WO2020230793A1 - Technology for controlling migration of hollow nanoparticles to brain from blood - Google Patents

Abstract

The present invention provides a technology for controlling migration of hollow nanoparticles to the brain from blood. More specifically, the present invention provides a technology for controlling migration of hollow nanoparticles to the cerebrospinal fluid and to the cerebral parenchyma from blood.

Description

Technology for controlling the migration of hollow nanoparticles from the blood to the brain

The present invention relates to a technique for controlling the migration of hollow nanoparticles from blood to the brain. More specifically, the present invention relates to a technique for controlling the transfer of hollow nanoparticles from blood to cerebrospinal fluid and to the brain parenchyma.

It is known that there is a blood-brain barrier that limits substance exchange between the blood and the brain. It is considered that this is because the cerebrovascular endothelial cells form tight junctions, the gaps between the cells are extremely narrow, and the cells themselves selectively take up and excrete substances.

The permeation selectivity of the blood-brain barrier is high, and it can hardly pass through except for some substances (for example, alcohol, caffeine, nicotine and glucose). This has made it difficult to treat brain diseases with brain therapeutic agents, diagnose brain diseases with brain diagnostic agents, or image the brain with contrast media.

In Patent Document 1, a technique for delivering vesicles to the brain parenchyma has been developed. In Patent Document 1, when an animal to which a glucose-coated vesicle is administered is preliminarily placed in a hypoglycemic state by fasting, and the vesicle is administered to the animal and glucose is administered, the vesicle is effectively transferred to the brain. It is disclosed that it will be delivered.

US2016 / 0287714A

The present invention provides a technique for controlling the migration of hollow nanoparticles from blood to the brain. More specifically, the present invention provides techniques for controlling the transfer of hollow nanoparticles from blood to cerebrospinal fluid and to the brain parenchyma.

In the process of transporting particles from the cerebrovascular lumen to the brain parenchyma, the present inventors use the glial limiting membrane as a barrier to inhibit the transfer of nanoparticles having a particle size of 80 nm or more to the brain parenchyma, and to cerebrospinal fluid. I found it to be fastened. The present inventors have also found that even such particles having a particle size of 80 nm or more easily pass through the glial limit membrane and easily migrate to the brain parenchyma when the rigidity is low.

That is, according to the present invention, the following invention is provided.
(1) Hollow particles having an average particle size of 80 to 200 nm measured by a dynamic light scattering method and a polydispersity index (PDI) of less than 0.2, and having a surface rigidity of 10 pN / nm or more. A composition comprising hollow particles that are.
(2) The above-mentioned (1), wherein the surface rigidity includes hollow particles having the first predetermined value, and the first predetermined value is a value in the range of 10 pN / nm to 40 pN / nm. Composition.
(3) The composition according to (1) above, wherein the surface rigidity contains hollow particles having a second predetermined value, and the second predetermined value is a value of 35 pN / nm or more.
(4) The composition according to any one of (1) to (3) above, wherein the hollow particles are polyion complex type polymersomes.
(5) The composition according to any one of (1) to (4) above, wherein the hollow particles contain an aqueous solution.
(6) The composition according to (2) above, for use in delivering the encapsulated aqueous solution to the brain parenchyma.
(7) The composition according to (3) above, for use in delivering the encapsulated aqueous solution to cerebrospinal fluid.
(8) A method of delivering hollow particles to cerebrospinal fluid in a subject.
A method comprising administering to the subject the composition according to any one of (1) to (7) above.
(9) A method of administering hollow particles to a subject.
The step of providing the composition according to any one of (1) to (7) above, and
A step of administering the composition to the subject, whereby the hollow particles are transferred to the cerebrospinal fluid, and the hollow particles are stably present in the cerebrospinal fluid for 60 minutes or more.
Method.
(10) The method according to (9) above.
A method, wherein the step of administration is a step of administering the composition to cerebrospinal fluid.
(11) A method for producing a composition containing hollow particles having a particle size of 80 nm to 200 nm.
To prepare hollow particles with an average particle size of 80 nm or more measured by the dynamic light scattering method.
Including adjusting the stiffness of the hollow particles to make the stiffness 10 pN / nm to 35 pN / nm or 35 pN / nm to 50 pN / nm.
Here, the method, wherein the polydispersity index (PDI) of the hollow particles is less than 0.2.
(12) A method for adjusting the permeability of hollow particles having a particle size of 80 nm to 200 nm to a glial limit film.
A method comprising adjusting the stiffness of the hollow particles.
(13) A method for adjusting the permeability of hollow particles having a particle size of 80 nm to 200 nm to a glial limit film.
A method comprising adjusting the stiffness of the hollow particles to make the stiffness 35 pN / nm to 50 pN / nm.
(14) A method for adjusting the permeability of hollow particles having a particle size of 80 nm to 120 nm to a glial limit film.
A method comprising adjusting the stiffness of the hollow particles to make the stiffness 10 pN / nm to 35 pN / nm.

FIG. 1 shows a polyion complex type micelle (PIC / m) having a particle size of 30 nm and a glucose modification rate of 25% and a glucose modification rate of 0%, 25%, 50% and 100% having a particle size of 100 nm administered to mice. The amount of polymersome (PIC / some) accumulated in the brain tissue is shown. FIG. 2 is an in vivo imaging image of a mouse brain tissue intraperitoneally administered with a mixture of Cy5-labeled 25% Gluc-PIC / some (100 nm) and Dlyght488-labeled 25% Gluc-PIC / m (30 nm). FIG. 3 shows mice intraperitoneally administered 25% Gluc-PIC / some (100 nm) containing β-galactosidase, and the substrate fluorescein di (β-D-galactopyranoside) was intracerebrospinal fluidly administered. The results of in vivo imaging of brain tissue are shown. Panel (a) is the result of the 25% Gluc-PIC / some (100 nm) administration group containing β-galactosidase, and panel (b) is the result of the free β-galactosidase administration group (negative control). FIG. 4 shows the results of measuring the synthesis of PIC / some using a cantilever of an atomic force microscope. FIG. 5 is a diagram showing the relationship between the rigidity of PIC / some having a particle size of 100 nm and the half-life in blood. FIG. 6 shows the distribution of PIC / some having a particle size of 100 nm and having various rigidity administered intraperitoneally to mice in the brain tissue. FIG. 7 shows an outline of the present invention. FIG. 8 shows a polyion complex type micelle (PIC / m) having a particle size of 30 nm and a glucose modification rate of 25% and a glucose modification rate of 0%, 25%, 50% and 100% having a particle size of 100 nm administered to mice. A mixture of polymersomes (PIC / some) accumulated in brain tissue (left) and Cy5-labeled 25% Gluc-PIC / some (100 nm) and In vivo-labeled 25% Gluc-PIC / m (30 nm). An in vivo imaging image (right) of intraperitoneally administered mouse brain tissue is shown. FIG. 9 shows a schematic diagram of the tissue structure from blood vessels to the brain parenchyma in the brain tissue, and a consideration of the causes causing the difference in the localization of particles of different sizes. FIG. 10 shows an outline of the properties of PIC / some. FIG. 11 shows that a clinical trial of enzyme replacement therapy using an AAV1 vector has been started, and an alternative enzyme replacement therapy using the hollow particles of the present invention (allylsulfatase A was used as an example in the figure). The explanation of is shown. FIG. 12 shows a scheme of proof of principle of enzyme replacement therapy using hollow particles (second embodiment) of the present invention. FIG. 13 shows that the substrate fluorescein di (β-D-galactopyranoside) was intraperitoneally administered to mice intraperitoneally administered with 25% Gluc-PIC / some (100 nm) containing β-galactosidase. The results of in vivo imaging of brain tissue are shown. The upper panel is the result of the 25% Gluc-PIC / some (100 nm) administration group containing β-galactosidase, and the lower panel is the result of the free β-galactosidase administration group (negative control).

Specific description of the invention

As used herein, the term "subject" refers to mammals, especially primates such as humans and monkeys, rodents such as mice, rats, rabbits and guinea pigs, cats, dogs, sheep, pigs, cows and horses. Examples include donkeys, goats, ferrets, etc.

In the present specification, the “nanoparticle” refers to a particle having an average particle size (D50) of 1000 nm or less as measured by a dynamic light scattering method. There are hollow nanoparticles and non-hollow nanoparticles in the nanoparticles. Hollow nanoparticles are particles that have cavities inside and can contain a solution in the cavities. "Internal capsule" means enclosing a substance inside. Encapsulation can reduce the rate of diffusion of substances in the encapsulated solution out of the particles. Encapsulation can protect the encapsulating substance in the body from the extraparticle immune system. Preferably, it means that the inside is spatially separated from the outside by the membrane. However, the inclusion allows the membrane to be material permeable.

In the present specification, the "hollow particle" means a particle-shaped object including an internal aqueous solution and a film structure covering the internal aqueous solution. Hollow particles can contain various substances by dissolving or suspending the various substances in the aqueous solution inside. Examples of the substance contained in the hollow particles include a water-soluble compound, a physiologically active substance (for example, an enzyme, for example, a proteinaceous enzyme), and a contrast medium. Hollow particles include vesicles, such as lipid vesicles (eg, liposomes). Hollow particles also include PIC / some. As used herein, the term "drug transport particles" refers to particles containing a drug and suitable for transporting the drug in the body. The drug transport particles are preferably hollow particles containing an aqueous solution containing a drug in the internal space.

