Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
  • C08G83/002—Dendritic macromolecules
  • C08G83/003—Dendrimers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/10—Organic compounds
  • A61K49/12—Macromolecular compounds
  • A61K49/124—Macromolecular compounds dendrimers, dendrons, hyperbranched compounds
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
  • C08G73/02—Polyamines
  • C08G73/028—Polyamidoamines
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
  • C08L101/00—Compositions of unspecified macromolecular compounds
  • C08L101/02—Compositions of unspecified macromolecular compounds characterised by the presence of specified groups, e.g. terminal or pendant functional groups
  • C08L101/04—Compositions of unspecified macromolecular compounds characterised by the presence of specified groups, e.g. terminal or pendant functional groups containing halogen atoms

Definitions

• the present invention relates to a fluorine-containing dendrimer particle with high contrast ability, which can be used in a magnetic resonance imaging (MRI) using fluorine as a detection nucleus (hereinafter, referred to as F-MRI) , a contrast medium using the same, and a method of manufacturing a dendrimer particle.
• MRI magnetic resonance imaging
• F-MRI fluorine as a detection nucleus
• MRI magnetic nuclear resonance
• H-MRI that uses proton ( 1 H) as a detection nucleus and captures a magnetic environment of water molecules in the living body to produce an image.
• Water molecules are present in almost all regions in the living body.
• H-MRI can susceptibly capture a difference in magnetic property of protons due to a change in environment around water molecules . Therefore, the H-MRI is suitable for whole-body imaging.
• the H-MRI is used as a diagnosis method with more information contents because the difference appears as a change in image to provide disease information.
• 19 F has a characteristic of being a stable nucleide which can be detected by NMR-spectroscopy has a detection sensitivity as high as 83% of 1 H is a stable element with a natural abundance ratio of 100%, and is applicable to imaging with a conventional H-MRI apparatus. Therefore, F-MRI has been expected to be a next-generation diagnostic method following H-MRI.
• F-MRI Fluorescence MRI
• diagnostic imaging for obtaining tracer information, which utilizes a feature that fluorine atoms are not present in the living body.
• positional information about an affected area can be obtained by recognizing an endogenous change attributable to a disease and using a fluorine compound accumulated thereto as a contrast medium.
• the method can be advantageously used in diagnosis of a morphologically unchanged lesion part which has not been detected by the conventional diagnostic imaging method.
• PET Positron Emission Tomography
• SPECT Single Photon Emission Computed Tomography
• the resonant frequency shifts and the chemical shift can be a physical property to be used as a probe for finding out the structure of a molecule containing the 19 F.
• the nuclear spin of an atomic nucleus excited by irradiation of RF pulses in MRI measurement shows uniform phases at the beginning. However, the phases become nonuniform rapidly by relaxation and the vector sizes decrease rapidly. This is called the T2 relaxation. Simultaneously, some of the downward spins return to upward spins and the vector size in the vertical direction recovers slowly. This is called the Tl relaxation.
• the Tl relaxation is a process by which a magnetization vector in the vertical direction recovers exponentially with time and the Tl value (Tl relaxation time) is defined as a time (time constant) to return to 1-1/e (63.2%) of the original value.
• the T2 relaxation is a process by which a magnetization vector in the horizontal direction decreases exponentially with time.
• the T2 value (T2 relaxation time) is defined as a time (time constant) until a signal attenuates from the maximum (initial value) to 1/e (36.8%) .
• the diffusion of object molecules for measurement affects on the MRI signal intensity. For instance, strong diffusion leads to nonuniform phases, causing a decrease in MRI signal intensity. Thus, information about the Brownian movement of molecules in the tissue can be obtained based on the decrease in MRI signal intensity.
• useful diagnostic information in which anatomical information (information about the coordinate axis in the living body) coexists with functional information (information about the lesion part) may be obtained by simultaneously taking F-MRI and H-MRI images at a single diagnosis and then superimposing one on another.
• the PFC emulsion has a large number of fluorine atoms, and hence it can be the most advantageous compound among the present fluoro compounds with respect to contrast sensitivity. Contrasting experiments on the blood system, the reticuloendothelial system, and the like using the commercial PFCs have been reported. Also, neoangiogenesis is actively occurred in a tumor cell site. The newborn blood vessel does not have a fine structure compared with the vessel of the normal tissue blood vessel. Thus, a phenomenon of allowing even fine particles with certain sizes to permeate the blood vessel to the outside (Enhanced Permeability and Retention: EPR effect) has been known.
• EPR effect Enhanced Permeability and Retention
• the quality of the imaging thereof changes basically depending on the pharmacokinetics of fine particles on the bloodstream.
• Various kinds of barriers have been found in the body, even in the blood capillary and the retina. As their barrier permeabilities are different depending on their sizes, and naturally, changes in pharmacokinetics occur .
