001 to 1000 mg per day can be administered. The preparation containing the drug carrier of the present invention is preferably administered parenterally, specifically administered by intravascular (intraarterial or intravenous) injection, continuous infusion, subcutaneous administration, local administration, intramuscular administration, and the like. be able to. The preparation containing the drug carrier of the present invention is useful as a platelet aggregation inducer, a platelet aggregation inhibitor, a vasoconstrictor, a vasodilator and an anti-inflammatory agent, and also a platelet replacement agent, antiplatelet agent, vascular disorder, Drugs such as preventive and therapeutic agents for vascular damage and thrombosis, diagnostic agents for platelet dysfunctions such as asthenia gravis, biological or medical reagents, reagents for screening platelet substitutes and antiplatelet agents It is also useful as a diagnostic agent or therapeutic agent for examination of vascular injury sites and angiogenesis sites.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to these Examples. Unless otherwise specified in the examples, the concentration of ADP or CF inclusion indicates the concentration (mM) in which ADP or CF is included in 10 mg / mL of lipid.

Example 1 ADP-containing endoplasmic reticulum
1. Synthesis of Glu2C18 Glutamic acid (2.96 g, 20 mmol) and p-toluenesulfonic acid monohydrate (4.56 g, 24 mmol) were dissolved in 150 mL of benzene, and the generated water was removed at 105 ° C. using a Dean-Stark apparatus. The mixture was refluxed for 1 hour. Stearyl alcohol (11.9 mg, 44 mmol) was added, and the mixture was further refluxed at 105 ° C. for 14 hours while removing generated water. After the solvent was distilled under reduced pressure, the residue was dissolved in 150 mL of chloroform and washed twice with 150 mL of a saturated aqueous solution of sodium carbonate and twice with 150 mL of water. The chloroform layer was collected, dehydrated with 5 g of sodium sulfate, and the solvent was removed under reduced pressure. The residue was dissolved in 400 mL of methanol at 60 ° C. and filtered if there were insoluble components, recrystallized at 4 ° C., filtered and dried to obtain white powder Glu2C18 (13.3 g, yield 85%).

2. Synthesis of H12-MAL-PEG-Glu2C18 After adding Glu2C18 (115.1 mg, 176 μmol) and triethylamine (TEA) (24.6 μL, 176 μmol) in 5 mL of chloroform, α-malemidyl-ω-N-hydroxysuccinimidyl was added. Polyethylene glycol (MAL-PEG-NHS) (Mw = 3400) (300 mg, 58.8 μmol) was dissolved and stirred at room temperature for 12 hours. The reaction solution was added dropwise to 250 mL of diethyl ether to recover insoluble components. The recovered product was dissolved in benzene and freeze-dried to obtain white powder MAL-PEG-Glu2C18 (264.8 mg, yield 64%).
MAL-PEG-Glu2C18 (n = 71) (100 mg, 25.37 μmol) and fibrinogen γ-chain C-terminal 400 to 411st amino acid sequence (Cys-H12) (CHHLGGAKQAGDV, SEQ ID NO: 2) with cysteine introduced at the C-terminus ( 32.8 mg, 25.37 μmol) was dissolved in 5 mL of dimethylformamide (DMF) and stirred at room temperature for 12 hours. The reaction solution was added dropwise to 250 mL of diethyl ether to recover insoluble components. After adding 250 mL of water to remove insoluble components, the solvent was removed by a freeze dryer to obtain a pale yellow powder H12-MAL-PEG-Glu2C18 (62.8 mg, 47% yield).