In the present specification, the "polyion complex" (hereinafter, also referred to as "PIC") is a copolymer of a non-charged hydrophilic polymer block such as PEG and an anionic block, and a non-charged hydrophilic polymer block such as PEG. It is an ionic layer formed between the cationic block and the anionic block of both block copolymers when the copolymer of the copolymer and the cationic block is mixed in an aqueous solution so as to neutralize the charge. The significance of binding PEG to the above-mentioned charged chain is to prevent the polyion complex from aggregating and precipitating, thereby forming a monodisperse core-shell structure with a particle size of several tens of nm. It is to form the nanoparticles to have. At this time, since the uncharged hydrophilic polymer block such as PEG covers the outer shell (shell) of the nanoparticles, it is also known to be highly biocompatible and convenient in improving the retention time in blood. It is also clear that in polyion complex formation, one charged block copolymer does not require a non-charged hydrophilic polymer block moiety such as PEG and may be replaced with homopolymers, detergents, nucleic acids and / or enzymes. It has become. Then, in the formation of the polyion complex, at least one of the anionic polymer and the cationic polymer forms a copolymer with a non-charged hydrophilic polymer block such as PEG, and both of them form a non-charged hydrophilic polymer such as PEG. A copolymer with the block may be formed. Further, it is known that PIC micelles are likely to be formed by increasing the content of the uncharged hydrophilic polymer block such as PEG, and the content of the uncharged hydrophilic polymer block such as PEG is decreased or the copolymer concentration is increased. It is known that polyion complex type polymersomes (PIC / some), which are hollow particles having an average particle size of about 80 nm to 1,000 nm (particularly about 100 nm to 1,000 nm), are likely to be formed. There is. Since PIC / some is a hollow particle having a film structure formed by PIC and an internal cavity, an aqueous solution (for example, an aqueous solution containing a substance such as a physiologically active substance or a contrast agent) is contained in the internal cavity. Can be done. PIC / some are particles formed by ionic interactions and are permeable to ions, small molecules (lipids and amino acids), and water (see FIG. 10).

In the present specification, the "average particle size measured by the dynamic light scattering method" is based on the median diameter (D50), which is a particle size indicating a 50% integrated value of the integrated distribution curve of the particle size distribution based on the number.

In the present specification, the "multidispersity index" (PDI) is a dimensionless index indicating the spread of particle size distribution. The PDI is a numerical value of 0 or more and 1 or less, and the smaller the numerical value, the more uniform the particle size of the measured particles. When the PDI is 0.3 or less, 0.2 or less, or 0.1 or less, it is expected that the pharmacokinetics of each particle becomes homogeneous due to the high homogeneity of the particle size of the particles. Therefore, when the PDI is 0.3 or less, 0.2 or less, or 0.1 or less, it becomes more important to adjust the characteristics of the homogeneous particles to those suitable for the purpose.

In the present specification, "rigidity" is a force (pN) required to generate a deformation displacement (nm). The rigidity of the nanoparticles can be measured, for example, using a cantilever of an atomic force microscope. The elasticity can be obtained by applying a force so as to press the particles on the solid phase plane against the plane using a cantilever and measuring the displacement of the deformation of the particles and the force at that time.

As used herein, "lower alkyl" and "lower alkylene" mean C _1-6 alkyl and C _1-6 alkylene, respectively. Lower can mean C _1-2 , C _1-3 , C _1-4 , or C _1-5 .

As shown in FIG. 9, the important structures in the transport of substances from the blood vessels of the brain to the parenchyma of the brain are the blood vessels of the brain, cerebrospinal fluid endothelial cells (cerebrospinal fluid barrier, BCSFB), cerebrospinal fluid, and glia limit membrane. (Boundary membrane), and brain parenchyma are present. It is considered that the blood-brain barrier (BBB) is composed of cerebrovascular endothelial cells and glial limit membrane. In the case of hollow particles having a size of 100 nm under the initial conditions, the particles existing in the cerebral blood vessels can pass through the glia limit membrane even if they pass through the cerebral vascular endothelial cells. It is retained in the cerebrospinal fluid. On the other hand, it was revealed that micelles having a small size (for example, 30 nm) easily pass through the glia limit membrane and migrate to the parenchyma of the brain when they pass through the cerebrovascular endothelial cells. Further, the present inventors have found that the passability of the glial limit film changes depending on the rigidity even for hollow particles having a size of 100 nm. That is, it was clarified that even particles having a size that does not pass through the glial limit film (for example, PIC / some) are more likely to pass through the glial limit film as the rigidity becomes lower.

Compositions Containing Hollow Particles of the First Embodiment According to the present invention, the average particle size measured by a dynamic light scattering method is 80 to 120 nm, and the polydispersity index (PDI) is less than 0.2. A composition comprising hollow particles, wherein the surface rigidity is the first predetermined value, is provided.

In some aspects of the invention, the hollow particles can be hollow vesicles. In some aspects of the invention, the hollow particles can be, for example, hollow lipid particles, for example, liposomes. In some aspects of the invention, the hollow particles can be PIC / some.

In some aspects of the invention, the average particle size of the particles can be 80-120 nm, 90-120 nm, 90-110 nm, 95-105 nm, 80-100 nm, 80-200 nm, 80-150 nm, or 100-120 nm. The particle size of PIC / some can be controlled by the degree of polymerization of the uncharged hydrophilic polymer block and the concentration of cationic and anionic polymers.

In some aspects of the invention, the polydispersity index (PDI) of the particles can be 0.3 or less, 0.25 or less, 0.2 or less, 0.15 or less, or 0.1 or less. In a preferred embodiment of the invention, the PDI of the particles can be, for example, 0.04 to 0.1, for example 0.04 to 0.08.

In one aspect of the present invention, the surface rigidity of the hollow particles is the first predetermined value. The first predetermined value can be in the range of 10 pN / nm to 40 pN / nm. For example, the first predetermined value can be, for example, 15 pN / nm to 35 pN / nm. The first predetermined value can also be between 35 pN / nm and 45 pN / nm.

The lower limit of the range of values that the surface rigidity of the hollow fine particles of the first embodiment can take is, for example, more than 10 pN / nm, 15 pN / nm, 20 pN / nm, 25 pN / nm, 30 pN / nm, 35 pN / nm. Or it can be 40 pN / nm. The upper limit of the range of values that the surface rigidity of the hollow fine particles of the first embodiment can take is, for example, 50 pN / nm, 45 pN / nm, 40 pN / nm, 35 pN / nm, 30 pN / nm, 25 pN / nm, or 20 pN. Can be / nm.

In some preferred embodiments, the hollow particles may have a surface stiffness of 10 pN / nm or more and 35 pN / nm or less.

The composition containing the hollow nanoparticles of the first embodiment can be preferably used to deliver the encapsulated aqueous solution to the brain parenchyma.

Composition Containing Hollow Particles of Second Embodiment According to the present invention, the average particle size measured by a dynamic light scattering method is 80 to 200 nm, and the polydispersity index (PDI) is less than 0.2. A composition comprising hollow particles, wherein the surface rigidity is a second predetermined value, is provided.

In some aspects of the invention, the hollow particles can be hollow vesicles. In some aspects of the invention, the hollow particles can be, for example, hollow lipid particles, for example, liposomes. In some aspects of the invention, the hollow particles can be PIC / some.

In some aspects of the invention, the average particle size of the particles can be 80-200, 80-150 nm, 80-120 nm, 90-120 nm, 90-110 nm, 95-105 nm, 80-100 nm, or 100-120 nm. The particle size of PIC / some can be controlled by the degree of polymerization of the uncharged hydrophilic polymer block and the concentration of cationic and anionic polymers.

In some aspects of the invention, the polydispersity index (PDI) of the particles can be 0.3 or less, 0.25 or less, 0.2 or less, 0.15 or less, or 0.1 or less. In a preferred embodiment of the invention, the PDI of the particles can be, for example, 0.04 to 0.1, for example 0.04 to 0.08.

In one aspect of the present invention, the surface rigidity of the hollow particles is a second predetermined value. The second predetermined value can be in the range of 30 pN / nm to 50 pN / nm. For example, the second predetermined value can be, for example, 30 pN / nm to 35 pN / nm. The second predetermined value can also be 35 pN / nm to 45 pN / nm, 45 pN / nm to 50 pN / nm.

The lower limit of the surface rigidity of the hollow fine particles of the second embodiment can be, for example, 30 pN / nm, 35 pN / nm, 36 pN / nm, 37 pN / nm, 38 pN / nm, 39 pN / nm, or 40 pN / nm. The upper limit of the surface rigidity of the hollow fine particles of the second embodiment can be, for example, 50 pN / nm, 45 pN / nm, or 40 pN / nm. In one preferred embodiment, the surface stiffness of the hollow particles of the second embodiment can be 35 pN / nm or more and 50 pN / nm or less.