• the fine particles cannot permeate the newborn blood vessel of the tumor portion if the particle size is too large.
• the particle size is too small, the fine particles can also permeate through the normal blood vessel walls other than the newborn blood vessel of the tumor portion, so that selective imaging only for the tumor portion cannot be performed.
• the emulsion is a molecular self assembly, and the numbers of accumulated molecules among the particles cannot be exactly equalized. Thus, it is difficult to adjust the particle sizes precisely.
• an object of the present invention is to provide dendrimer particle containing fluorine atoms with precisely controlled sizes.
• an object of the present invention is to provide an F-MRI contrast medium with improved contrast sensitivity and an improved uniformity of size.
• an object of the present invention is to provide a method of manufacturing dendrimer particles containing fluorine atoms having precisely controlled sizes .
• the first aspect of the present invention provides a dendrimer particle having a unit that contains a fluorine atom at a plurality of branch ends of an aliphatic highly regular branched polymer.
• the second aspect of the present invention provides an MRI contrast medium containing the dendrimer particle.
• the third aspect of the present invention is a method of manufacturing a dendrimer particle including providing a unit that contains a fluorine atom to a plurality of branch ends of an aliphatic highly regular branched polymer with.
• FIGS. IA and IB each illustrate an H-NMR spectrum of a polyamide amine dendrimer, in which FIG. IA illustrates that of a raw material (before reaction) , PAMAM-NH 2 ; and FIG.
• FIG. 2 illustrates an H-NMR spectrum
• FIG. 3 is a graph illustrating a relationship among polymerization time, molecular weight (Mn) , and molecular weight distribution (Mw/Mn) in polymerization of PAMAM-g-PTFEMA.
• FIG. 4 illustrates an F-NMR spectrum of PAMAM-g-PTFEMA.
• FIG. 5 is a graph illustrating a relationship between reaction time and molecular weight (Mn) in polymerization reinitiation reaction of PAMAM-g-PTFEMA.
• FIGS. 6A and 6B each illustrate a TEM image of the PAMAM-g-PTFEMA, in which FIG. 6A is of a 25.5-hour polymerization sample and FIG. 6B is of a 4-hour polymerization sample (each of the scale bars represents 10 nm) .
• FIG. 7 is a graph illustrating a relationship among polymerization time, molecular weight (Mn) , and molecular weight distribution (Mw/Mn) in the polymerization of PAMAM-g-PTFPMA.
• FIGS. 1OA and 1OB each illustrate an F-NMR spectrum obtained in measuring Tl-relaxation times by an inversion recovery method, in which FIG. 1OA is of TFT in chloroform solution and FIG. 1OB is of PAMAM-g-PTFEMA in chloroform solution.
• the inventors of the present invention have arrived at the contrast medium of the present invention, which is capable of controlling the contrast sensitivity and sizes as a result of intensive studies on use of dendrimers .
• a compound with an increased content of fluorine atoms should be used.
• An increase in content of fluorine atoms may be attained by use of micelles, vesicles, or emulsion obtained by self-assembly of compounds with comparatively lower molecular weights. In this case, however, it is difficult to exactly control the size of the compound.
• the inventors of the present invention have paid attention to dendrimers to solve the problem, which are polymers with a structure of a branched molecular chain which is branched with high regularity (highly regular branched polymer) .
• the dendrimer is a general term for the branched polymer with regular dendric branches as described in Hawker, et al., J. Chem. Soc, Chem. Commun. 1990, (15), 1010-1013; D. A. Tomalia, et . al., Angew. Chem. Int. Ed. Engl., 29, 138-175 (1990); J. M. J. Frechet, Science, 263, 1710 (1994); Masaaki Kakimoto, Chemistry, vol. 50, page 608 (1995); and the like.
• Such a molecule has a polymer structure with regular branches extending from the center of the molecule.
• it becomes a globular molecular form because of extremely sterically crowded branch ends generated with increasing molecular weight .
• the dendrimer has an advantage in that the size thereof can be exactly controlled by its generation and has a characteristic in that the number of the outermost structures can be regularly changed.
• the inventors of the present invention have found that the use of many- partial structures arranged on the outermost of the dendrimer can effectively increase the content of fluorine and rigorously controls the size thereof.
• dendrimers with fluorine atoms include one having a substituent group with a fluorine atom in its benzene ring as disclosed in Japanese Patent Application Laid-Open No. 2002-220468 and one prepared by bonding a fluorine-atom-containing compound to a siloxane dendrimer as disclosed in Japanese Patent Application Laid-Open No. 2003-226611.
• the dendrimers used in the present invention should be of aliphatic compounds but not aromatic or siloxane compounds in terms of biocompatibility and solubility. Therefore, the dendrimer particle of the present invention has a structure in which a fluorine-containing unit is bound to a plurality of branch ends of an aliphatic dendrimer (highly regular branched polymer) .
