JP2021178779A - Polyion complex-type polymersome that has sufficient retentivity in blood and improved permeability to glia limitans - Google Patents

Abstract

SOLUTION: To provide a composition including polyion complex-type polymersome that has sufficient retentivity in blood and improved permeability to a glia limitans.EFFECT: A composition including polyion complex-type polymersome whose average particle diameter measured by a dynamic light scattering method is 80 nm or more, and a polydispersity index (PDI) is 0.2 or less, in which the polyion complex-type polymersome includes a first polymer including a cationic polymer and a second polymer including anionic polymer, either or both of the first polymer and the second polymer are a block copolymer further including a non-electric charge hydrophilic polymer block, and the polymers are cross-linked by a cleavable linker under a reduction environment.SELECTED DRAWING: None

Description

The present invention relates to a polyion complex type polymersome having sufficient blood retention and improved permeability to a glia limit membrane.

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

The blood-brain barrier is highly transparent and can hardly pass through except for some substances (eg, 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.

The blood-brain barrier has a first barrier formed by endothelial cells and pericytes, and a second barrier called the glial limit membrane formed by astroglia existing outside the first barrier. Cerebrospinal fluid is perfused in the perivascular space between the endothelial cells and the glial marginal membrane. The glial marginal membrane is a barrier that exists further outside the cerebral vascular endothelial cells, and even if a large macromolecular can reach the cerebrospinal fluid from the blood vessel through the vascular endothelial cells by the method of Patent Document 1, glia. It has low permeability to the marginal membrane and cannot be efficiently delivered to the brain parenchyma.

US2016 / 0287714A

The present invention provides a polyion complex type polymersome having sufficient blood retention and improved permeability to the glia limit membrane.

In order for the polyion complex type polymersome (PICsome) to exist stably under the blood salt concentration, cross-linking is required between the polymers constituting the PICsome. Further, although the crosslinked polyion complex polymersome (PICsome) having an average particle size of 80 nm or more measured by the dynamic light scattering method passes through the layer of vascular endothelial cells from the cerebral blood vessels, the glia limit is subsequently reached. It tends to stay in the cerebrospinal fluid without passing through the membrane.

We have developed a polyion complex polymersome (PICsome) having an average particle size of 80 nm or more as measured by a dynamic light scattering method, which has a bond (linker) that can be cleaved in a reducing environment at the cross-linking. We have found that the glial limit film can be permeated through the contained aqueous solution, and completed the present invention.