The composition containing the hollow nanoparticles of the second embodiment can be preferably used for delivering the encapsulated aqueous solution to the cerebrospinal fluid. By delivering to cerebrospinal fluid, it means that at least part of what was administered in cerebrospinal fluid some time after administration is retained. The fixed time can be, for example, 60 to 90 minutes.

In the present specification, the hollow particles of the first embodiment and the hollow particles of the second embodiment may be collectively referred to as the hollow particles of the present invention.

Aqueous solution contained in the hollow particles of the present invention The hollow particles of the present invention contain an aqueous solution inside. The aqueous solution may contain water and a pharmaceutically acceptable carrier and / or excipient. The aqueous solution can be, for example, water, saline, and a buffer. The aqueous solution may further contain a substance selected from the group consisting of bioactive substances and contrast agents (eg, biocompatible substances). Examples of the physiologically active substance include proteins such as antibodies (about 10 to 15 nm), antigen-binding fragments thereof, growth factors, enzymes and the like, and nucleic acids such as ribozymes, miRNA and non-coding RNA inhibitory nucleic acids. Be done. Enzymes include, for example, enzymes used in enzyme replacement therapy, such as arylsulfatase, islonate-2-sulfatase, α-galactosidase, agarsidase alpha, agarsidase beta, imiglucerase, taliglucerase alpha, velaglucerase alpha. , Alglucerase, Severipase Alpha, Laronidase, Izulfatase, and Erosulfase Alpha. When used for enzyme replacement therapy, the hollow particles of the present invention are preferably PIC / some. Since PIC / some has permeability to water and small molecules, if an enzyme is included inside, the substrate can permeate the membrane and access the enzyme inside PIC / some, and the substrate is returned to PIC / some. This is because it can be expected to be released to the outside of some. Physiologically active substances also include therapeutic agents for cranial nerve diseases. Drugs for treating neurological disorders include, for example, therapeutic agents for brain disorders such as anxiety, depression, sleep disorders, Alzheimer's disease, Parkinson's disease and multiple sclerosis. As a therapeutic agent for Alzheimer's disease, for example, Aβ antibody is well known, as a therapeutic agent for Parkinson's disease, for example, dopamine receptor agonist and L-dopa are well known, and as a therapeutic agent for multiple sclerosis, for example, adrenal gland. Steroids, interferon β (IFNβ), and immunosuppressants are well known and these therapeutic agents can be used in the present invention. Peripheral neuropathy includes peripheral neuropathy that can be treated by passing a therapeutic agent for peripheral neuropathy across the blood-brain barrier, such as Guillain-Barré syndrome, Miller syndrome and chronic inflammatory demyelinating multiple neuropathy. Examples of the contrast medium include ultrasonic waves, contrast media for MRI and CT, and radioisotopes. The biocompatible substance may be an anti-cancer agent and may not be an anti-cancer agent. As the ribozyme, any of a hammer head type, a hairpin type, and a lasso type ribozyme may be used. As nucleic acids that inhibit non-coding RNA, microRNA, etc., miRNA sponge (Nature Methods 6,897-903 (2009)), RNA decoy (NAR 2009 Apr; 37 (6): e43, NAR 2012 Appr; 40 (8) :. e58) may be used. When the molecular weight of the biocompatible substance is about 5000 Da or more, about 6000 Da or more, about 7000 Da or more, about 8000 Da or more, about 9000 Da or more, or about 10000 Da or more, the biocompatible substance contained in PIC / some is contained. It can stay inside the PIC / some stably. A bulky structure may be added as needed to stably enclose the hollow particles.

Rigidity of Hollow Particles of the Present Invention In the present invention, the rigidity of the surface of the hollow particles can be appropriately adjusted by, for example, the type of the raw material of the hollow particles and the cross-linking thereof. For example, in PIC / some, it is selected from the group consisting of the degree of polymerization of polycation as a raw material of PIC / some, the degree of polymerization of polyanion, the degree of polymerization of uncharged hydrophilic polymer block, and the degree of intermolecular cross-linking thereof. It can be adjusted by controlling one or more. For example, the rigidity of PIC / some can be imparted by increasing the degree of polymerization of the polycation and the polyanion. By increasing the degree of polymerization of the uncharged hydrophilic polymer block, the rigidity of PIC / some can be reduced. In addition, rigidity can be imparted to the PIC / some by forming an intermolecular crosslink on the ionic polymer constituting the PIC / some. As described above, in the present invention, the rigidity of the particles can be controlled by using various methods for imparting the rigidity to the hollow particles. Since the PIC / some membrane is permeable to moisture, it can be advantageously used to adjust the rigidity.

Preparation of hollow particles of the present invention Examples of raw materials for PIC / some include a combination of a copolymer and a homopolyanion containing a non-charged hydrophilic polymer block and a polycation block, and a non-charged hydrophilic polymer block and a polyanion block. Examples thereof include a combination of a copolymer containing and a homopolycation. Here, the uncharged hydrophilic polymer block can be a polymer containing monomeric units that are both uncharged and hydrophilic. Non-charged hydrophilic polymer blocks include, for example, polyalkylene glycol (eg, polyethylene glycol), and polyoxazoline (eg, poly-C _1-4 alkyloxazoline, eg, poly (2-ethyl-2-oxazoline), poly (eg, poly). 2-Isopropyl-2-oxazoline). The uncharged hydrophilic polymer block may contain branches in the molecule, but preferably does not include branches. The uncharged hydrophilic polymer block may contain branches. For example, the number average molecular weight may be 1,000 to 15,000, for example, 2,000 to 12,000. Further, the polycation block and the polyanion block have a number average degree of polymerization of 40 to 150, 50 to 130, or. Can have 60-100.

Examples of polycation blocks and homopolycations include non-natural polycations and natural polycations. The polycation block and homopolycation are also represented by-(NH- (CH ₂ ) ₂ ) _p- NH ₂ with a polymer of naturally cationic amino acids such as polylysine and polyornithine and a polypeptide as the main chain. Examples thereof include polymers of unnatural cationic amino acids having a group (where p is 2, 3 or 4) as a side chain. The main chain polypeptide has, for example, a polymer of amino acids selected from the group consisting of aspartic acid and glutamic acid as the main chain, and is a group represented by-(NH- (CH ₂ ) ₂ ) _p- NH ₂ { Here, a polymer of an unnatural cationic amino acid having (p is 2, 3 or 4) as a side chain can be mentioned. Those skilled in the art will be able to obtain such a polymer by reacting a polymer containing aspartic acid or glutamic acid as a monomer unit with diethyltriamine, triethyltetraamine or tetraethylpentamine.

In a preferred embodiment of the present invention, the cationic polymer is composed of the following general formula (I):

{In the formula,
R ¹ can be a hydrogen atom, a lower alkyloxy group, or an optionally substituted linear or branched C _1-12 alkyl group, or polyethylene glycol, in this case adjacent to polyethylene glycol. Amino acids may be linked via a linker,
R ² is a lower alkylene and
R ³ is a group represented by-(NH- (CH ₂ ) ₂ ) _p- NH ₂ or -NH- (CH ₂ ) _q- NH ₂ , p is 2, 3 or 4, q Is 2, 3, 4, or 5
R ⁴ is hydrogen, a protecting group, a hydrophobic group or polymerizable group, for example, a hydrogen atom, an acetyl group, trifluoroacetyl group which may be an acryloyl group or a methacryloyl group,
X is a cationic amino acid and
n is an integer of 2 to 5000,
n ₁ is an integer from 0 to 5000,
n ₃ is an integer from 0 to 5000,
nn _1- n ₃ is an integer of 0 or more, and each repeating unit in the formula is shown in a specific order for convenience of description, but each repeating unit can exist in random order, and each repeating unit can exist in any order. The repeating units may exist randomly, and each repeating unit may be the same or different}.

In the formula (I), the protecting groups include C _1-6 alkylcarbonyl group, preferably an acetyl group, a hydrophobic group, a benzene, naphthalene, anthracene, pyrene and derivatives thereof, or C _{1 -6} Alkyl group is mentioned, and the polymerizable group includes a methacryloyl group and an acetylloyl group. Methods of introducing these protecting, hydrophobic and polymerizable groups into block copolymers are well known to those of skill in the art.

In the above formula (I), polyethylene glycol (PEG) has an average degree of polymerization of 5 to 20000, preferably 10 to 5000, and more preferably 40 to 500, unless the formation of a polyion complex between the block copolymer and mRNA is inhibited. Not particularly limited. The PEG ends of the cationic polymer may be protected by hydroxyl groups, methoxy groups or protecting groups.

In the above formula (I), the linker is, for example, − (CH ₂ ) r-NH- {where r is an integer of any one of 1 to 5. } Or-(CH ₂ ) s-CO- {where s is an integer of any of 1-5. }, And preferably it can be bound to an adjacent amino acid of the formula (I) by a peptide bond. Further, the linker may preferably be bonded to PEG via the O atom of PEG on the methylene side of PEG.

In the above formula (I), n is an integer of 0 to 5000, for example, an integer of 0 to 500, and m is an integer of 0 to 5000, for example. It is an integer of 0 to 500, and m + n is an integer of 2 to 5000, for example, an integer of 2 to 500.