3. Endoplasmic reticulum preparation method 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) (100 mg, 136 μmol), cholesterol (52.7 mg, 136 μmol) and 1,5-dihexadecyl-N-succinyl-L- Glutamate (DHSG) (19.0 mg, 13.6 μmol) mixed lipid (in the description of lipid concentration in this specification, even if it is mixed lipid, it is simply described as lipid), and PEG-Glu2C18 (PEG- DSPE, manufactured by NOF Corporation, 4.74 mg, 0.817 μmol) or H12-MAL-PEG-Glu2C18 (4.34 mg, 0.817 μmol) was dissolved in 5 mL of benzene and freeze-dried for 3 hours. After drying, 8.5 mL of adenosine diphosphate (ADP) solution (0, 1, 10, 25 or 100 mM) was added and stirred for 12 hours, and then a filter (3000 nm → 800 nm → 650 nm → 450 nm → 300 nm → The particle size was controlled by passing 220 nm × 2). Ultracentrifugation (33000 rpm, 30 minutes) was performed twice and dispersed in 5 mL of phosphate buffered saline (PBS) to obtain an endoplasmic reticulum dispersion. Further, the endoplasmic reticulum dispersion was subjected to gel filtration (Sephadex G25) to completely remove ADP remaining in a trace amount in the outer aqueous phase.
By the above procedure, ADP-encapsulated H12-PEG vesicles (average particle size 250 ± 80 nm, average number of layers 1.6; Glu2C18 is a part of constituents of vesicles; the same applies hereinafter), ADP-unencapsulated H12- PEG vesicles (average particle size 230 ± 70 nm, average layer number 1.8), ADP-encapsulated PEG vesicles (average particle size 240 ± 90 nm, average layer number 1.5), ADP non-encapsulated PEG vesicles (average particle size A dispersion liquid of 250 ± 90 nm and an average number of layers of 1.8) was obtained.
When the lipid concentration of the collected endoplasmic reticulum dispersion was quantified (phospholipid C-Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.), the lipid concentration was 18 ± 5 mg / mL. In the animal experiments described below, PBS was used to adjust the lipid concentration of each endoplasmic reticulum dispersion to 0.25, 1.0, 2.5, 5.0, 10 mg / mL at 4 mL / kg. The dose was 1, 4, 10, 20, 40 mg / kg (in terms of lipid amount).
The number of endoplasmic reticulum layers (average number of layers) was calculated as follows.
A dispersion of non-ADP-encapsulated H12-PEG vesicles (0.5 wt%, 0, 20, 30, 50, 70, 90 μL) was diluted with PBS to a constant volume of 3 mL. 100 μL of TNS aqueous solution (70 μM) or pure water was added, and shaken at room temperature for 12 hours. Fluorescence measured (λex = 321nm, λem = 445nm ), to calculate the proportional expression of lipid concentration and fluorescence intensity was the inclination _{K 1.} Similarly, in the dispersion of PEG vesicles (0.5 wt%, 0, 20, 30, 50, 70, 90 μL) that has passed through a filter with a pore size of 0.05 μm, an aqueous TNS solution (70 μM) or pure water is added. After shaking for 12 hours at room temperature, the slope K ₂ was calculated, and the average number of layers N = K ₁ / K ₂ was calculated. The average number of layers was also calculated for ADP-unencapsulated H12-PEG vesicles, ADP-encapsulated PEG vesicles, and ADP-unencapsulated PEG vesicles.
Hereinafter, an ADP-encapsulated H12-PEG vesicle and an ADP-unencapsulated H12-PEG vesicle prepared using H12-MAL-PEG-Glu2C18 are referred to as an H12-PEG (ADP) vesicle and an H12-PEG vesicle, respectively. In addition, ADP-encapsulated PEG vesicles and ADP-unencapsulated PEG vesicles prepared using PEG-Glu2C18 are referred to as PEG (ADP) vesicles and PEG vesicles, respectively.

4). Quantification of ADP inclusion concentration using HPLC H12-PEG (ADP) vesicles (1 mg / mL) were solubilized with 2% lauryl ether (C ₁₂ E ₁₀ ), and ADP was quantified using HPLC (Abs. 260 nm). went.
FIG. 2 shows the relationship between the ADP concentration at the time of hydration (preparation) (0, 10, 25, 100 mM) and the concentration of ADP encapsulated in 10 mg / mL lipid. The encapsulated ADP concentration was proportional to the ADP concentration at the time of hydration, and it was confirmed that the encapsulated concentration could be controlled. Further, when the ADP concentration of the inner aqueous phase was calculated from the particle size (250 ± 80 nm), it was almost equal to the ADP concentration at the time of hydration.