｛式中、
Ｒ¹は、水素原子、低級アルキルオキシ基、もしくは置換されていてもよい直鎖もしくは分岐鎖のＣ_1-12アルキル基であり得、またはポリエチレングリコールであり得、この場合、ポリエチレングリコールと隣り合うアミノ酸とはリンカーを介して結合してもよく、
Ｒ²は、低級アルキレンであり、
Ｒ³は、−（ＮＨ−（ＣＨ₂）₂）_p−ＮＨ₂または−ＮＨ−（ＣＨ₂）_q−ＮＨ₂で表される基であり、ｐは、２、３または４であり、ｑは、２、３、４、または５であり、
Ｒ⁴は、水素、保護基、疎水基、または重合性基であり、
Ｘは、カチオン性アミノ酸であり、
ｎは、２〜５０００のいずれかの整数であり、
ｎ₁は、０〜５０００のいずれかの整数であり、
ｎ₃は、０〜５０００のいずれかの整数であり、
ｎ−ｎ₁−ｎ₃は、０以上の整数であり、式中の各繰り返し単位は記載の都合上特定の順で示されているが、各繰り返し単位は順不同に存在することができ、各繰り返し単位はランダムに存在してもよく、また、各繰り返し単位は同一であっても異なっていてもよい｝で表される、上記（１）〜（６）のいずれかに記載の組成物。
（８）薬剤を含む水溶液を内包した、上記（１）〜（７）のいずれかに記載の組成物。
（９）非経口投与される、上記（１）〜（８）のいずれかに記載の組成物。
（１０）脳脊髄液に投与される、上記（９）に記載の組成物。
（１１）腹腔内投与または静脈内投与される、上記（９）に記載の組成物。
（１２）その必要のある対象に薬剤を投与する方法であって、
上記（８）に記載の組成物を当該対象に投与することを含む、方法。 According to the present invention, the following inventions are provided.
(1) A composition containing polyion complex type polymersomes having an average particle size of 80 nm or more and a polydispersity index (PDI) of 0.2 or less as measured by a dynamic light scattering method.
Polyion complex polymersomes include a first polymer containing a cationic polymer and a second polymer containing an anionic polymer, and either or both of the first polymer and the second polymer are uncharged hydrophilic. A block copolymer that further contains polymer blocks,
A composition in which the polymers are crosslinked by a linker that can be cleaved in a reducing environment.
(2) The first polymer has a biocompatible main chain and a side chain having an amino group, and the cross-linking is N-hydroxysuccinimide (NHS) -L _1- S-S-L _2- NHS {formula. Among them, L ₁ and L ₂ are independently bonded and substituted lower alkylene, substituted lower alkynyl, optionally substituted lower alkenyl, and optionally substituted poly. An alkylene glycol, wherein one carbon atom of the lower alkylene, lower alkynyl and lower alkenyl may be replaced by a heteroatom selected from the group consisting of O, S, and N}.
The composition according to (1) above, made between the first polymers according to the above.
(3) The composition according to (1) or (2) above, wherein the uncharged hydrophilic polymer block is modified with a GLUT1 ligand at the terminal opposite to the cationic polymer and / or the anionic polymer.
(4) The composition according to (3) above, wherein the GLUT1 ligand is glucose.
(5) The composition according to any one of (1) to (4) above, wherein the polycation in the first polymer is a polymer of a natural cationic amino acid or a polymer of a non-natural cationic amino acid. ..
(6) The composition according to any one of (1) to (5) above, wherein the first polymer contains a non-charged hydrophilic polymer, and the non-charged hydrophilic polymer is polyalkylene glycol and polyoxazoline. ..
(7) The first polymer is
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 bound via a linker and may be linked.
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 , where p is 2, 3 or 4 and q. Is 2, 3, 4, or 5,
R ⁴ is a hydrogen, protecting group, hydrophobic group, or polymerizable group.
X is a cationic amino acid and
n is an integer of 2 to 5000,
n ₁ is an integer of 0 to 5000,
n ₃ is an integer of 0 to 5000,
n − n _{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 any order, and each repeat unit. The composition according to any one of (1) to (6) above, wherein the repeating units may exist at random, and each repeating unit may be the same or different.
(8) The composition according to any one of (1) to (7) above, which comprises an aqueous solution containing a drug.
(9) The composition according to any one of (1) to (8) above, which is orally administered.
(10) The composition according to (9) above, which is administered to cerebrospinal fluid.
(11) The composition according to (9) above, which is administered intraperitoneally or intravenously.
(12) A method of administering a drug to a subject who needs it.
A method comprising administering to the subject the composition according to (8) above.

FIG. 1 shows the effect of cross-linking on the stability of PICsome in the presence of salt. Panel A in FIG. 1 shows the results for PICsome (A), and panel B shows the results for PICsome (B). FIG. 2 shows the stability of PICsome crosslinked in a reducing environment so that it can be cleaved. Panel A in FIG. 2 shows the results for PICsome (A), and panel B shows the results for PICsome (B). FIG. 3 shows the relationship between the residual amount (%) in blood and the cross-linking strength in the blood 1 hour after administration of PICsome that was cleavably cross-linked in a reducing environment. FIG. 4 shows the distribution of PICsome with cleaved crosslinks and PICsome with non-cleavable crosslinks administered to the tail vein in the brain. The right panel of FIG. 4 is the result of PICsome with cleavage-type cross-linking, and the left panel is the result of PICsome with non-cleavable cross-linking. In the left panel of FIG. 4, the labeled antibody remains around the vascular endothelial cells, whereas in the right panel of FIG. 4, the labeled antibody is widely distributed in the brain parenchyma and is a polymer. It showed another localization.

Detailed 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. Donkeys, goats, ferrets and the like can be mentioned.

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.

As used herein, the term "polyion complex" (hereinafter, also referred to as "PIC") refers to 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 ion layer formed between the cationic block and the anionic block of both block copolymers when the copolymer of and the cationic block is mixed in an aqueous solution so as to neutralize the charge. The significance of binding PEG to the charged chain described above 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. It may form a copolymer with a block. It is also known that increasing the content of uncharged hydrophilic polymer blocks such as PEG tends to form PIC micelles, reducing the content of uncharged hydrophilic polymer blocks such as PEG or increasing the copolymer concentration. It is known that a polyion complex type polymersome (PICsome), which is a hollow particle having an average particle size of about 80 nm to 1,000 nm (particularly, about 100 nm to 1,000 nm), is likely to be formed. Since PICsome is a hollow particle having a membrane 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 medium) can be contained in the internal cavity. .. Examples of the contained substance include a water-soluble compound, a physiologically active substance (for example, an enzyme, for example, a proteinaceous enzyme, an antibody, a fragment thereof (for example, an antigen-binding fragment), another protein preparation, etc.), and a contrasting agent. Be done.