• the aliphatic dendrimer which can be used in the present invention preferably includes one having a PAMAM skeleton (polyamideamine) or one having a MPA skeleton.
• Lagmuir, 23, 8299-8303, 2007 discloses a study case in which the terminal end of dendrimers is chemically attached to a substrate temporarily and a fluorine-containing compound is chemically bonded to the dendrimer terminal end using the supercritical condition of carbon dioxide.
• the state shown in this study case is such that it is fixed on the substrate by chemical bonding, and hence this case cannot be used for a contrast medium required to be administered to the living body.
• the dendrimer particle of the present invention is characterized by including a fluorine-atom-containing unit at a plurality of branch ends of an aliphatic highly regular branched polymer.
• polymers with repeating units that contain fluorine atoms may be used as the fluorine-atom-containing unit .
• repeating unit may include those represented by the following general formulae (1) and (2) :
• Rfi and Rf 2 each represent a monomer or an oligomer of a linear or branched alkyl group that contains a fluorine atom, a linear or branched oxyalkyl group that contains a fluorine atom, or a linear or branched oxyalkylene group that contains a fluorine atom, in which hydrogen in the alkyl group, the oxyalkyl group, and the oxyalkylene group may be substituted with an atom or an atomic group other than hydrogen, and -CH 2 - in the alkyl group and the oxyalkyl group may be substituted with -0-, -CO-, -NH-, or -C00-.
• the polymerization degree of the oligomer of the linear or branched oxyalkylene group that contains a fluorine atom is preferably 10 or less.
• Li and L 2 each represent a single bond or a divalent linking group selected from -0-, an alkylene group, an alkylene group having a hydroxyl group, an oxyalkylene group, and -NRiR 2 -, in which Ri represents hydrogen or an alkyl group and R 2 representsa single bond or a divalent linking group selected from an alkylene group, an alkylene group having a hydroxyl group, and an oxyalkylene group.
• fluorine-free repeating units may include those represented by the following general formulae (Ia) and (2a) :
• Rf 4 and Rf 5 each represent a monomer or an oligomer of a linear or branched alkyl group that does not contain a fluorine atom, a linear or branched oxyalkyl group that does not contain a fluorine atom, or a linear or branched oxyalkylene group that does not contain a fluorine atom, in which hydrogen in the alkyl group, the oxyalkyl group, and the oxyalkylene group may be substituted with an atom or an atomic group other than hydrogen, and -CH 2 - in the alkyl group and the oxyalkyl group may be substituted with -0-, -CO-, -NH-, or -C00-.
• the polymerization degree of the oligomer of the linear or branched oxyalkylene group that does not contain a fluorine atom is preferably 10 or less.
• L 3 and L 4 each represent a single bond or a divalent linking group selected from -O-, an alkylene group, an alkylene group having a hydroxyl group, an oxyalkylene group, and -NR 3 R 4 -, in which R3 represents hydrogen or an alkyl group and R 4 represents a single bond or a divalent linking group selected from an alkylene group, an alkylene group having a hydroxyl group, and an oxyalkylene group.
• examples thereof may include one represented by the following general formula (3) : where Rf3 represents an alkyl group that contains a fluorine atom and may be linear or branched or a linear or branched oxyalkyl group that contains a fluorine atom, in which hydrogen in the alkyl group and the oxyalkyl group may be substituted with an atom or an atomic group other than hydrogen, and -CH 2 - in the alkyl group and the oxyalkyl group may be substituted with -O-, -CO-, -NH-, or -COO-.
• L represents a single bond or a divalent linking group selected from an alkylene group, an alkylene group having a hydroxyl group, an oxyalkylene group, a phenylene group, an oxyphenylene group, and -NR 3 R 4 -, in which R 3 represents hydrogen or an alkyl group and R 4 represents a single bond or a divalent linking group selected from an alkylene group, an alkylene group having a hydroxyl group, and an oxyalkylene group.
• Rf 3 of the above formula (3) can increase the content of fluorine atoms in a dendrimer particle.
• the use of a perfluoroalkyl group or perfluoroalkylene group with many fluorine atoms causes a decrease in molecular mobility of a fluorine-containing portion, and may lead to a decrease in detection sensitivity in F-MRI. Therefore, insofar as considering the use of the perfluoroalkyl group or perfluoroalkylene group as an F-MRI contrast medium, it is preferable to appropriately control the content of fluorine atoms in the group represented by Rf 3 . More specifically, the content of fluorine atoms per unit in the perfluoroalkyl group or perfluoroalkylene group is preferably 1 to 50, more preferably 3 to 30.
• the compound of the present invention is preferably modified to increase its water solubility (more precisely, solubility in body fluids such as the blood) .