In one aspect of the present invention, as the cationic polymer, a compound defined by any of the formulas (II) to (V) described later can be used.

In some aspects of the invention, the cationic polymer is a block copolymer of a PEG-linker-polycation block {where the PEG, linker and polycation block are as defined above. } Can be. In some aspects of the invention, the cationic polymer may be a homopolycation.

In some aspects of the invention, the anionic polymer block and the anionic polymer can be polymers formed by peptide bonds of natural or unnatural amino acids. Anionic polymer blocks and anionic polymers include, for example, polyglutamic acid and polyaspartic acid.

In one aspect of the present invention, as the anionic polymer, a compound defined by any of the formulas (VI) to (IX) described later can be used.

When the hollow vesicles are lipid particles, for example, phospholipids can be used as the lipids. Examples of phospholipids include phosphatidylcholine (for example, dioleoil phosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, etc.), phosphatidylglycerol (eg, dioleoil phosphatidylglycerol, dilauroylphosphatidylglycerol, etc.). Dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, etc.), phosphatidylethanolamine (eg, diolaylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine) Stearoyl phosphatidylethanolamine, etc.), phosphatidylserine, phosphatidylinositol, phosphatidylic acid, cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramidephosphorylglycerol, ceramidephosphorylglycerol phosphate, 1,2-dipalmitoyl-1,2-deoxyphosphatidylcholine, Examples thereof include plasmalogen, egg yolk lecithin, soy lecithin, and hydrogenated additives thereof.

PIC / some is a solution containing a combination of a copolymer containing a non-charged hydrophilic polymer block and a polycation block and a homopolyanion in an aqueous solution, or a copolymer containing a non-charged hydrophilic polymer block and a polyanion block. And can be obtained by mixing solutions containing a combination of homopolycations. When the biocompatible substance is introduced into the contained aqueous solution, the biocompatible substance may be contained in the solution to be mixed above.

Methods of forming intermolecular crosslinks in hollow vesicles are well known to those skilled in the art. Cross-linking can be performed after the vesicles have been formed, for example, using a cross-linking agent. The cross-linking agent can be appropriately selected and used. For example, 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) is a water-soluble cross-linking agent capable of cross-linking intermolecular carboxylic acid groups and amino groups. The degree of cross-linking can be controlled by the amount of the cross-linking agent added and the reaction time between the cross-linking agent and the hollow vesicles.

Modification of Hollow Particles of the Present Invention The hollow particles of the present invention can be coated with a substance having an affinity for the surface of cerebrovascular endothelial cells. Thereby, the hollow fine particles can be imparted with transferability to the brain. Examples of substances having an affinity for the surface of cerebral vascular endothelial cells include molecules that bind to molecules expressed on the surface of cerebral vascular endothelial cells (for example, antibodies (for example, antibodies that bind to transferrin) and GLUT1 ligands). Be done. The GLUT1 ligand is a molecule that binds to GLUT1. GLUT1 ligands include various molecules that bind to GLUT1, such as glucose, 2-N-4- (1-azi-2,2,2-trifluoroethyl) benzoyl-1,3-bis (D). -Mannose-4-yloxy) -2-propylamine (ATB-BMPA), 6- (N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino) -2-deoxyglucose ( 6-NBDG), 4,6-O-ethylidene-α-D-glucose, 2-deoxy-D-glucose and 3-O-methylglucose also include molecules that bind to GLUT1. The GLUT1 ligand can be exposed on the surface of hollow particles so that it can be recognized by GLUT1. For example, glucose is a polymer (particularly the tip of an uncharged hydrophilic polymer block) that constitutes hollow particles via carbon atoms at the 2-, 3-, or 6-position (and preferably O atoms bonded to the carbon atom). Hollow particles can be coated, for example, by covalently linking to polyethylene glycol (O atom), and the coated particles can be preferably recognized by GLUT1. The coating of particles with GLUT1 increases the tendency of the particles to stay in the vascular endothelial cells as the amount increases, and increases the tendency of the particles to pass through the vascular endothelial cells (transitosis) and reach the parenchyma of the brain when the amount decreases. From this point of view, the degree of coating of the particles with the GLUT1 ligand can be adjusted. When the particles are coated with glucose, depending on the particles, for example, 10 to 40 mol% (for example, 15 to 35 mol%, for example, 20 to 30 mol%) of the polymer or polymer constituting the vesicle is glucose. Can be connected (introduced).

Method of Administration of Hollow Particles of the Present Invention The composition containing the hollow particles of the present invention can be administered into the cerebrospinal fluid of the subject. By doing so, the hollow particles of the first embodiment of the present invention can be transferred to the brain parenchyma, and the hollow particles of the second embodiment can be retained in the cerebrospinal fluid. Whether to transfer to the brain parenchyma or to stay in the cerebrospinal fluid may be appropriately selected according to the purpose at that time. In this administration method, the hollow particles of the present invention do not need to have a molecule having an affinity for the surface of cerebrovascular endothelial cells.

The composition containing the hollow particles of the present invention can be orally administered to a subject. Parenteral administration includes, for example, intravenous administration and intraperitoneal administration. In this administration method, the hollow particles of the present invention preferably have a molecule having an affinity for the surface of cerebrovascular endothelial cells, and it is particularly preferable to express the GLUT1 ligand so as to be recognizable by GLUT1. preferable.

Nanoparticles expressing the GLUT1 ligand can further increase the transfer rate to the brain by administering the nanoparticles according to the administration plan of the present invention. Specifically, the dosing regimen of the invention comprises administering the composition to a fasted or hypoglycemic subject and inducing an increase in blood glucose levels in the subject. This method is as detailed in WO 2015/075942A, which is incorporated herein by reference in its entirety. In the dosing regimen according to the invention, the composition can be administered to the subject continuously or sequentially at the same time as inducing an increase in blood glucose level in the subject. The dosing regimen may or may not have an interval between administration of the composition to the subject and induction of elevated blood glucose levels in the subject. If the composition is administered simultaneously with the induction of an increase in blood glucose level in the subject, the composition may be administered to the subject in a mixed form with an agent that induces an increase in blood glucose level. , It may be administered in a form different from the drug that induces an increase in blood glucose level in the subject. Also, when the composition is administered to the subject continuously or sequentially, the composition is prior to the induction of an increase in blood glucose level in the subject. May be administered to the subject or later, but preferably the composition can be administered to the subject prior to inducing an increase in blood glucose level in the subject. When inducing an increase in blood glucose level in the subject prior to administration of the composition to the subject, within 1 hour, within 45 minutes, within 30 minutes after inducing the increase in blood glucose level in the subject. It is preferred to administer the composition to the subject within, within 15 minutes or within 10 minutes. In addition, when inducing an increase in blood glucose level in the subject after administration of the composition to the subject, within 6 hours, within 4 hours, or 2 hours after the administration of the composition to the subject. Within 1 hour, within 45 minutes, within 30 minutes, within 15 minutes or within 10 minutes, it is preferable to induce an increase in blood glucose level in the subject. The administration planning cycle described above may be performed more than once. The context of glucose administration and sample administration can be determined by the timing of crossing the blood-brain barrier.

In the present specification, "inducing hypoglycemia" means lowering the blood glucose level in the subject than the blood glucose that would have been indicated if the treatment was not performed. Examples of the method for inducing hypoglycemia include administration of a diabetic drug. For example, when inducing hypoglycemia, it is permissible to take, for example, other drugs or drink beverages such as water, as long as the purpose of inducing hypoglycemia is achieved. Inducing hypoglycemia may be accompanied by other treatments that have no substantial effect on blood glucose.

In the present specification, "fasting" means fasting to a subject, for example, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more. 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 20 hours or more, 21 hours or more, 22 hours or more, 23 hours 24 hours or more, 25 hours or more, 26 hours or more, 27 hours or more, 28 hours or more, 29 hours or more, 30 hours or more, 31 hours or more, 32 hours or more, 33 hours or more, 34 hours or more, 35 hours or more, 36 hours or more, 37 hours or more, 38 hours or more, 39 hours or more, 40 hours or more, 41 hours or more, 42 hours or more, 43 hours or more, 44 hours or more, 45 hours or more, 46 hours or more, 47 hours or more or 48 hours It means to fast as above. The subject causes hypoglycemia by fasting. The fasting period is determined by a doctor or the like in consideration of the health condition of the subject, and is preferably a period longer than the time when the subject reaches fasting blood glucose, for example. The fasting period may be, for example, longer than the expression of GLUT1 on the inner surface of blood vessels of cerebrovascular endothelial cells increases or reaches a plateau. The fasting period can be, for example, the above period of 12 hours or more, 24 hours or more, or 36 hours or more. Fasting may also be accompanied by other treatments that do not substantially affect blood glucose levels or expression of GLUT1 on the inner surface of blood vessels. The time for maintaining the blood glucose level of the subject in a low blood glucose state is, for example, 0 hours or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 Hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 20 hours or more , 21 hours or more, 22 hours or more, 23 hours or more, 24 hours or more, 25 hours or more, 26 hours or more, 27 hours or more, 28 hours or more, 29 hours or more, 30 hours or more, 31 hours or more, 32 hours or more, 33 Hours or more, 34 hours or more, 35 hours or more, 36 hours or more, 37 hours or more, 38 hours or more, 39 hours or more, 40 hours or more, 41 hours or more, 42 hours or more, 43 hours or more, 44 hours or more, 45 hours or more , 46 hours or more, 47 hours or more, 48 hours or more. After that, the blood sugar level can be raised. As used herein, "maintaining blood glucose" is permitted, for example, to take other agents or drink beverages such as water, as long as the subject achieves the purpose of maintaining hypoglycemia. Inducing hypoglycemia may be accompanied by other treatments that have no substantial effect on blood glucose.