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.

As used herein, 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 higher the homogeneity of the particle size of the measured particles, and it is expected that the pharmacokinetics of each particle becomes homogeneous. .. 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. However, if the particles are homogeneous, there is an increased need for the particles to provide the desired physical and / or chemical properties.

According to the present invention, there is provided a polyion complex type polymersome having an average particle size of 80 nm or more as measured by a dynamic light scattering method. The polyion complex polymersome may have a polydispersity index (PDI) of 0.2 or less.

According to the present invention, the polyion complex type polymersome may include a first polymer containing a cationic polymer and a second polymer containing an anionic polymer.

According to the present invention, either or both of the first polymer and the second polymer can be block copolymers further comprising a non-charged hydrophilic polymer block.

Polyion complex polymersomes can be formed by mixing a first polymer with a second polymer. However, the polyion complex polymersome thus formed disintegrates in the presence of 150 mM sodium chloride. 150 mM corresponds to the sodium ion concentration in the blood. Therefore, cross-linking is required to stabilize polyion complex polymersomes in blood (see, eg, FIG. 1).

However, in the case of cross-linking, the polyion complex type polymersome having an average particle size of 80 nm or more measured by a dynamic light scattering method has low permeability to the glia limit film.

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, glia limit membrane (boundary membrane), and brain parenchyma. Exists. It is considered that the blood-brain barrier (BBB) is composed of cerebrovascular endothelial cells and glial limit membranes. Due to the large particle size, polyion complex polymersomes do not pass through the glial limiting membrane and are retained in the cerebrospinal fluid even if they pass through the cerebral vascular endothelial cells (see, for example, FIG. 4). In contrast, micelles with a small size (eg, 30 nm) are more likely to pass through the glial limiting membrane and migrate to the parenchyma of the brain when they pass through cerebrovascular endothelial cells (eg, by quoting them). See WO 2015/075942A, which is incorporated herein in its entirety). Therefore, it was considered that PICsome with a large particle size is not suitable for penetrating the glial limit membrane.

The present inventors, on the other hand, enhance the permeability of the polyion complex polymersome to the glia limit membrane by making the crosslink in the polyion complex polymersome a crosslink containing a cleavage type linker, or the cerebrospinal. It was revealed that polyion complex polymersomes can be disrupted in liquid, thereby delivering an aqueous solution containing a drug contained in polyion complex polymersomes, which originally has low permeability to the glia limit membrane, to the brain parenchyma. Clarified that it can be done.

According to the present invention, there is provided a polyion complex type polymersome having an average particle size of 50 nm or more as measured by a dynamic light scattering method.
According to the present invention, the polyion complex type polymersome having an average particle size of 50 nm or more and a polydispersity index of 0.3 or less, preferably 0.2 or less as measured by a dynamic light scattering method. Is provided.

The average particle size of the polyion complex type polymersome can be, for example, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 95 nm or more, or 100 nm or more. The average particle size is also 1000 nm or less, 900 nm or less, 800 nm or less, 700 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, 190 nm or less, 180 nm or less, 170 nm or less, 160 nm or less, 150 nm or less, 140 nm or less. , 130 nm or less, 120 nm or less, or 110 nm or less.

The polydispersity index (PDI) of the polyion complex type polymersome is, for example, 0.3 or less, 0.25 or less, 0.2 or less, 0.19 or less, 0.18 or less, 0.17 or less, 0. It can be 16 or less, 0.15 or less, 0.14 or less, 0.13 or less, 0.12 or less, 0.11 or less, or 0.10 or less. The polydispersity index (PDI) can be, for example, 0.05 or greater, 0.06 or higher, 0.07 or higher, 0.08 or higher, 0.09 or higher, or 0.1 or higher. The smaller the polydispersity index, the more preferable.