5). Interaction analysis by flow cytometry (FACS) Whole blood (1/10 (v / v) 3.8% sodium citrate) was centrifuged (600 rpm, 15 minutes) to obtain platelet-rich plasma (PRP). The precipitated component was further centrifuged (2500 rpm, 10 minutes) to obtain platelet poor plasma (PPP). H12-PEG (ADP) vesicles having different ADP inclusion concentrations were added to PRP ([platelet] = 1.0 × 10 ⁵ / μL, 50 μL) whose platelet count was adjusted with PPP ([lipid] = 1 mg / mL 10 μL) and stirred at 37 ° C. for 10 minutes. After adding FITC-labeled PAC-1 (20 μL) as a platelet activation marker, the mixture was shaken at 37 ° C. for 10 minutes and fixed with formaldehyde (fc 1%). Samples were tested by flow cytometry. The positive control group was at the time of ADP stimulation, and the negative control group was a PEG vesicle.
In a system in which PEG (ADP) vesicles having an ADP encapsulation concentration of 1.5 mM or less or H12-PEG (ADP) vesicles were added to platelets, the PAC-1 binding rate was higher than that in the ADP non-encapsulating vesicle addition system. It was almost the same, and it was confirmed that platelets were not activated (FIG. 3). On the other hand, it was found that PEG (ADP) endoplasmic reticulum with the highest inclusion concentration and H12-PEG (ADP) endoplasmic reticulum (6 mM) were added to platelets and caused platelet activation.

6). Functional evaluation by platelet aggregometer In the following experiments, PEG (ADP) endoplasmic reticulum and A12-PEG (ADP) endoplasmic reticulum having an ADP inclusion concentration of 1.5 mM, which were found not to activate platelets by FACS measurement, In addition, PEG vesicles and H12-PEG vesicles were used. After adding the endoplasmic reticulum dispersion (10 μL) to PRP ([platelet] = 2.0 × 10 ⁵ / μL, 180 μL) whose platelet count was adjusted with PPP, collagen (fc 0.4 μg / mL, 10 μL), platelet aggregation was induced, and the change in permeability was measured (FIG. 4).
When PEG vesicle (a) was added, it was confirmed that there was no effect on platelet aggregation as compared with the PBS addition system. When H12-PEG vesicle (b) was added instead of PEG vesicle (a), the permeability increased and the effect of promoting platelet aggregation could be confirmed. This is thought to be because the H12-PEG vesicles were multipoint bonded to platelets and promoted platelet aggregation. In PEG (ADP) endoplasmic reticulum (c), aggregation was enhanced as compared with (b), and in H12-PEG (ADP) endoplasmic reticulum (d), it was more than the promoting effect of (c). On the other hand, it was also confirmed that platelet aggregation was not induced only by the presence of (d) (that is, (e) in the absence of collagen). Therefore, it is considered that platelet aggregation was induced by collagen, and the vesicle was immediately physically involved in the aggregate, and the vesicle was deformed to release ADP. The effect of (d) is considered to be due to the synergistic effect of multipoint bonding and ADP release by supporting H12.
Further, about 1 minute after the addition of collagen, a gradual decrease in permeability is observed with changes in platelet shape caused by collagen stimulation ((a) and (b)). However, in the ADP inclusion systems (c) and (d), an increase in permeability is observed immediately after the addition of collagen, which is a phenomenon peculiar to ADP stimulation. This suggested that platelet aggregation caused the release of ADP from the H12-PEG (ADP) endoplasmic reticulum (d).

7). Evaluation of effects of H12-PEG vesicle and H12-PEG (ADP) vesicle on tail bleeding time using thrombocytopenia model rats Wistar male rats (8 weeks old, 250-270 g) were treated with busulfan (20 mg / day). kg) was administered via the tail vein, and the thrombocytopenia model rat ([platelet] = 20 × 10 ⁴ / μL) was formed on the 10th day after administration. After sevofuran anesthesia, the sample was administered, and 5 minutes after administration, a wound 1 mm in length and 2.5 mm in length was made in a site 1 cm from the tip of the tail, and that part was immersed in physiological saline, and the bleeding time was measured.
When physiological saline was administered to thrombocytopenia model rats (4 mL / kg), the bleeding time was 682 ± 198 seconds, and the bleeding time (178 ± 56) of normal rats ([platelet] = 80 × 10 ⁴ / μL). (About 5 seconds) (Fig. 5). When H12-PEG endoplasmic reticulum dispersion adjusted to a lipid concentration of 2.5 and 10 mg / mL was administered (4 mL / kg), the bleeding time was shortened depending on the dose, and 10, 40 mg / kg (in terms of lipid amount) ) Bleeding times were 573 ± 127 and 335 ± 96 seconds, respectively. Therefore, when H12-PEG (ADP) endoplasmic reticulum dispersion liquid (ADP inclusion concentration 1 mM) adjusted to a lipid concentration of 0.25, 1, 2.5 mg / mL was administered (4 mL / kg), 1, 4, 10 mg / mL Bleeding time in kg (lipid amount conversion) was 543 ± 134, 521 ± 88, 349 ± 49 seconds, respectively, and it was found that it could be shortened with a dose of 1/4 that was confirmed to have a shortening effect with H12-PEG vesicles. did. From the above, it was confirmed in vivo that the hemostatic effect of H12-PEG vesicles by ADP encapsulation was improved.