• Such a modification may be the introduction of a hydrophilic group into a dendrimer particle (preferably into the vicinity of the surface thereof) .
• a hydrophilic group such as -OH, -COOH, -NH 2 , -0-, or -NH- may be introduced.
• those hydrophilic groups may be introduced into the main chain thereof or may be introduced into the side chain thereof.
• a hydrophilic group can be introduced into any of Li, Rfi, L 2 , Rf 2 , L, and Rf 3 . More preferably, the hydrophilic group is added to the end of the fluorine-containing unit.
• a fluorine-free unit may be also arranged on the outside of the fluorine-containing unit and a hydrophilic group may be then introduced into the fluorine-free unit.
• the molecular designing is desired to be carried out in consideration of the degree of solvation of the fluorine atoms themselves.
• the compound of the present invention when used as a tumor-specific F-MRI contrast medium or an inflamed-site-specific F-MRI contrast medium, it is desirable to adjust the particle sizes of the compound within an appropriate range.
• the particle size is preferably in the range of 10 nm or more and 200 nm or less because the compound of the present invention can enter the tissue of the tumor or inflamed site.
• the particles with sizes of 10 nm or more and 200 nm or less can facilitate the entry to the tissue of the tumor or inflamed site from the blood vessel while making it difficult to enter to the normal tissue from the normal capillary vessel.
• the particle size is preferably 20 nm or more and 200 nm or less, more preferably 50 nm or more and 100 nm or less.
• the particle size is preferably 2 nm or more and 100 nm or less.
• a method of adjusting the particle size may be appropriately selecting the generation of dendrimers to be provided as a core.
• the particle size can be easily adjusted by controlling the polymerization reaction time.
• Method of manufacturing dendrimer particles As a method of providing a fluorine-containing unit at the terminal end of a dendrimer, there are two methods.
• One is a method of covalently bonding a fluorine-atom-containing molecule with a comparatively low molecular weight to the terminal end of a dendrimer.
• the other one is a method of polymerizing fluorine-atom-containing molecules as monomers from the terminal end of a dendrimer.
• fluorine-atom-containing low-molecular-weight compound means a fluorine-atom-containing molecule as a material to be bonded to the terminal end of a dendrimer without using a polymerization method.
• fluorine-atom-containing monomer means a fluorine-atom-containing molecule as a material to be bonded to the terminal end of a dendrimer using the polymerization method.
• an aliphatic dendrimer reacts with a molecule that contains a fluorine atom (fluorine-atom-containing low-molecular-weight compound) represented by the following general formula (6) : where Rf 3 represents an alkyl group that contains a fluorine atom and may be linear or branched or a linear or branched oxyalkyl group that contains a fluorine atom, in which hydrogen in the alkyl group and the oxyalkyl group may be substituted with an atom or an atomic group other than hydrogen, and -CH2- in the alkyl group and the oxyalkyl group may be substituted with -0-, -CO-, -NH-, or -COO-.
• a fluorine atom fluorine-atom-containing low-molecular-weight compound represented by the following general formula (6) : where Rf 3 represents an alkyl group that contains a fluorine atom and may be linear or branched or a linear or branched oxyal
• Rf 3 may include a group for enhancing its hydrophilic property.
• an OH group or a COOH group can be added to the terminal end opposite to L.
• a group as an OH group and a COOH group be protected by a protective group.
• Xi is a functional group to form a covalent bond with a functional group at the terminal end of the dendrimer.
• an amino group, a carboxyl group, a hydroxyl group, a halogen, carboxylic chloride, and carboxylic fluoride are favorably used.
• L is a linker to bond the functional group Xi and the group Rf 3 containing a fluorine atom.
• L include a single bond (the linker is not substantially present) , an alkylene group, an alkylene group having a hydroxyl group, a phenylene group, and an oxyphenylene group.
• a single bond, an alkylene group, and an alkylene group substituted with a hydroxy group can be favorably used.
• Particularly preferred is a single bond or a divalent linking group selected from -0-, an alkylene group, an alkylene group having a hydroxyl group, and an oxyalkylene group.
• fluorine-atom-containing low-molecular-weight compound represented by the general formula (6) Specific examples of the fluorine-atom-containing low-molecular-weight compound represented by the general formula (6) are illustrated below. However, the present invention is not limited to these exemplified compounds.
• the number of fluorine atoms in the fluorine-atom-containing low-molecular-weight compound be comparatively low.
• a higher generation of a dendrimer to be provided as a core and an increased number of bonded fluorine-atom-containing low-molecular-weight compounds are effective.
• the terminal end of the dendrimer should have a reactive functional group.
• an amino group, a carboxyl group, and a hydroxyl group are preferable.