Raw materials for PICsome include, for example, a combination of a copolymer and a homopolyanion containing a non-charged hydrophilic polymer block and a polycation block, and a copolymer and a homopolycation containing a non-charged hydrophilic polymer block and a polyanion block. The combination of. Here, the uncharged hydrophilic polymer block can be a polymer containing monomeric units that are uncharged and hydrophilic. Examples of the uncharged hydrophilic polymer block include polyalkylene glycol (eg, polyethylene glycol), and polyoxazoline (eg, poly-C _1-4 alkyloxazoline, eg, poly (2-ethyl-2-oxazoline), poly (eg, poly (2-ethyl-2-oxazoline)). 2-Isopropyl-2-oxazoline). The uncharged hydrophilic polymer block may contain branches in the molecule, but preferably does not contain branches. The uncharged hydrophilic polymer block may contain branches. For example, it may have a number average molecular weight of 1,000 to 15,000, for example, 2,000 to 12,000, and the polycation block and the polyanion block may 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. In certain preferred embodiments, the polycation block and homopolycation have a biocompatible backbone (eg, starch, cellulose, chitosan, chitin, polylactic acid, polyglycolide, polycaprolactone) and a side chain having an amino group. obtain. The polycation block and homopolycation also have a polymer of a natural cationic amino acid such as polylysine and polyornithine and a polypeptide as the main chain, and are represented by-(NH- (CH ₂ ) ₂ ) _p- NH ₂ . 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 is, 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, p is 2, 3 or 4} as a side chain, and examples thereof include polymers of unnatural cationic amino acids. 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.

｛式中、
Ｒ¹は、水素原子、低級アルキルオキシ基、もしくは置換されていてもよい直鎖もしくは分岐鎖のＣ_1-12アルキル基であり得、またはポリエチレングリコールであり得、この場合、ポリエチレングリコールと隣り合うアミノ酸とはリンカーを介して結合してもよく、
Ｒ²は、低級アルキレンであり、
Ｒ³は、−（ＮＨ−（ＣＨ₂）₂）_p−ＮＨ₂または−ＮＨ−（ＣＨ₂）_q−ＮＨ₂で表される基であり、ｐは、２、３または４であり、ｑは、２、３、４、または５であり、
Ｒ⁴は、水素、保護基、疎水基、または重合性基であり、例えば、水素原子、アセチル基、トリフルオロアセチル基、アクリロイル基またはメタクリロイル基であり得、
Ｘは、カチオン性アミノ酸であり、
ｎは、２〜５０００のいずれかの整数であり、
ｎ₁は、０〜５０００のいずれかの整数であり、
ｎ₃は、０〜５０００のいずれかの整数であり、
ｎ−ｎ₁−ｎ₃は、０以上の整数であり、式中の各繰り返し単位は記載の都合上特定の順で示されているが、各繰り返し単位は順不同に存在することができ、各繰り返し単位はランダムに存在してもよく、また、各繰り返し単位は同一であっても異なっていてもよい｝で表されるものとすることができる。 In a preferred embodiment of the invention, the cationic polymer is 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 bound via a linker and may be linked.
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 , where p is 2, 3 or 4 and q. Is 2, 3, 4, or 5,
R ⁴ can be a hydrogen, protecting group, hydrophobic group, or polymerizable group, eg, a hydrogen atom, an acetyl group, a trifluoroacetyl group, 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 of 0 to 5000,
n ₃ is an integer of 0 to 5000,
n − n _{1 − n 3} is an integer of 0 or more, and each repeating unit in the equation is shown in a specific order for convenience of description, but each repeating unit can exist in random order, and each repeat unit. The repeating units may exist randomly, and each repeating unit may be the same or different}.

In the above formula (I), the protecting group includes a C _1-6 alkylcarbonyl group, preferably an acetyl group, and the hydrophobic group includes benzene, naphthalene, anthracene, pyrene and derivatives thereof or C _{1 -6} Alkyl groups are mentioned, and the polymerizable group includes a methacryloyl group and an acryloyl 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, 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 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 first polymer, a compound defined by any of the formulas (II) to (V) and (XIII) to (XI) described later can be used.

（上記式中、
Ｒ₁が、−（ＣＨ₂）₃ＮＨ₂または−ＣＯＮＨ（ＣＨ₂）_s−Ｘを表し、かつｓが０〜２０であり、ここで、Ｘが、−ＮＨ₂、ピリジル基、モルホリル基、１−イミダゾリル基、ピペラジニル基、４−（Ｃ_1-6アルキル）−ピペラジニル基、４−（アミノＣ_1-6アルキル）−ピペラジニル基、ピロリジン−１−イル基、Ｎ−メチル−Ｎ−フェニルアミノ基、ピペリジニル基、ジイソプロピルアミノ基、ジメチルアミノ基、ジエチルアミノ基、−（ＣＨ₂）_tＮＨ₂、または−（ＮＲ₉（ＣＨ₂）_o）_pＮＨＲ₁₀からなる群から選択される少なくとも一つのものであり、ここで、Ｒ₉が水素原子またはメチル基を表し、Ｒ₁₀が、水素原子、アセチル基、トリフルオロアセチル基、ベンジルオキシカルボニル基またはｔｅｒｔ―ブトキシカルボニル基を表し、ｏが１〜５であり、ｐが１〜５であり、ｔが０〜１５であり、
Ｒ２が、水素原子、アセチル基、トリフルオロアセチル基、アクリロイル基またはメタクリロイル基を表し、
ａが０〜５，０００であり、ｂが０〜５，０００であり、かつａ＋ｂが２〜５，０００である。）