In the present specification, "inducing an increase in blood glucose level" means increasing a blood glucose level in a subject who has induced hypoglycemia or a subject who has maintained a hypoglycemic state. Blood glucose levels can be elevated by a variety of methods well known to those skilled in the art, for example, administration of one that induces elevated blood glucose levels, eg, induction of elevated blood glucose levels such as glucose, fructose (fructose), galactose, etc. It can be increased by administration of monosaccharides, administration of polysaccharides such as maltose that induce an increase in blood glucose level, intake of carbohydrates that induce an increase in blood glucose level such as starch, or diet.

Method for Controlling Permeability of Hollow Particles to Glia Limit Film According to the present invention, there is provided a method for controlling the permeability of hollow particles to the glare limit film. According to the present invention, by controlling the particle size (average particle size) and the rigidity, the hollow particles changed the permeability to the glial limit film. The permeability to the glial limit film decreases as the particle size increases for particles that are resistant to deformation by external force (that is, high rigidity), but this decrease in permeability decreases the rigidity of the particles. It can be suppressed by this. Thereby, the distribution in the brain of the aqueous solution (for example, the aqueous solution containing a drug) contained in the hollow particles can be controlled.

In particular, hard hollow particles having a particle size of 80 nm or more have low permeability of the glial limit film. Therefore, according to the present invention, it is a method of increasing the permeability of hollow particles having a particle size of 80 nm or more, 90 nm or more, or 100 nm or more to the glial limit film, and the rigidity of the hollow particles is adjusted to increase the rigidity. A method is provided, including setting the value below the first predetermined value. In this embodiment, the permeability of the glial limit film of the hollow particles can be increased based on the disclosure of the hollow particles of the first embodiment {for example, as compared with the hollow particles having the rigidity of the second predetermined value. }. In this embodiment, the rigidity can be set to a value exceeding 10 pN / nm. By doing so, blood stability can be enhanced.

Further, according to the present invention, it is a method of reducing the permeability of hollow particles having a particle size of 80 nm or more, 90 nm or more, or 100 nm or more to a glial limit film, and the rigidity of the hollow particles is adjusted to increase the rigidity. Is provided, the method including setting the value to a second predetermined value or more. In this embodiment, the permeability of the glial limit film of the hollow particles can be reduced based on the disclosure of the hollow particles of the second embodiment {for example, as compared with the hollow particles having the rigidity of the first predetermined value. hand}.

According to the present invention, a method for producing a composition containing hollow particles having a particle size of 80 nm to 120 nm, in which hollow particles having a particle size of 80 nm or more are prepared and the rigidity of the hollow particles is determined. A method is provided that includes adjusting the stiffness to be less than or equal to the first predetermined value. For example, the PDI of hollow particles can be 0.3 or less, 0.25 or less, 0.2 or less, 0.15 or less, or 0.1 or less. In a preferred embodiment of the invention, the PDI of the particles can be, for example, 0.04 to 0.1, for example 0.04 to 0.08. In this aspect, a composition containing hollow particles can be produced in the same manner as in the method for producing hollow particles of the first embodiment based on the disclosure of the hollow particles of the first embodiment.

According to the present invention, it is also a method for producing a composition containing hollow particles having a particle size of 80 nm to 200 nm, in which hollow particles having a particle size of 80 nm or more are prepared and the rigidity of the hollow particles is determined. A method is provided that includes adjusting the stiffness to a second predetermined value or higher. For example, the PDI of hollow particles can be 0.3 or less, 0.25 or less, 0.2 or less, 0.15 or less, or 0.1 or less. In a preferred embodiment of the invention, the PDI of the particles can be, for example, 0.04 to 0.1, for example 0.04 to 0.08. In this aspect, a composition containing hollow particles can be produced in the same manner as in the method for producing hollow particles of the second embodiment based on the disclosure of the hollow particles of the second embodiment.

According to the present invention, a method for administering a hollow vesicle to a subject, comprising providing a composition containing the hollow particles of the first embodiment and administering the composition to the subject. Is provided. In this embodiment, the hollow vesicles may contain, for example, an aqueous solution containing a biocompatible substance. In this embodiment, the hollow vesicles may be administered according to the above dosing regimen. This allows hollow vesicles that are administered and reside in blood vessels to cross the blood-brain barrier and be delivered to the brain parenchyma.

According to the present invention, a method for administering a hollow vesicle to a subject, comprising providing a composition containing the hollow particles of the second embodiment and administering the composition to the subject. Is provided. In this embodiment, the hollow vesicles may contain, for example, an aqueous solution containing a biocompatible substance.
In this embodiment, the hollow vesicles may be administered without following the above dosing regimen. If the dosing regimen is not followed, administration may preferably be directed to cerebrospinal fluid.
Further, in the above embodiment, the hollow vesicles may be administered according to the above administration plan. This allows hollow vesicles that are administered and are present in the blood vessels to be delivered to the cerebrospinal fluid across the blood-spinal fluid barrier. Then, it may stay in cerebrospinal fluid for, for example, 60 minutes or longer, 70 minutes or longer, 80 minutes or longer, or 90 minutes or longer.

According to the present invention, a method of administering a physiologically active substance selected from the group consisting of a protein, an enzyme, a nucleic acid, and a water-soluble drug to a subject, the first embodiment containing the physiologically active substance in the subject. A method comprising administering the hollow particles of the above or the hollow particles of the second embodiment is provided. In this aspect, the hollow particles of the first embodiment or the hollow particles of the second embodiment containing the bioactive substance can be administered according to the administration plan of the present invention. According to this method, the hollow particles of the first embodiment accumulate in the brain parenchyma, and the hollow particles of the second embodiment accumulate in the cerebrospinal fluid at least in part.

According to the present invention, the following inventions may also be provided.
Item 1: A method of setting drug transport particles that pass through a glial limit membrane by controlling particle size and elastic modulus (rigidity).

Item 2: The method according to item 1 above, wherein the GLUT-1 ligand is added to the surface of the particles.

Item 3: The method according to Item 1 or 2, wherein the particles are covered with a block copolymer containing a nonionic uncharged hydrophilic segment and a charged segment.

Item 4: The method according to item 3 above, wherein the elastic modulus is controlled by adjusting the cross-linking rate between block copolymers.

Item 5: A first block copolymer having a nonionic uncharged hydrophilic segment, a positively charged charged segment, a nonionic uncharged hydrophilic segment, and a negative ion charged. The method according to item 1 or 2 above, which comprises a film formed by a second block copolymer having a charged segment.

Item 6: The method according to any one of items 2 to 5 above, wherein the uncharged hydrophilic segment is polyethylene glycol and / or poly (2-isopropyr-2-oxazoline).

Item 7: The method according to item 5 or 6 above, wherein the charged segment of the first block copolymer is represented by the formula (II).

(In the above formula,
R ₁ represents − (CH ₂ ) ₃ NH ₂ or −CONH (CH ₂ ) _s − X, and s is 0 to 20, where X is −NH ₂ , pyridyl group, morpholic group, 1-imidazolyl group, piperazinyl group, 4- (C _1-6 alkyl) -piperazinyl group, 4- (amino C _1-6 alkyl) -piperazinyl group, pyrrolidine-1-yl group, N-methyl-N-phenylamino At least one selected from the group consisting of group, piperidinyl group, diisopropylamino group, dimethylamino group, diethylamino group,-(CH ₂ ) _t NH ₂ or-(NR ₉ (CH ₂ ) _o ) _p NHR ₁₀ Where R ₉ represents a hydrogen atom or a methyl group, R ₁₀ represents a hydrogen atom, an acetyl group, a trifluoroacetyl group, a benzyloxycarbonyl group or a tert-butoxycarbonyl group, and o represents 1-5. , P is 1 to 5, t is 0 to 15, and
R2 represents a hydrogen atom, an acetyl group, a trifluoroacetyl group, an acryloyl group or a methacryloyl group.
a is 0 to 5,000, b is 0 to 5,000, and a + b is 2 to 5,000. )

Item 8: R ₁ represents -CONH (CH ₂ ) _S- NH ₂ , and s is 2 to 5.
R ₂ represents a hydrogen atom
The method according to item 7, wherein a is 0 to 200, b is 0 to 200, and a + b is 10 to 200.

Item 9: The method according to any one of items 5 to 8 above, wherein the charged segment of the second block copolymer is represented by the formula (III).