• spacers with a constant low molecular weight may be used for effectively bonding fluorine-atom-containing low-molecular-weight compounds to dendrimers.
• the following functional group may be newly introduced into an amino group terminal end of a dendrimer, whereby the terminal end of the dendrimers is provided with a functional group other than an amino group.
• the polymerization of fluorine-atom-containing monomers from a terminal end of a dendrimer can provide the terminal end of the dendrimer with a polymer including a fluorine-atom-containing repeating unit.
• a functional group suitable for the starting point of a polymerization reaction may be bonded to the terminal end of dendrimer in advance.
• a preferable polymerization method is a living radical polymerization method as described later, particularly an atom transfer radical polymerization method, because this method can control the size of a dendrimer particle precisely, because of a wide range of usable raw materials and easiness of molecular weight control.
• the use of this technique can increase the content of fluorine atoms in the finally obtained dendrimer particle.
• Reactive monomers which can be used herein include those with double bonds and triple bonds; substituents such as a carboxyl group, an amino group, and a hydroxyl group, which are condensation polymerizable; an epoxy group which is ring-opening polymerizable; and an isocyanate group and a thioisocyanate group, which are addition polymerizable.
• the sizes of the dendrimer particle may vary as a result of a broaden distribution of molecular weights depending on the polymerization method. In terms of preventing the generation of this problem, any monomer with a double bond, particularly a monomer with an acryl group or a methacryl group is preferable.
• acrylic monomers represented by the following general formulae (4) and (5) can be used preferably.
• Rfi and Rf 2 each represent a monomer or an oligomer of a linear or branched alkyl group that contains a fluorine atom, a linear or branched oxyalkyl group that contains a fluorine atom, or a linear or branched oxyalkylene group that contains a fluorine atom, in which hydrogen in the alkyl group, the oxyalkyl group, and the oxyalkylene group may be substituted with an atom or an atomic group other than hydrogen, and -CH2- in the alkyl group and the oxyalkyl group may be substituted with -0-, -CO-, -NH-, or -COO-.
• the polymerization degree of the oligomer of the linear or branched oxyalkylene group that contains a fluorine atom is preferably 10 or less.
• Li and L 2 each represent a single bond or a divalent linking group selected from -0-, an alkylene group, an alkylene group having a hydroxyl group, an oxyalkylene group, and -NRiR 2 -, in which Ri represents hydrogen or an alkyl group and R 2 represents a single bond or a divalent linking group selected from an alkylene group, an alkylene group having a hydroxyl group, and an oxyalkylene group.
• fluorine-atom-containing monomers are illustrated below. However, the present invention is not limited to these exemplified compounds. For instance, some of H atoms present on the side chain of the exemplified compound below may be further replaced with F atoms.
• Hydrophilic groups can be introduced into the side chains of these monomers. Some of the examples thereof are exemplified above.
• the other monomers may be subjected to the replacement of CF 3 or CHF 2 on the side chain with CF 2 OH or the like.
• the side chain may be changed to a polyethylene glycol derivative such as COO (C 1n F 2 InO) n R 5
• n each independently represent an integer of 1 or more, preferably 10 or less, and R 5 represents H or an alkyl group.
• a single kind of monomers or two or more kinds of monomers may be introduced.
• the introduction of two or more monomers may be carried out by random copolymerization where two or more monomers are polymerized randomly, or may be carried out by block copolymerization where each monomer forms a domain.
• a combination of fluorine-atom-containing monomers and fluorine-atom-free monomers can be used.
• additional functions such as solubility and stability can be provided by the fluorine-atom-free monomers.
• the fluorine-free monomers which can provide the solubility function is desirably located on the outer side than the fluorine-atom-containing monomers.
• fluorine-atom-free monomers preferably those with hydrophilic groups
• the hydrophilic groups can be arranged near the surface of the particles.
• an acrylic monomer represented by one of the following formulae (4a) and (5a) can be preferably used as the fluorine-atom-free monomers:
• Rf 4 and Rf 5 each represent a monomer or an oligomer of a linear or branched alkyl group that does not contain a fluorine atom, a linear or branched oxyalkyl group that does not contain a fluorine atom, or a linear or branched oxyalkylene group that does not contain a fluorine atom, in which hydrogen in the alkyl group, the oxyalkyl group, and the oxyalkylene group may be substituted with an atom or an atomic group other than hydrogen, and -CH 2 - in the alkyl group and the oxyalkyl group may be substituted with -O- , -CO-, -NH-, or -COO-.
• L 3 and L 4 each represent a single bond or a divalent linking group selected from -0-, an alkylene group, an alkylene group having a hydroxyl group, an oxyalkylene group, and -NR 3 R 4 -, in which R 3 represents hydrogen or an alkyl group and R 4 represents a single bond or a divalent linking group selected from an alkylene group, an alkylene group having a hydroxyl group, and an oxyalkylene group.