(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, morpholyl 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 a 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. )

（上記式中、
Ｒ₂が、水素原子、アセチル基、トリフルオロアセチル基、アクリロイル基またはメタクリロイル基を表し、
Ｒ₃がそれぞれ独立してメチレン基またはエチレン基を表し、
ｃが０〜５，０００であり、ｄが０〜５，０００であり、かつｃ＋ｄが２〜５，０００である。）

(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. )

（上記式中、
Ｒ₁が、−（ＣＨ₂）₃ＮＨ₂または−ＣＯＮＨ（ＣＨ₂）_s−Ｘを表し、かつｓが０〜２０であり、ここで、Ｘが、−ＮＨ₂、ピリジル基、モルホリル基、１−イミダゾリル基、ピペラジニル基、４−（Ｃ_1-6アルキル）−ピペラジニル基、４−（アミノＣ_1-6アルキル）−ピペラジニル基、ピロリジン−１−イル基、Ｎ−メチル−Ｎ−フェニルアミノ基、ピペリジニル基、ジイソプロピルアミノ基、ジメチルアミノ基、ジエチルアミノ基、−（ＣＨ₂）_tＮＨ₂、または−（ＮＲ₉（ＣＨ₂）_o）_pＮＨＲ₁₀からなる群から選択される少なくとも一つのものであり、かつＲ₉が水素原子またはメチル基を表し、Ｒ₁₀が、水素原子、アセチル基、トリフルオロアセチル基、ベンジルオキシカルボニル基またはｔｅｒｔ―ブトキシカルボニル基を表し、ｏが１〜５であり、ｐが１〜５であり、ｔが０〜１５であり、
Ｒ₂が、水素原子、アセチル基、トリフルオロアセチル基、アクリロイル基またはメタクリロイル基を表し、
Ｒ₄が水素原子または置換されていてもよい直鎖もしくは分岐鎖のＣ_1-12アルキル基を表し、
Ｒ₅が−（ＣＨ₂）_gＮＨ−を表し、かつｇが０〜５であり、
ａが０〜５，０００であり、ｂが０〜５，０００であり、かつａ＋ｂが２〜５，０００であり、
ｅが５〜２，５００である。）

(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, morpholyl 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 a 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 a linear or branched C _1-12 alkyl group which may be substituted.
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. )

（上記式中、
Ｒ₁が、−（ＣＨ₂）₃ＮＨ₂または−ＣＯＮＨ（ＣＨ₂）_s−Ｘを表し、かつｓが０〜２０であり、ここで、Ｘが−ＮＨ₂、ピリジル基、モルホリル基、１−イミダゾリル基、ピペラジニル基、４−（Ｃ_1-6アルキル）−ピペラジニル基、４−（アミノＣ_1-6アルキル）−ピペラジニル基、ピロリジン−１−イル基、Ｎ−メチル−Ｎ−フェニルアミノ基、ピペリジニル基、ジイソプロピルアミノ基、ジメチルアミノ基、ジエチルアミノ基、−（ＣＨ₂）_tＮＨ₂、または−（ＮＲ₉（ＣＨ₂）_o）_pＮＨＲ₁₀からなる群から選択される少なくとも一つのものであり、ここで、Ｒ₉が水素原子またはメチル基を表し、Ｒ₁₀が、水素原子、アセチル基、トリフルオロアセチル基、ベンジルオキシカルボニル基またはｔｅｒｔ―ブトキシカルボニル基を表し、ｏが１〜５であり、ｐが１〜５であり、ｔが０〜１５であり、
Ｒ₂が、水素原子、アセチル基、トリフルオロアセチル基、アクリロイル基またはメタクリロイル基を表し、
Ｒ₆が、水素原子または置換されていてもよい直鎖もしくは分岐鎖のＣ_1-12アルキ ル基を表し、
Ｒ₇が−（ＣＨ₂）_hＮＨ−を表し、かつｈが０〜５であり、
Ｒ₈が直鎖または分岐鎖のＣ_1-12アルキル基を表し、
ａが０〜５，０００であり、ｂが０〜５，０００であり、かつａ＋ｂが２〜５，０００であり、
ｆが５〜２，５００である。）

(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, morpholyl 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. Here, 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 a linear or branched C _1-12 alkyl group which may be substituted.
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. )

In one aspect of the invention, the first polymer is a block copolymer of PEG-linker-polycation block {where PEG, linker and polycation block are as defined above. }.