8). Evaluation of the effect of H12-PEG (ADP) endoplasmic reticulum on ear bleeding time using a severe thrombocytopenia model rabbit New Zealand white rabbit (11 weeks old, 2.5 kg) tailed with busulfan (30 mg / kg) It was intravenously administered, and a thrombocytopenia model rabbit ([platelet] = 2.6 × 10 ⁴ / μL) was obtained on the 15th day after administration. After ketamine / cerectal anesthesia, the sample was administered at a rate of 0.5 mL / min. 30 minutes after administration, the periauric vein was injured with a length of 6 mm, and the part was immersed in physiological saline to measure the bleeding time.
When physiological saline was administered to a thrombocytopenia model rabbit (4 mL / kg), the bleeding time was 1695 ± 197 seconds, and the bleeding time (112 ± 24) of a normal rabbit ([platelet] = 41 × 10 ⁴ / μL). Compared to the second) (FIG. 6). As a positive control group, rabbit platelets were administered at 0.4 × 10 ⁹ , 2.0 × 10 ⁹ , 4.0 × 10 ⁹ / kg, and the bleeding time was shortened depending on the platelet count (each 1505 ± 410). 863 ± 440, 505 ± 257 seconds). Therefore, when H12-PEG (ADP) vesicle dispersion (4 mL / kg) adjusted to a lipid concentration of 2.5 and 5.0 mg / mL was administered, bleeding times were 881 ± 303 and 433 ± 52 seconds, respectively. Yes, the bleeding time was significantly shortened in a dose-dependent manner compared with the physiological saline group, comparable to that of the platelet administration group. Therefore, it was confirmed that H12-PEG (ADP) endoplasmic reticulum efficiently shortens the bleeding time of a thrombocytopenic model rabbit.

9. Comparison of the effect of PEG (ADP) vesicles and H12-PEG (ADP) vesicles on bleeding time Dispersion of PEG (ADP) vesicles or H12-PEG (ADP) vesicles (10 mg / kg (lipid amount conversion) ), The bleeding time when ADP inclusion concentration 0, 1, 10 mM) was administered. Bleeding time was measured using normal rats and thrombocytopenia model rats. As well as. The results are shown in FIG. The H12-PEG vesicle (that is, ADP inclusion concentration 0) administration system does not show an effect of shortening the bleeding time, but the H12-PEG (ADP) vesicle has an effect compared with the physiological saline administration system of a thrombocytopenia model rat. The bleeding time was significantly shortened, and a similar shortening was obtained at a dose of 1/10 at which the shortening effect could be confirmed with the PEG (ADP) endoplasmic reticulum. From the above, it was confirmed that the H12-PEG (ADP) vesicle had a superior hemostatic effect than the PEG (ADP) vesicle.

10. Measurement of ADP inclusion effect using a platelet aggregometer PEG vesicles and H12-PEG vesicles were prepared with different ADP inclusion amounts (PEG (ADP) vesicles, H12-PEG (ADP) vesicles). The ADP inclusion concentration was calculated by HPLC (260 nm) after solubilizing the endoplasmic reticulum with 2% lauryl ether. The effect of ADP encapsulation was evaluated using a platelet aggregometer. Prepare platelet-decreased plasma (PLT) ([platelet] = 10 × 10 ⁴ / μL, 180 μL) with platelet count adjusted by PPP, add 10 μL of ER dispersion, then add ADP (30-45 μM, 10 μL). It was added and the transmittance was measured. The added vesicles were PEG (ADP) vesicles or H12-PEG (ADP) vesicles having an ADP inclusion concentration of 0, 0.1, 0.5, 1.0, 2.0, or 10.0 mM. Evaluation was performed based on the difference from the transmittance when PEG vesicles were added. FIG. 8 shows the results when platelet aggregation was induced using ADP. In the H12-PEG (ADP) vesicle addition system, the inclusion effect could be confirmed at an ADP inclusion concentration of 1 mM or more, and the inclusion effect was almost the same even in the higher concentration inclusion vesicles. Moreover, the inclusion effect was not confirmed about the PEG (ADP) endoplasmic reticulum addition system.