• the conventional polymerization method known in the art can be used as a method of polymerizing these monomers from the terminal end of dendrimer.
• a preferable type of polymerization is one using anionic polymerization, cationic polymerization, or a living radical polymerization method.
• the reason of selecting the former anionic polymerization is to facilitate the generation of molecular chains- with a uniform length because the reaction rate at the time of growth is shorter than one at the time of initiating the reaction.
• the latter living radical polymerization has been actively investigated in recent years.
• the living radical polymerization is a more preferable polymerization method because of a broader choice of monomers and easiness of setting reaction conditions.
• the living radical polymerization is based on the establishment of a quick equilibrium between a small amount of growing radical (free radicals) species and a large amount of resting radicals (dormants) species in the growth reaction.
• Various types of the living radical polymerization have been proposed according to the resting (dormant) chains.
• the ATRP atom transfer radical polymerization
• the RAFT reversible addition fragmentation chain transfer
• the NMP nitroxide mediated polymerization
• the optical iniferter method using a dithiocarbamate compound have been proposed.
• the ATRP method is a method of polymerizing vinyl monomers using both a polymerization initiator with a highly reactive carbon-halogen bond and a transition metal complex serving as a polymerization catalyst.
• the RAFT method is a method of polymerizing vinyl monomers by adding a chain transfer agent (so-called RAFT agent) with a high chain transfer constant made of dithioesters to a normal radical polymerization system.
• the NMP method is a method including thermally cleaving the carbon-oxygen bond of an alkoxy amine, generating stable nitroxyl radicals and polymer radicals, and polymerizing vinyl monomers to polymer radicals. Under the cleavage, the nitroxyl radicals do not initiate polymerization, but react with only carbon-centered free radicals. The polymer radicals react with the monomers to extend the molecular chain and are then recombined by a coupling reaction with nitroxyl radicals, thereby being present stably as dormant species.
• the optical iniferter method uses an optical iniferter group such as an N,N-diethyldithiocarbamate group as an initiator and a polymerization reaction is then initiated by ultraviolet radiation.
• the living radical polymerization in the present invention may employ any of the above methods.
• the ATRP method is preferably used in terms of, for example, a wide range of usable raw materials but the living radical polymerization is not specifically limited thereto.
• a polymerization initiator in the ATRP method is not specifically limited as far as it is a compound having at least one of a chlorine atom, a bromine atom, and an iodine atom to be provided as a polymerization initiation point.
• a compound having one or two of chlorine, bromine, or iodine atoms to be provided as polymerization initiation points is used.
• ⁇ -haloesters, ⁇ -haloalkylamides, benzyl halides, halogenated alkanes, ⁇ -haloketones, ⁇ -halonitriles, sulfonyl halides, and the like are used.
• ⁇ -haloesters are preferred from the viewpoint of easy availability of the raw material.
• ⁇ -haloesters ethyl 2-bromoisobutyrate or ethyl 2-bromopropionate is exemplified.
• 2-chloropropione amide or 2-bromopropione amide is exemplified.
• benzyl halides 1-phenylethyl chloride or 1-bromoethyl benzene is exemplified.
• halogenated alkanes chloroform or carbon tetrachloride is exemplified.
• ⁇ -haloketones ⁇ -bromoacetone or ⁇ -bromoacetophenone is exemplified.
• ⁇ -halonitriles 2-bromopropionitorile is exemplified.
• the polymer formed from monomers should be bonded to each terminal end of a dendrimer.
• two methods are conceivable: one involving carrying out monomer polymerization from each terminal end of a dendrimer as an initiation point of the polymerization reaction and the other one involving polymerizing monomers to have a desired length to be bonded to each terminal end of a dendrimer.
• the use of the former technique is preferable.
• the polymerization reaction with monomers of interest is preferably carried out after bonding a polymerization initiator to be provided as a polymerization initiation point as described above to the terminal end of a dendrimer.
• the transition metal complex serving as a polymerization catalyst is not particularly limited, and a metal complex containing, as a central metal, a transition metal (M) selected from the metals belonging to the Group 7 to Group 11 in the periodic table is exemplified.
• a metal complex containing, as a central metal, a transition metal (M) selected from the metals belonging to the Group 7 to Group 11 in the periodic table is exemplified.
• Specific examples thereof include copper compounds having a monovalent copper metal such as cuprous chloride, cuprous bromide, cuprous iodide, and cuprous cyanide; nickel compounds having divalent nickel such as nickel dichloride, nickel dibromide, and nickel diiodide; iron compounds having a divalent iron such as iron dichloride, iron dibromide, and iron diiodide; and ruthenium compounds having a divalent ruthenium such as ruthenium dichloride, ruthenium dibromide, and ruthenium diiodide.