(In the above formula,
R ₂ represents a hydrogen atom, an acetyl group, a trifluoroacetyl group, an acryloyl group or a methacryloyl group.
R ₃ independently represents a methylene group or an ethylene group, respectively.
c is 0 to 5,000, d is 0 to 5,000, and c + d is 2 to 5,000. )

Item 10: R ₂ represents a hydrogen atom
R ₃ represents a methylene group
9. The method according to item 9, wherein c is 0 to 200, d is 0 to 200, and c + d is 10 to 200.

Item 11: The method according to item 5 above, wherein the first block copolymer is represented by the formula (IV).

(In the above formula,
R ₁ represents − (CH ₂ ) ₃ NH ₂ or −CONH (CH ₂ ) _s − X, and s is 0 to 20, where X is −NH ₂ , pyridyl group, morpholic group, 1-imidazolyl group, piperazinyl group, 4- (C _1-6 alkyl) -piperazinyl group, 4- (amino C _1-6 alkyl) -piperazinyl group, pyrrolidine-1-yl group, N-methyl-N-phenylamino At least one selected from the group consisting of group, piperidinyl group, diisopropylamino group, dimethylamino group, diethylamino group,-(CH ₂ ) _t NH ₂ or-(NR ₉ (CH ₂ ) _o ) _p NHR ₁₀ And R ₉ represents a hydrogen atom or a methyl group, R ₁₀ represents a hydrogen atom, an acetyl group, a trifluoroacetyl group, a benzyloxycarbonyl group or a tert-butoxycarbonyl group, and o is 1-5. , P is 1-5, t is 0-15,
R ₂ represents a hydrogen atom, an acetyl group, a trifluoroacetyl group, an acryloyl group or a methacryloyl group.
R ₄ represents a hydrogen atom or an optionally substituted linear or branched C _1-12 alkyl group.
R ₅ represents − (CH ₂ ) _g NH-, and g is 0 to 5.
a is 0 to 5,000, b is 0 to 5,000, and a + b is 2 to 5,000.
e is 5 to 2,500. )

Item 12: R ₁ represents -CONH (CH ₂ ) _S- NH ₂ , and s is 2 to 5.
R ₂ represents a hydrogen atom
R ₄ represents a methyl group
a is 0 to 200, b is 0 to 200, and a + b is 10 to 200.
The method according to item 11, wherein e is 10 to 300.

Item 13: The method according to item 5 above, wherein the first block copolymer is represented by the formula (V).

(In the above formula,
R ₁ represents − (CH ₂ ) ₃ NH ₂ or −CONH (CH ₂ ) _s − X, and s is 0 to 20, where X is −NH ₂ , pyridyl group, morpholic group, 1 -Imidazolyl group, piperazinyl group, 4- (C _1-6 alkyl) -piperazinyl group, 4- (amino C _1-6 alkyl) -piperazinyl group, pyrrolidine-1-yl group, N-methyl-N-phenylamino group , Piperidinyl group, diisopropylamino group, dimethylamino group, diethylamino group,-(CH ₂ ) _t NH ₂ , or-(NR ₉ (CH ₂ ) _o ) _p NHR ₁₀ at least selected from the group. Yes, where R ₉ represents a hydrogen atom or a methyl group, R ₁₀ represents a hydrogen atom, an acetyl group, a trifluoroacetyl group, a benzyloxycarbonyl group or a tert-butoxycarbonyl group, and o is 1-5. Yes, p is 1-5, t is 0-15,
R ₂ represents a hydrogen atom, an acetyl group, a trifluoroacetyl group, an acryloyl group or a methacryloyl group.
R ₆ represents a hydrogen atom or an optionally substituted linear or branched C _1-12 alkyl group.
R ₇ represents − (CH ₂ ) _h NH-, and h is 0 to 5.
R ₈ represents a linear or branched C _1-12 alkyl group.
a is 0 to 5,000, b is 0 to 5,000, and a + b is 2 to 5,000.
f is 5 to 2,500. )

Item 14: R ₁ represents − (CH ₂ ) ₃ NH ₂
R ₂ represents a hydrogen atom
R ₆ represents a methyl group
R ₈ represents -CH (CH ₃ ) ₂ and represents
a is 0 to 200, b is 0 to 200, and a + b is 10 to 200.
Item 13. The method according to item 13, wherein f is 10 to 300.

Item 15: The method according to item 5 above, wherein the second block copolymer is represented by the formula (VI).

(In the above formula, R ₂ represents a hydrogen atom, an acetyl group, a trifluoroacetyl group, an acryloyl group or a methacryloyl group.
R ₃ independently represents a methylene group or an ethylene group, respectively.
R ₄ represents a hydrogen atom or an optionally substituted linear or branched C _1-12 alkyl group.
R ₅ represents − (CH ₂ ) _g NH-, and g is 0 to 5.
c is 0 to 5,000, d is 0 to 5,000, and c + d is 2 to 5,000.
i is 5 to 2,500. )

Item 16: R ₂ represents a hydrogen atom
R ₃ represents a methylene group
R ₄ represents a methyl group
c is 0 to 200, d is 0 to 200, and c + d is 10 to 200.
The method according to item 15, wherein i is 10 to 300.

Item 17: The method according to item 5 above, wherein the second block copolymer is represented by the formula (VII).

(In the above formula,
R ₂ represents a hydrogen atom, an acetyl group, a trifluoroacetyl group, an acryloyl group or a methacryloyl group.
R ₃ independently represents a methylene group or an ethylene group, respectively.
R ₆ represents a hydrogen atom or an optionally substituted linear or branched C _1-12 alkyl group.
R ₇ represents − (CH ₂ ) _h NH-, and h is 0 to 5.
R ₈ represents a linear or branched C _1-12 alkyl group.
c is 0 to 5,000, d is 0 to 5,000, and c + d is 2 to 5,000.
j is 5 to 2,500. )

Item 18: R ₂ represents a hydrogen atom
R ₃ represents a methylene group
R ₆ represents a methyl group
R ₈ represents -CH (CH ₃ ) ₂ and represents
c is 0 to 200, d is 0 to 200, and c + d is 10 to 200.
The method according to item 17, wherein j is 10 to 300.

Item 19: The nonionic uncharged hydrophilic segment and the charged segment are represented by the following formula (VIII) or (IX):

R ¹ represents a hydrogen atom or an unsubstituted or substituted linear or branched C _1-12 alkyl group.
L ¹ is-(CH ₂ ) _b -NH-, b is an integer of 1 to 5, L ² is-(CH ₂ ) _c -CO-, and c is an integer of 1 to 5.
R ² represents a methylene group or an ethylene group, R ³ represents a hydrogen atom, a protecting group, a hydrophobic group or a polymerizable group.
R ⁴ is the same as R ⁵ or is -NH-R ⁹ , where R ⁹ represents an unsubstituted or substituted linear or branched C _1-20 alkyl group.
R ⁵ is independently a hydroxyl group, an oxybenzyl group, or an amine group, except that 85% or more of R ⁵ is an amine group.
Here, the amine group is a group represented by the following formula (X):

Selected from, a is an integer from 1 to 5, m is an integer from 5 to 20,000, and
n is an integer of 2 to 5,000 and x is an integer of 0 to 5,000, but x is not greater than n)
The method according to item 3 above, which is a block copolymer represented by (1) or a salt thereof.

Item 20: Drug transport particles designed by the method according to any one of items 1 to 19 above.

Pharmaceutical Use According to the present invention, the composition containing the hollow particles of the first embodiment and the composition containing the hollow particles of the second embodiment are subjected to enzyme replacement therapy (particularly, the hollow particles of the first embodiment are applied to the brain parenchyma. For enzyme replacement, the hollow particles of the second embodiment can be used for enzyme replacement of cerebrospinal fluid).
More specifically, for example, the composition containing the hollow particles of the first embodiment and the composition containing the hollow particles of the second embodiment contain an aqueous solution containing an enzyme in the hollow particles, and the enzyme is introduced into a living body. Can be replenished. Here, the hollow particles can be PIC / some. PIC / some stably encloses an enzyme having a molecular weight of 10,000 Da or more in particles, but the substrate has permeability to the membrane of PIC / some. Therefore, the enzyme can convert the substrate outside the PIC / some by its original enzyme activity by being provided in the PIC / some.

In some embodiments, the composition comprising the hollow particles of the first embodiment and the composition comprising the hollow particles of the second embodiment contain an agar cedar zebeta in the hollow particles, and the hollow fine particles are PIC / some. , Can be used to treat Fabry disease.

In some embodiments, the composition comprising the hollow particles of the first embodiment and the composition comprising the hollow particles of the second embodiment contain agarsidase alpha in the hollow particles, and the hollow fine particles are PIC / some. , Can be used to treat Fabry disease.

In some embodiments, the composition comprising the hollow particles of the first embodiment and the composition comprising the hollow particles of the second embodiment contain imiglucerase in the hollow particles, and the hollow fine particles are PIC / some and a goucher. It can be used to treat the disease.

In some embodiments, the composition comprising the hollow particles of the first embodiment and the composition comprising the hollow particles of the second embodiment contain taligulcerase alpha in the hollow particles, and the hollow fine particles are PIC / some. And can be used to treat goucher disease.