11. Synthesis of Bio-PEG-Glu2C18 N- [6- ( Biotinamide ) hexyl] -3 ′-(2′-pyridyldithio) propionamide (Bio-HPDP, 10 mg, 18.5 μmol) was dissolved in 5 mL of DMF, and dithiosley. 20 μL of a tall aqueous solution (1M) was added and stirred at room temperature for 30 minutes, then MAL-PEG-Glu2C18 (73.0 mg, 18.5 μmol) was added and stirred at room temperature for 12 hours. The reaction solution was added dropwise to 250 mL of diethyl ether to recover insoluble components. After adding 250 mL of water and removing insoluble components, the solvent was removed with a freeze dryer to obtain a light yellow powder Bio-MAL-PEG-Glu2C18 (Bio: biotin).

12 Electron Microscopic Observation of H12-PEG (ADP) Endoplasmic Reticulum Engaged in Platelet Aggregate For the purpose of observation of endoplasmic reticulum in H12-PEG (ADP) endoplasmic reticulum addition system, Bio-MAL was applied to H12-PEG (ADP) endoplasmic reticulum. -PEG-Glu2C18 was introduced, and the aggregate was frozen and sliced and observed with an immunoelectron microscope using a transmission electron microscope (FIG. 9). It is confirmed that the endoplasmic reticulum is involved between platelets, and Bio-MAL-PEG-Glu2C18 is scattered at each site of the platelet, suggesting that the endoplasmic reticulum has collapsed in the aggregate. It was.

Example 2 CF-containing endoplasmic reticulum
1. Production of CF-encapsulated PEG vesicles and CF-encapsulated H12-PEG vesicles ~ 3. PEG vesicles and H12-PEG vesicles encapsulating 10 mM 5 (6) -carboxyfluorescein (CF) were prepared according to the procedure described in 1. The amount of CF inclusion is the same as that in Example 1. Quantified in the same manner as above.
CF-encapsulated PEG vesicles (average particle size 230 ± 80 nm, average number of layers 1.8)
CF-encapsulated H12-PEG vesicles (average particle size 240 ± 50 nm, average number of layers 1.6)
2. Quantification of amount of endoplasmic reticulum release accompanying platelet aggregation CF (10 mM) -encapsulated PEG vesicle or CF (10 mM) -encapsulated H12-PEG vesicle was PRP ([platelet] = 2.0 × 10 ⁵ / μL, [vesicle] = 0.05 mg / mL), platelet aggregation was induced by ADP ([ADP] = fc 2 μM), and the permeability change was measured with a platelet aggregometer. After completion of the measurement, aggregates were removed by centrifugation (1200 rpm, 5 minutes). At this time, in the ADP non-addition system, only the platelets were removed, and the fluorescence intensity (A) when the vesicle was solubilized with 2% lauryl ether (C ₁₂ E ₁₀ ) was measured and defined as 100%. The fluorescence intensity (B) in the supernatant (endoplasmic reticulum dispersion) was measured, and the rate of endoplasmic reticulum uptake into the platelet aggregate was calculated. Further, the supernatant was centrifuged (33,000 rpm, 45 minutes) to remove the endoplasmic reticulum, the fluorescence intensity (C) of the supernatant was measured, and the CF release rate from the endoplasmic reticulum incorporated into the platelet aggregate was measured. Was calculated.