• the ligand of the transition metal complex is not particularly limited.
• examples thereof include 2, 2 ' -bipyridine and derivatives thereof (for example, 4, 4 ' -dinonyl-2, 2 ' -bipyridine and 4,4' -di (5-nonyl) -2,2' -bipyridine) , 1, 10-phenanthroline and derivatives thereof (4, 7-diphenyl-l, 10-phenanthroline and 2, 9-dimethyl-4, 7-diphenyl-l, 10-phenanthroline) , tetramethylethylenediamine, pentamethyldiethylene triamine, and hexamethyl (2-aminoethyl) amine .
• any of the dendrimer particles with the aforementioned configurations can be used as a material for an MRI contrast medium.
• N, N-dimethylformamide (manufactured by Wako Pure Chemical Industries) was added followed by evaporation at 90 0 C under reduced pressure to completely distill the solvent off, resulting in 791 mg (0.24 mmol) of a polyamideamine dendrimer as a pale yellow, viscous product.
• H-NMR result of the pale yellow, viscous product H-NMR (d ppm, in D 2 O) 2.3 (-CH 2 CH 2 CONH-), 2.5 (-CH 2 CH 2 N ⁇ ) , 2.6 (-CH 2 CH 2 NH 2 ), 2.7 (-NCH 2 CH 2 CO-), 3.1-3.2 (-CONHCH 2 CH 2 -) (refer to FIG. IA) .
• the thus obtained polyamideamine dendrimer was dissolved in 10.7 ml of N, N-dimethylformamide.
• reaction temperature was returned to room temperature and the mixture was then stirred for 2 hours, followed by further reaction in a hot bath at 60 0 C for 48 hours.
• the resultant was subjected to evaporation at 90 0 C and then dried under vacuum, resulting in a brown viscous product.
• This product was dissolved in methanol and then repeatedly precipitated three times in acetone as a solvent. Further, the resultant was washed with hexane, resulting in a white powder.
• N,N-dimethylformamide as a solvent.
• a polymerization solution was prepared by subjecting these agents to three cycles of freeze-pump-thaw degassing with each of a rotary pump and a diffusion pump and then mixing the agents. The solution was loaded into a degassed polymerization tube, followed by sealing. Then, the polymerization reaction was carried out in an oil bath at 90 0 C for 25 minutes. After terminating the polymerization, the obtained product was re-precipitated in methanol as a solvent, resulting in a white solid product.
• the molecular weight of the polymer was confirmed by GPC (gel permeation chromatography) to be a number average molecular weight (Mn) of 120,700 g/mol and a weight average molecular weight (Mw) of 153,500 g/mol.
• Mn number average molecular weight
• Mw weight average molecular weight
• a fluorine-atom-containing dendrimer was synthesized in a manner similar to Example 1 except that the polymerization reaction time of 2, 2, 2-trifluoroethyl methacrylate was set to 1 hour.
• a fluorine-atom-containing dendrimer was synthesized in a manner similar to Example 1 except that the polymerization reaction time of 2, 2, 2-trifluoroethyl methacrylate was set to 4 hours.
• the obtained white solid product (PAMAM-g-PTFEMA, 25.5-hour polymerization: Example 5) was subjected to measurement with a 600-MHz NMR device (manufactured by JEOL, JNM-ECA 600) to obtain an F-NMR spectrum thereof.
• the used solvent was CDCI 3 and the used reference substance was C 6 H 5 CF 3 .
• the result was shown in FIG. 4. As shown in FIG. 4, a single peak was observed at approximately -74.5 ppm.
• a polymerization solution was prepared by subjecting these agents to three cycles of freeze-pump-thaw degassing with a rotary pump and a diffusion pump and then mixing the agents.
• the solution was loaded into a degassed polymerization tube, followed by sealing.
• the polymerization reaction was carried out in an oil bath at 90 0 C for 4 hours (11 hours in total with the reaction time of 2,2, 2-trifluoroethyl methacrylate in Example 4) .
• the obtained product was re-precipitated in methanol as a solvent, resulting in a white solid product.
• the molecular weight of the polymer was confirmed by GPC (gel permeation chromatography) to be a number average molecular weight (Mn) of 602,000 g/mol and a weight average molecular weight (Mw) of 1,050,000 g/mol. (Example 9)
• a fluorine-atom-containing dendrimer was synthesized in a manner similar to Example 8 except that the polymerization reaction time of 2, 2, 2-trifluoroethyl methacrylate was set to 5 hours (12 hours in total with the reaction time of 2, 2, 2-trifluoroethyl methacrylate in Example 4) .
• Example 10 A fluorine-atom-containing dendrimer was synthesized in a manner similar to Example 8 except that the polymerization reaction time of 2, 2, 2-trifluoroethyl methacrylate was set to 7 hours (14 hours in total with the reaction time of 2, 2, 2-trifluoroethyl methacrylate in Example 4) .