In some embodiments, the composition comprising the hollow particles of the first embodiment and the composition comprising the hollow particles of the second embodiment contain velaglucerase alfa in the hollow particles, and the hollow fine particles are PIC / some. And can be used to treat Goucher's disease.

In some embodiments, the composition comprising the hollow particles of the first embodiment and the composition comprising the hollow particles of the second embodiment contain alglucerase in the hollow particles, the hollow fine particles are PIC / some, and I. It can be used to treat type goucher disease.

In some embodiments, the composition comprising the hollow particles of the first embodiment and the composition comprising the hollow particles of the second embodiment contain severipase alpha in the hollow particles, and the hollow fine particles are PIC / some. , Can be used to treat lysosomal lipase deficiency.

In some embodiments, the composition comprising the hollow particles of the first embodiment and the composition comprising the hollow particles of the second embodiment contain laronidase in the hollow particles, and the hollow fine particles are PIC / some and mucopolysaccharidans. It can be used to treat polysaccharidosis (MPS) type I.

In some embodiments, the composition comprising the hollow particles of the first embodiment and the composition comprising the hollow particles of the second embodiment contain isul sulphase in the hollow particles, and the hollow fine particles are PIC / some. It can also be used to treat mucopolysaccharidosis (MPS) type II.

In some embodiments, the composition comprising the hollow particles of the first embodiment and the composition comprising the hollow particles of the second embodiment contain elosulfase alfa in the hollow particles, and the hollow fine particles are PIC / some. It can also be used to treat mucopolysaccharidosis (MPS) type IVA.

In some embodiments, the composition comprising the hollow particles of the first embodiment and the composition comprising the hollow particles of the second embodiment contain galsulfase in the hollow particles, the hollow fine particles are PIC / some, and muco. It can be used to treat polysaccharidosis (MPS) type VI.

In some embodiments, the composition comprising the hollow particles of the first embodiment and the composition comprising the hollow particles of the second embodiment contain alglucosidase alfa in the hollow particles, and the hollow fine particles are PIC / some. , Can be used to treat Pombe's disease.

In some embodiments, the composition comprising the hollow particles of the first embodiment and the composition comprising the hollow particles of the second embodiment contain arylsulfatase in the hollow particles, the hollow fine particles are PIC / some, and the hollow particles are PIC / some. It can be used to treat lysosomal storage diseases, such as metachromatic leukodystrophy (MLD).

According to the present invention, it is a method of supplementing an enzyme in a subject.
The subject is a composition containing hollow particles of the first embodiment and a composition containing hollow particles of the second embodiment for use in enzyme replacement therapy, wherein the composition contains an enzyme to be supplemented into the hollow particles. Encapsulating, hollow microparticles are PIC / some, including administering the composition.
The method is provided. The relationship between the enzyme and the indicated disease of enzyme replacement therapy is as defined in the above composition.

According to the present invention, the use of a supplementing enzyme in the production of a composition containing hollow particles of the first embodiment and a composition containing hollow particles of the second embodiment for use in enzyme replacement therapy is provided. The relationship between the enzyme and the indicated disease of enzyme replacement therapy is as defined in the above composition.

According to the present invention, a composition containing hollow particles of the first embodiment and a composition containing hollow particles of the second embodiment for use in enzyme replacement therapy, the composition being supplemented in the hollow particles. A composition is provided in which the hollow fine particles are PIC / some. The relationship between the enzyme and the indicated disease of enzyme replacement therapy is as defined in the above composition.

The composition of the present invention may be a pharmaceutical composition and is pharmaceutically acceptable in addition to the hollow particles selected from the group consisting of the composition comprising the hollow particles of the first embodiment and the hollow particles of the second embodiment. It may contain possible excipients. Excipients include, but are not limited to, buffering agents, tonicity agents, pharmaceutically acceptable salts, dispersants, antioxidants, preservatives, and soothing agents.

Example 1: Relationship between BBB passage and particle size of nanoparticles 1. Preparation of Gluc-Modified Polyion Complex (PIC) -Type Polymer Aggregates of Different Diameters PEG-poly (α, β-) in which glucose is bound to the α-terminal of polyethylene glycol (PEG) segment via the OH group at position C6. Aspartic acid) (Gluc-PEG-PAsp) (Mn of PEG is 2000, DP of P (Asp) is 80) and PEG-PAsp without glucose (Mn of PEG is 2000, DP of P (Asp) is 80) ) Was dissolved in 10 mM phosphate buffer (pH 7.4, 0 mM NaCl) (10 mM PB) at 1 mg / mL and mixed at a ratio of 1: 1 (volume ratio) to prepare a polyanion solution. .. In addition, CH ₃ O-PEG-P (Asp-AP) -Cy5 (DP of P (Asp) is 80) in which a fluorescent dye (Cy5) is introduced at the ω end is also adjusted to 1 mg / mL in 10 mM PB. (Polycation solution), mix so that the charge ratio of the polyanion / cation solution is 1: 1 and add 10 equal amounts of 1-ethyl-3- (3-dimethyl) to the carboxyl group of PEG-PAsp. Aminopropyl) carbodiimide hydrochloride (EDC) was added and allowed to stand overnight. Then, it was purified by limit filtration to prepare 25% Gluc-PIC / m (diameter 30 nm). Here, "X%" immediately before Gluc means that the proportion of the polymer having Gluc modification in the total polymer mixed when preparing the PIC vesicle (PIC micelle; PIC / m) is X%. Shown.
In addition, Gluc-PIC / some having a diameter of 100 nm is a DP of Gluc-PEG-PAsp, PEG-PAsp, and Homo-P (Asp-AP) -Cy5 (P (Asp-AP)) having no PEG as described above. 75) was mixed in the same manner, EDC crosslinked, and purified to prepare. Here, a solution (1: 3, 1: 1 and 1: 0) in which PEG-PAsp carrying no Gluc was mixed at an arbitrary ratio was used to prepare Gluc-PIC / some in which the ligand density of the surface layer was controlled. .. Here, "X%" immediately before Gluc indicates that the proportion of the polymer having Gluc modification in all the polymers mixed when preparing the PIC vesicle (PICsome; PIC / some) is X%. .. In this example, X% was 0% (null), 25%, 50%, or 100%.
The particle size and polydispersity index (PDI) were measured by dynamic light scattering measurements, respectively (Table 1). The results are as shown in Table 1 below.

Table 1: Glucose-modified micelles (Gluc-PICs) and glucose-modified PIC / some particle size and PDI

As shown in Table 1, the glucose-modified micelle had a particle size of about 30 nm and a PDI of 0.08. The particle size of the glucose-modified PIC / some had a particle size of about 100 to 105 nm regardless of the glucose modification rate, and the PDI was about 0.04 to 0.08.

2. Evaluation of brain accumulation and intracerebral distribution of nanocarriers of different diameters Prepared Cy5-labeled 0, 25, 50, 100% Gluc-PIC / some (100 nm), 25% Gluc-PIC / m (30 nm) mice ( Brain accumulation in BALB / c mice (female, 9 weeks old) was evaluated. The above nanocarriers were used in mice (blood glucose level about 100 mg / dL) whose blood glucose level was lowered within the normal range by removing food for 24 hours. Is administered to the tail vein (iv), and 30 minutes later, a glucose solution is intraperitoneally administered (ip) to raise the blood glucose level, and the amount accumulated in the brain 90 minutes after the nanocarrier administration. As for Gluc-PIC / some, 0% Gluc-PIC / some, which does not carry a glucose ligand, hardly accumulated in the brain (0.06% dose / g-brain), whereas 25 , 50, 100% Gluc-PIC / some (100 nm) was confirmed to accumulate in the brain. As with the previously reported glucose-modified PIC micelles (30 nm), the accumulation varies greatly depending on the ligand density, especially 25% Gluc-. PIC / some (100 nm) was the most efficient, and accumulation of 3.2% / g-brain was confirmed (Figs. 1 and 8), and 25% Gluc-PIC / m (30 nm) was the same as previously reported. A high accumulation in the brain of 5.9% dose / g-brain was confirmed.
In order to confirm the distribution in the brain, Cy5-labeled 25% Gluc-PIC / some (100 nm) and Fluorescent 488-labeled 25% Gluc-PIC / m (30 nm) were mixed and administered to the tail vein on the same schedule as above. Then, 90 minutes later, the observation was performed using an in-vivo confocal laser scanning microscope (IVRT-CLSM). As a result, the Dlyght488-labeled 25% Gluc-PIC / m (30 nm) was uniformly distributed in the brain, whereas the Cy5-labeled 25% Gluc-PIC / some (100 nm) accumulated in the vicinity of the cerebrovascular endothelial cells. It was confirmed that this was done (FIGS. 2 and 8).
25% Gluc-PIC / m (30 nm) can penetrate from cerebrospinal fluid (CSF) into the brain parenchyma after passing through the BBB, whereas 25% Gluc-PIC / some (100 nm) is localized only around blood vessels. This suggests that 25% Gluc-PIC / some (100 nm) was unable to cross the glial borderan covering the cerebral vascular endothelial cells, which indicates that the glial borderan covering the brain parenchyma It was suggested that it is a size-dependent barrier.