In certain aspects of the invention, the second polymer can be a polymer in which natural or unnatural amino acids are linked by peptide bonds. Anionic polymer blocks and anionic polymers include, for example, polyglutamic acid and polyaspartic acid.

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

（上記式中、Ｒ₂が、水素原子、アセチル基、トリフルオロアセチル基、アクリロイル基またはメタクリロイル基を表し、
Ｒ₃がそれぞれ独立してメチレン基またはエチレン基を表し、
Ｒ₄が水素原子または置換されていてもよい直鎖もしくは分岐鎖のＣ_1-12アルキル 基を表し、
Ｒ₅が−（ＣＨ₂）_gＮＨ−を表し、かつｇが０〜５であり、
ｃが０〜５，０００であり、ｄが０〜５，０００であり、かつｃ＋ｄが２〜５，０００で あり、
ｉが５〜２，５００である。）

(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 a linear or branched C _1-12 alkyl group which may be substituted.
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. )

（上記式中、
Ｒ₂が、水素原子、アセチル基、トリフルオロアセチル基、アクリロイル基またはメタクリロイル基を表し、
Ｒ₃がそれぞれ独立してメチレン基またはエチレン基を表し、
Ｒ₆が水素原子または置換されていてもよい直鎖もしくは分岐鎖のＣ_1-12アルキル 基を表し、
Ｒ₇が−（ＣＨ₂）_hＮＨ−を表し、かつｈが０〜５であり、
Ｒ₈が直鎖または分岐鎖のＣ_1-12アルキル基を表し、
ｃが０〜５，０００であり、ｄが０〜５，０００であり、かつｃ＋ｄが２〜５，０００で あり、
ｊが５〜２，５００である。）

(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 a linear or branched C _1-12 alkyl group which may be substituted.
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. )

Ｒ¹は水素原子または未置換もしくは置換された直鎖もしくは分枝のＣ₁-₁₂アルキル基を表し、
Ｌ¹は−（ＣＨ₂）_b−ＮＨ−であり、ｂは１〜５の整数であり、Ｌ²は−（ＣＨ₂）_c−ＣＯ−であり、ｃは１〜５の整数であり、
Ｒ²はメチレン基またはエチレン基を表し、Ｒ³は水素原子、保護基、疎水性基または重合性基を表し、
Ｒ⁴はＲ⁵と同一であるかまたは−ＮＨ−Ｒ⁹であり、ここでＲ⁹は未置換又は置換された直鎖又は分枝のＣ_1-20アルキル基を表し、
Ｒ⁵はそれぞれ独立して水酸基、オキシベンジル基、又はアミン基であり、但しＲ⁵の８５％以上がアミン基であり、
ここでアミン基は、下記式（Ｘ）に記載の基：

であり、ａは１〜５の整数であり、ｍは５〜２０，０００の整数であり、
ｎは２〜５，０００の整数であり、ｘは０〜５，０００の整数であるが、ｘはｎより大きくないとする）

R ¹ is C ₁ linear or branched which is unsubstituted or substituted or a hydrogen atom - represents an ₁₂ alkyl group,
L ¹ is − (CH ₂ ) _b −NH−, b is an integer from 1 to 5, L ² is − (CH ₂ ) _c −CO−, and c is an integer from 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 ^{identical to R 5} or -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):

A is an integer of 1 to 5, m is an integer of 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 formed PICsome can be cross-linked with a cross-linking agent. The cross-linking agent may contain a cleavable binding (or linker) in a reducing environment (eg, in the presence of 1 mM to 2 mM glutathione, eg, intracerebral environment). A bond (or linker) that can be cleaved in a reducing environment can be appropriately selected and used by those skilled in the art, and may be, for example, an SS (disulfide) bond. Polyion complex polymersomes cross-linked with a cross-linking agent having a cleavable bond in a reducing environment cleaved the cross-linked moiety in cerebrospinal fluid, thereby initiating and encapsulating the polyion complex polymersome. An aqueous solution (an aqueous solution that may contain a drug) is passed through the glial limit membrane and delivered to the brain parenchyma. For example, the drug can be a drug having a molecular weight of 500 Da or more, 1000 Da or more, 2000 Da or more, 3000 Da or more, 4000 Da or more, 5000 Da or more, 6000 Da or more, 7000 Da or more, 8000 Da or more, 9000 Da or more, or 10 kDa or more, for example, an antibody or It can be a protein such as its antigen-binding fragment. For example, a polyion complex type polymersome having an average particle size of about 100 nm can contain one or more (for example, 1 to 5 molecules) of antibody molecules. The antibody can be, for example, IgG.