• the solid circle represents Mn of the polymer obtained in each of Examples 1 to 5 and the solid triangle represents Mn of the polymer obtained by the reinitiation reaction in each of Examples 8, 9, and 10.
• the abscissa axis represents the reaction time of the reaction with 2, 2, 2-trifluoroethyl methacrylate.
• the sum of the reaction time in Example 4 and the reaction time of 2, 2, 2-trifluoroethyl methacrylate in each of Examples 8, 9, and 10 is used.
• a fluorine-atom-containing dendrimer was synthesized in a manner similar to Example 11 except that the polymerization reaction time of 2, 2, 3, 3-tetrafluoropropyl methacrylate was set to 20 minutes.
• a fluorine-atom-containing dendrimer was synthesized in a manner similar to Example 11 except that the polymerization reaction time of 2, 2, 3, 3-tetrafluoropropyl methacrylate was set to 30 minutes.
• a fluorine-atom-containing was synthesized in a manner similar to Example 11 except that the polymerization reaction time of 2, 2, 3, 3-tetrafluoropropyl methacrylate was set to 1 hour.
• FIG. 8Al to FIG. 8A ⁇ illustrate F-MRI images of trifluorotoluene (TFT: reference substance, F atom concentration: 50 mM) as a reference.
• FIG. 8Bl to FIG. 8B6 illustrate F-MRI images of 54 ⁇ lO 4 g mol "1 of PAMAM-g-PTFEMA
• the brightness of the respective images of FIG. 8Al to FIG. 8A6 were normalize with the brightness of FIG. 8Al and that of FIG. 8Bl to FIG. 8B6 were normalized with the brightness of FIG. 8Bl, thereby adjusting the brightness of FIG. 8Al and the brightness of FIG. 8Bl.
• FIG. 9A illustrates a 1 H-MRI image.
• a schematic diagram illustrated on the right side of FIG. 9A illustrates the arrangement of samples.
• FIG. 9Bl illustrates a 19 F-MRI image of PAMAM-g-PTFEMA.
• FIG. 9B2 illustrates a 19 F-MRI image of TFT.
• the 19 F-MRI images of PAMAM-g-PTFEMA are surrounded with broken-line circles
• the 19 F-MRI images of TFT provided as a reference substance are surrounded with solid-line circles. Numbers in the circles represent concentrations (unit: inM) .
• the center one is TFT with an F atom concentration of 0.01 mM
• the others, in the counterclockwise direction from the upper left are TFT with an F atom concentration of 0.1 mM
• PAMAM-g-PTFEMA with a F atom concentration of 0.01 mM
• PAMAM-g-PTFEMA with an F atom concentration of 1 mM
• PAMAM-g-PTFEMA with an F atom concentration of 0.1 mM.
• chloroform was used as a solvent.
• 9Bl and the 19 F-MRI image represented in FIG. 9B2 are those obtained by two-signal simultaneous measurement utilizing a difference in resonant frequencies of PAMAM-g-PTFEMA and TFT.
• the 19 F-MRI image of TFT cannot be obtained if the F atom concentration of TFT is not 1 mM.
• the 19 F-MRI image of PAMAM-g-PTFEMA can be clearly obtained even if the F atom concentration thereof is 0.1 mM. This indicates that PAMAM-g-PTFEMA has an excellent sensitivity for 19 F-MRI contrast mediums.
• the Tl measurement was carried out by the inversion recovery method and the T2 measurement was carried out by the spin echo method.
• FIG. 1OA and FIG. 1OB illustrates an F-NMR spectrum obtained when the Tl relaxation time measurement was carried out by the inversion recovery method.
• FIG. 1OA illustrates the spectrum of TFT
• the inversion times are 0.1, 0.25, 0.5, 0.8, 1.4, 2.0, 3.0, 4.0, 8.0, and 15.0 seconds from the front side.
• the results of the relaxation time measurement are listed in Table 3 below. Table 3
• MRI has advantages in that the shorter the Tl relaxation time is, the more the number of accumulations can be increased for a measurement with a certain measurement time, and the longer the T2 relaxation time is, the more the peak of a signal becomes sharp, resulting in a clear image.
• the PAMAM-g-PTFEMA as prepared had the shorter Tl relaxation time and the shorter T2 relaxation time, compared with those of TFT as a reference. Signals were slightly broadened by polymerization, and the relaxation times were shortened. However, as is evident from the F-MRI images of FIG. 9Bl, there was no problem in imaging of signals at all.
• the use of the dendrimer particle with a fluorine-atom-containing unit on the terminal end of the dendri ⁇ ier can provide an F-MRI contrast medium with high contrast sensitivity and an exactly controlled size.

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