Example 2: Enzyme replacement therapy in CSF 1. Preparation of Enzyme-Encapsulated Gluc-PIC / Some In the same manner as above, an aqueous solution (1 mg / ml) of Gluc-PEG-PAsp, PEG-PAsp, and Homo-P (Asp-AP) -Cy5 is adjusted so that the positive and negative charge ratios are balanced. Was mixed and stirred to form a hollow 25% Gluc-Cy5-PIC / some through self-association by electrostatic interaction. Β-galactosidase (β-Gal) was added thereto, and the reversible dissociation / reassociation behavior of PIC was induced by stirring, so that β-Gal was encapsulated inside the obtained PIC / some. To the carboxyl group of PEG-PAsp, 10 equivalents of EDC was added and allowed to stand overnight. Then, purification (EDC, removing unencapsulated β-Gal) by limit filtration was performed to prepare 25% Gluc-β-Gal @ Cy5-PIC / some (diameter 100 nm).

2. Evaluation of enzyme activity in the brain (in CSF) 25% Gluc-β-Gal @ Cy5-PIC / some (diameter 100 nm) and unencapsulated β-Gal were administered tail vein to mice subjected to the same glycemic procedure as above. Fluorescein (β-D-galactosidase) (FDG), which is a substrate for β-Gal after 24 hours and emits fluorescence (Ex / Em = 490/514 nm) when decomposed by β-Gal. Was administered into cerebrospinal fluid (CSF) and observed with IVRT-CLSM (FIGS. 3, 12, and 13). As a result, fluorescence generated by an enzymatic reaction in the brain was confirmed in the 25% Gluc-β-Gal @ Cy5-PIC / some administration group, whereas most of the fluorescence was confirmed in the unencapsulated β-Gal administration group. Not confirmed. These results show that unencapsulated β-Gal did not induce an enzymatic reaction in the brain, whereas 25% Gluc-β-Gal @ Cy5-PIC / some induced an enzymatic reaction in the brain. Suggests.

Example 3: Gluc-PIC / some with different rigidity and distribution in the brain 1. Preparation of Gluc-PIC / some with different rigidity Similar to the above, the positive and negative charge ratios of Gluc-PEG-PAsp, PEG-PAsp, and Homo-P (Asp-AP) -Cy5 aqueous solution (1 mg / ml) are balanced. As described above, the mixture was mixed and stirred to form a hollow 25% Gluc-Cy5-PIC / some through self-association by electrostatic interaction. For the purpose of changing the rigidity of PIC / some, an amount of EDC different from 0.3, 0.5, 1, 3, 5, 10, 20 equivalents is added to the carboxyl group of PEG-PAsp. It was allowed to stand at night. Then, it was purified by limit filtration to prepare 25% Gluc-Cy5-PIC / some. By DLS measurement, it was confirmed that both PIC / some had a monodisperse (PDI <0.1) with a diameter of 100 nm.

2. Rigidity evaluation method For the sample prepared above, the rigidity known as a physical quantity representing deformability was evaluated. Here, the measurement was performed using the Quantitative Imaging mode of an atomic force microscope (AFM, NanoWizard ULTRA SpeedTM (JPK)). The measurement was carried out at room temperature of 25 ° C. Prior to the measurement, a cantilever (OLYMPUS: BL-AC 40 TS-C2, Biolever mini; Nominal Spring constant 0.09 N / m) mounted on the AFM head was calibrated by the Thermal noise method. The cantilever was immersed in the prepared liquid sample for about 10 minutes for thermal equilibrium and stabilization, and then AFM measurement was performed under the following condition settings in the Quantitative Imaging (QI) mode of NanoWizard ULTRA SpeedTM. set point: 100-140 pN; cantilever speed: 15 μm / s; scanner 1 μm × 1 μm / 128 × 128 pixels.

For AFM image data acquired in QI mode so that the decomposition is 8 nm / pixel or less, 25% Gluc-PIC / some deformation-stress curve slope (rigidity pN / nm) from the force curve stored for each pixel. ) Was linearly fitted in a linear region where stress is applied (a stress region of about 10 to 20% or more of the maximum stress). The obtained AFM image is obtained from JPK Data Processing Software v.I. The area-equivalent diameter, maximum height, and volume of the 25% Gluc-PIC / some whose rigidity was analyzed by correcting the image tilt according to 6.0 are determined by Gwydion software v. Calculated according to 2.47. For statistical analysis, Turkey's multiple comparison test was performed after confirming the statistically significant difference between the groups by One-way ANOVA.

As a result, it was confirmed that the rigidity of 25% Gluc-PIC / some increases as the amount of EDC added increases (Fig. 4). In addition, the amount of EDC added is significantly significant in rigidity (pN / nm) between 0.3 equivalent and 0.5 equivalent of 25% Gluc-PIC / some and between 10 equivalent and 20 equivalent of 25% Gluc-PIC / some. No significant difference was confirmed.

3. 3. Evaluation of blood stability of PIC / some with different rigidity The 25% Gluc-Cy5-PIC / some with different rigidity prepared above was administered iv to mice, and the half-life was based on the blood circulation and the information obtained there. Was calculated. As a result, it was clarified that 25% Gluc-PIC / some showing a rigidity of 10 pN / nm or less has low blood stability and shows a high half-life in blood above that (Fig. 5).

4. Intracerebral distribution of PIC / some with different stiffness 18, 32, 40 pN / nm 25% Gluc-Cy5-PIC / some with guaranteed blood stability was administered to mice on the same schedule as above. , 3 hours after administration, the brain was excised, a brain section was prepared, vascular endothelial cells were stained with PECAM-1, and the nucleus was stained with DAPI, and observed with a confocal microscope. As a result, 40 pN / nm, which shows high rigidity, cannot pass through the glial limit film and stays in the CSF as in the result shown above (FIG. 5 (c)), whereas the rigidity is controlled. It was confirmed that the temperature was lowered (softened) (18, 32 pN / nm) to pass through the glial limit film (FIGS. 6 (a) and 6 (b)).

Thus, particles with a particle size of 30 nm can pass through the glial limit membrane and migrate from the vascular endothelium to the brain regardless of stiffness, while particles with a particle size of 100 nm have low stiffness (eg,). In the case of less than 40 pN), it can pass through the glial limit membrane and transfer from the vascular endothelium to the brain, but it has been clarified that the permeability of the glial limit membrane decreases when the rigidity is high (for example, 40 pN or more). It was.

Claims (14)

1. Hollow particles having an average particle size of 80 to 200 nm measured by dynamic light scattering and a polydispersity index (PDI) of less than 0.2, with a surface rigidity of 10 pN / nm or more. A composition comprising particles.
2. The composition according to claim 1, wherein the surface rigidity includes hollow particles having a first predetermined value, and the first predetermined value is a value in the range of 10 pN / nm to 40 pN / nm.
3. The composition according to claim 1, wherein the composition contains hollow particles having a surface rigidity of a second predetermined value, and the second predetermined value is a value of 35 pN / nm or more.
4. The composition according to any one of claims 1 to 3, wherein the hollow particles are polyion complex type polymersomes.
5. The composition according to any one of claims 1 to 4, wherein the hollow particles contain an aqueous solution.
6. The composition according to claim 2, for use in delivering the encapsulated aqueous solution to the brain parenchyma.
7. The composition according to claim 3, for use in delivering the encapsulated aqueous solution to cerebrospinal fluid.
8. A method of delivering hollow particles into the cerebrospinal fluid in a subject.
   A method comprising administering to the subject the composition according to any one of claims 1-7.
9. A method of administering hollow particles to a subject,
   The step of providing the composition according to any one of claims 1 to 7.
   A step of administering the composition to the subject, whereby the hollow particles are transferred to the cerebrospinal fluid, and the hollow particles are stably present in the cerebrospinal fluid for 60 minutes or more.
   Method.
10. The method according to claim 9.
    A method, wherein the step of administration is a step of administering the composition to cerebrospinal fluid.
11. A method for producing a composition containing hollow particles having a particle size of 80 nm to 200 nm.
    To prepare hollow particles having an average particle size of 80 nm or more as measured by the dynamic light scattering method.
    Including adjusting the stiffness of the hollow particles to make the stiffness 10 pN / nm to 35 pN / nm or 35 pN / nm to 50 pN / nm.
    Here, the method, wherein the polydispersity index (PDI) of the hollow particles is less than 0.2.
12. A method for adjusting the permeability of hollow particles having a particle size of 80 nm to 200 nm to a glial limit film.
    A method comprising adjusting the stiffness of the hollow particles.
13. A method for adjusting the permeability of hollow particles having a particle size of 80 nm to 200 nm to a glial limit film.
    A method comprising adjusting the stiffness of the hollow particles to make the stiffness 35 pN / nm to 50 pN / nm.
14. A method for adjusting the permeability of hollow particles having a particle size of 80 nm to 120 nm to a glial limit film.
    A method comprising adjusting the stiffness of the hollow particles to make the stiffness 10 pN / nm to 35 pN / nm.
    
    

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