When an amino group is present in the side chain of the cationic polymer constituting PICsome, a cross-linking agent having an N-hydroxysuccinimide group that reacts with the amino group to form a covalent bond can be used. In one aspect of the invention, an amino group is present in the side chain of the cationic polymer constituting the PICsome, the cross-linking agent has N-hydroxysuccinimide groups (NHS groups) at both ends, and the two NHS groups are disulfides. It can be a molecule linked via a bond. Such cross-linking agents include, for example,
N-Hydroxysuccinimide (NHS) -L _1- S-S-L _2- NHS {In the formula, L ₁ and L ₂ are each independently bonded and optionally substituted lower alkylene, substituted. It may be a lower alkynyl, a lower alkenyl which may be substituted, a polyalkylene glycol which may be substituted, wherein one carbon atom of the lower alkylene, the lower alkynyl and the lower alkenyl is O, S, and N. May be replaced by a heteroatom selected from the group consisting of}
Can be mentioned.

In some embodiments, L ₁ and L ₂ can be independently bonded, substituted or unsubstituted lower alkylenes, respectively. As used herein, the term "lower" means that the number of carbon atoms is 1 to 4. For example, lower alkylene _{means C 1-4} alkylene.

In some embodiments, L ₁ and L ₂ can be independently substituted or unsubstituted polyalkylene glycols, respectively. In this embodiment, L ₁ and L ₂ can be polyethylene glycol independently of each other.

Where lower alkylenes, lower alkynyls and lower alkenyl are substituted, the substituents on these can be lower alkyl groups, amino groups, hydroxyl groups, or halogens.

In certain preferred embodiments, L ₁ and L ₂ can be bonds, respectively. That is, in one preferred embodiment, the cross-linking agent may be NHS-S-S-NHS.

In one preferred embodiment, the cross-linking agent is 0.2 equal to 2 equals, eg 0.2 equal to 1.5 equals, 0.2 etc. with respect to _{the NH 2 groups on the side chain of the cationic polymer.} Amount ~ 1.4 equal amount, 0.2 equal amount ~ 1.3 equal amount, 0.2 equal amount ~ 1.2 equal amount, 0.2 equal amount ~ 1.1 equal amount, 0.2 equal amount ~ 1.0 equal amount, 0.2 equal amount to 0.9 equal amount, 0.2 equal amount to 0.8 equal amount, 0.2 equal amount to 0.7 equal amount, 0.2 equal amount to 0. It can be added in an amount of 6 equal or 0.2 equal to 0.5 equal.

In one preferred embodiment, the first polymer is poly ([5-aminopentyl] -α, β-aspartamide) and the second polymer is polyaspartic acid, the first polymer and the second polymer. Either or both of these are block copolymers linked to PEG, and after the formation of polyion complex polymersomes, 0.2 equal to 1.0 equal amount with respect to _{2 NHs in the side chain of the cationic polymer.} 0.2 equal to 0.9 equal, 0.2 equal to 0.8 equal, 0.2 equal to 0.7 equal, 0.2 equal to 0.6 equal, or more It may be a polyion complex type polymersome obtained by adding 0.2 equal to 0.5 equal amounts of NHS-S-S-NHS. The polyion complex type polymersome has an average particle size of 80 nm or more and a PDI of 0.2 or less as measured by a dynamic light scattering method. Further, the polyion complex type polymersome contains an aqueous solution, for example, an aqueous solution containing a drug, for example, an aqueous solution containing an antibody or an antigen-binding fragment thereof.

According to the present invention, there is provided a method for administering to a subject an aqueous solution, for example, an aqueous solution containing a drug, for example, an aqueous solution containing an antibody or an antigen-binding fragment thereof. In the method of the invention, the composition comprising the polyion complex type polymersome of the present invention is administered to a subject, and the polyion complex type polymersome comprises an aqueous solution, for example, an aqueous solution containing a drug, for example, an antibody or an antigen-binding fragment thereof. Contains an aqueous solution. In the method of the invention, the encapsulated aqueous solution is delivered to the cerebrospinal fluid or brain parenchyma. In the present invention, the composition may be administered parenterally (eg, intravenously and intraperitoneally).