When CF-encapsulated PEG vesicles were added, it was confirmed that there was no effect on platelet aggregation compared to the PBS-added system. When CF-encapsulated H12-PEG vesicles were added instead of CF-encapsulated PEG vesicles, secondary aggregation was enhanced, the permeability was increased, and the platelet aggregation promoting effect was confirmed (FIG. 10). This is presumably because the CF-encapsulated H12-PEG vesicles were multipoint bonded to platelets and promoted platelet aggregation.
The incorporation rates of CF-encapsulated PEG-vesicles and CF-encapsulated H12-PEG vesicles into platelet aggregates were 13 ± 5 and 17 ± 5%, respectively, which were almost the same. Therefore, when the CF release rate from the incorporated endoplasmic reticulum was measured, they were 0.6 ± 0.5 and 10 ± 1%, respectively (Table 1). This is because the binding of the endoplasmic reticulum to platelets was strengthened by supporting H12 on the endoplasmic reticulum, and the CF release rate increased with physical stimulation when the endoplasmic reticulum was involved in platelet aggregation. Conceivable.

  The drug carrier of the present invention exhibits selective binding to activated platelets, vascular injury sites and / or inflamed tissues, and releases the drug carried only at these locations, so that adverse effects can occur other than at the desired location. Without effect, the effect of the carrier drug can be expressed only in activated platelets, vascular injury sites and / or inflamed tissues. Therefore, the preparation containing the drug carrier of the present invention is useful as a platelet aggregation inducer, a platelet aggregation inhibitor, a vasoconstrictor, a vasodilator, and an anti-inflammatory agent, and also a platelet substitute, antiplatelet, blood vessel For screening pharmaceuticals such as prophylactic / therapeutic agents for disorders, vascular injury and thrombosis, etc., diagnostic agents for platelet dysfunctions such as asthenia gravis, biological or medical reagents, platelet replacement agents and antiplatelet agents It is also useful as a reagent for the above, a diagnostic agent or a therapeutic agent for examination of vascular damage sites and angiogenesis sites.

2 is a synthesis scheme of Glu2C18 and H12-MAL-PEG-Glu2C18 described in Example 1. It is a graph which shows the relationship of the ADP density | concentration included in 10 mg / mL of lipid with respect to the ADP density | concentration (0, 10, 25, 100 mM) at the time of hydration. FIG. 6 is a graph showing the binding ratio of PAC-1 to platelets in the presence of H12-PEG (ADP) vesicles (black) or PEG (ADP) vesicles (white). It is a change in permeability showing the effect of promoting collagen-induced platelet aggregation by H12-PEG (ADP) vesicles. [Collagen]: f. c. 0.4 μg / mL, [platelet]: 2.0 × 10 ⁵ / μL, [lipid]: 0.05 mg / mL. (A) PEG vesicle, (b) H12-PEG vesicle, (c) PEG (ADP) vesicle, (d) H12-PEG (ADP) vesicle, (e) H12-PEG in the absence of collagen ( ADP) endoplasmic reticulum. It is a graph which shows the hemostatic effect of H12-PEG (ADP) endoplasmic reticulum administration with respect to tail bleeding time. H12-PEG (ADP) endoplasmic reticulum dosage: 1, 4, 10 mg / kg (in terms of lipid amount). ○: Platelet count (N = 10). ^* P <0.05. It is a graph which shows the hemostatic effect of H12-PEG (ADP) endoplasmic reticulum administration and PRP with respect to ear bleeding time. H12-PEG (ADP) endoplasmic reticulum dose: 10, 20 mg / kg (in terms of lipid amount). Rabbit platelet dosage: 0.4, 2.0, 4.0 × 10 ⁹ / kg. ○: Platelet count (N = 5-6). ^* P <0.05 vs. Saline group. It is the graph which compared the hemostatic effect of PEG (ADP) vesicle and H12-PEG (ADP) vesicle with respect to tail bleeding time. Endoplasmic reticulum dose: 10 mg / kg. ○: Platelet count (N = 6-10). It is a graph which shows the ADP inclusion effect to a PEG vesicle and an H12-PEG vesicle. It is a transmission electron microscope image of platelet aggregation. What is indicated by an arrow is an H12-PEG (ADP) endoplasmic reticulum. It is a change in permeability showing the effect of promoting ADP-induced platelet aggregation by CF-encapsulated H12-PEG vesicles.

SEQ ID NO: 1: Design peptide SEQ ID NO: 2: Design peptide

Claims (20)

  1) a drug carrier that is a conjugate of a drug-carrying molecular assembly, 2) a linker, and 3) a substance that recognizes activated platelets, vascular injury sites and / or inflammatory tissues.   The drug carrier according to claim 1, wherein the drug-carrying molecular assembly is a lipid bilayer vesicle in which a drug is encapsulated in an inner aqueous phase thereof.   The drug carrier according to claim 1 or 2, represented by (drug-carrying molecular assembly)-(linker)-(substance that recognizes activated platelets, vascular injury sites and / or inflammatory tissues).   The linker contains an amphiphilic molecule that becomes a part of the constituent components of the drug-carrying molecular assembly at the time of binding to the drug-carrying molecular assembly, and the linker and the drug-carrying molecular assembly are bonded via the amphiphilic molecule. The drug carrier according to claim 3.   The drug carrier according to claim 3, wherein the linker includes a hydrophobic molecule, and the linker and the drug-carrying molecular assembly are bonded to the drug-carrying molecular assembly through the hydrophobic molecule.   The drug carrier according to any one of claims 1 to 5, wherein the linker comprises a spacer moiety.   The drug carrier according to claim 6, wherein the spacer portion is polyoxyethylene.   The drug carrier according to any one of claims 1 to 7, wherein the drug is selected from the group consisting of a platelet aggregation inducer, a platelet aggregation inhibitor, a vasoconstrictor, a vasodilator, and an anti-inflammatory agent.   The drug delivery according to any one of claims 1 to 7, wherein the drug is selected from the group consisting of adenosine diphosphate, collagen, collagen-derived peptide, convulxin, serotonin, aspirin, dipyridamole, ticlopidine, cilostazol and beraprost. body.   Substances that recognize activated platelets, vascular injury sites and / or inflammatory tissues are integrin or selectin exposed to activated platelets, collagen exposed to vascular injury sites, collagen exposed to vascular injury sites A substance that recognizes bound von Willebrand factor, selectin exposed to inflamed tissue and / or selectin ligand exposed to leukocytes, and is taken up by aggregates of activated platelets and / or leukocytes, and / or The drug carrier according to any one of claims 1 to 9, which is a substance that accumulates at a vascular injury site and / or an inflamed tissue.   A substance that recognizes activated platelets, sites of vascular injury and / or inflamed tissue is selected from the group consisting of H12, GPIbα, GPIa / IIa, GPVI, MAC-1, fibrinogen, P-selectin and PSGL-1. Item 11. A drug carrier according to Item 10.   Lipid bilayer endoplasmic reticulum is composed of a mixed lipid containing 20-100% of cholesterol in a molar ratio with respect to phosphatidylcholine which is hydrogenated egg yolk lecithin, hydrogenated soybean lecithin, distearoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine, and a linker The drug carrier according to claim 2, wherein a conjugate with a substance that recognizes activated platelets, vascular injury sites and / or inflammatory tissues is contained in an amount of 0.001 to 20% relative to the phosphatidylcholine.   The drug carrier according to claim 12, wherein the lipid bilayer vesicle has a particle size of 50 to 300 nm and the lipid bilayer has 1 to 4 layers.   1) a drug-carrying molecular assembly, 2) a linker, and 3) a drug carrier which is a conjugate of activated platelets, a substance recognizing a vascular injury site and / or an inflammatory tissue, which is attached to a cell or biological tissue A drug carrier in which a drug is released from a drug-carrying molecular assembly by receiving physical stimulation from a cell or a living tissue when it reaches.   The drug carrier according to claim 14, wherein the cells are activated platelets or leukocytes, and the drug is selected from the group consisting of a platelet aggregation inducer, a platelet aggregation inhibitor, a vasoconstrictor, a vasodilator, and an anti-inflammatory agent. .   The biological tissue is a vascular injury site or an inflammatory tissue, and the drug is selected from the group consisting of a platelet aggregation inducer, a platelet aggregation inhibitor, a vasoconstrictor, a vasodilator, and an anti-inflammatory agent. Drug carrier.   A diagnostic agent comprising the drug carrier according to any one of claims 1 to 16.   A reagent comprising the drug carrier according to any one of claims 1 to 16.   A platelet substitute comprising the drug carrier according to any one of claims 1 to 16.   An antiplatelet agent comprising the drug carrier according to any one of claims 1 to 16.