According to the present invention, a method for delivering an antibody or an antigen-binding fragment thereof to a subject's brain parenchyma, wherein the composition comprising the polyion complex type polymersome of the present invention is administered to the subject, and the polyion complex type polymersome is produced. , A method of encapsulating an aqueous solution containing an antibody or an antigen-binding fragment thereof is provided. In the present invention, the composition may be administered parenterally (eg, intravenously and intraperitoneally).

The antibody can be, for example, an antibody that binds to an antigen in the brain parenchyma (eg, intracellular antigen, cell surface antigen or extracellular antigen). In this embodiment, the antibody of the present invention can be an antibody that does not show significant binding to a component in blood. Examples of antigens in the brain parenchyma include amyloid β (Aβ, eg, Aβ _1-40 and Aβ _1-42 ), its soluble oligomers, its extracellular deposits, β-secretase 1 (BACE1), and aberrant prion proteins. (Misfolded prion protein), superoxide dismutase 1 (SOD1), and α-sinucrane.

When the antigen of the antibody is a surface antigen of a tumor in the brain parenchyma, the antibody can be an antibody having antibody-dependent cellular cytotoxicity (ADCC activity) and / or complement-dependent cytotoxic activity (CDC activity). ADCC activity can be enhanced, for example, by subclassing the antibody to IgG1. If the antigen of the antibody is a surface antigen of a tumor in the brain parenchyma, the antibody may be in the form of an antibody-drug conjugate (ADC) with a cytotoxic agent. The antibody may be, for example, in the form of a non-ADC.

According to the present invention, there is provided a pharmaceutical composition comprising the peptide or antibody of the present invention for treating or preventing a brain disease. Brain disorders include, for example, anxiety, depression, sleep disorders, Alzheimer's disease, Parkinson's disease and multiple sclerosis. Examples of the antibody include an antibody that binds to a causal factor of these diseases and neutralizes the activity thereof. For example, Aβ, an antibody of the invention that binds to its soluble oligomer and its extracellular deposits (eg, an antibody that binds to Aβ and / or its soluble oligomer and inhibits the formation or increase of extracellular deposits), Aβ. Antibodies of the invention that bind to cell deposits and suppress the increase in deposits or reduce deposits are administered, for example, to patients with Alzheimer's disease or patients at risk of developing Alzheimer's disease, thereby treating Alzheimer's disease. Can be used. An antibody of the invention that binds to an abnormal prion protein (eg, an antibody that binds to an abnormal prion protein and suppresses or reduces the accumulation of the abnormal prion protein) is, for example, Creutzfeldt-Jakob disease. It can be administered to patients with or at risk of developing Creutzfeldt-Jakob disease. Antibodies of the invention that bind to SOD1 (eg, antibodies that bind to and activate SOD1 variants found in amyotrophic lateral sclerosis (ALS), such as SOD1 and Derlin-1) are blocked. Anti-SOD1 antibody or anti-Derlin-1 antibody) can be administered, for example, to ALS patients or patients at risk of developing it, thereby being used in the treatment of ALS. Antibodies of the invention that bind to α-synuclein (eg, antibodies that bind to α-synuclein to suppress or decrease the accumulation of α-synuclein) include Lewy body dementias and Parkinson's disease. It can be administered to patients with or at risk of synucleinopathy and thereby used in the treatment of synucleinopathy. INDUSTRIAL APPLICABILITY According to the present invention, an antibody thus used for treating a disease is also provided. As used herein, "treatment of a disease" means prevention of a disease and treatment of a disease. Disease prevention is used to include preventing the onset of the disease, delaying the onset, and reducing the incidence. Treatment of a disease is used to include slowing the rate of exacerbation of the disease, delaying the exacerbation, preventing the exacerbation, reducing the symptoms of the disease, curing the disease, and relieving the disease.

The compositions of the invention can be administered according to a dosing regimen. Here, the administration plan is
Lowering the subject's blood sugar and then
Inducing an increase in blood glucose level and administering the composition of the present invention to the subject so that more antibody is transferred to the brain parenchyma as compared with the case where the increase in blood glucose level is not induced. Can include that.

In the dosing regimen according to the invention, the composition may be administered to the subject sequentially 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. , May be administered in a form different from the agent that causes the induction of an increase in blood glucose level in the subject. Also, if the composition is administered to the subject continuously or sequentially with the induction of an increase in blood glucose level in the subject, 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, in the case of inducing an increase in blood glucose level in the subject after the administration of the composition to the subject, within 6 hours or less, 4 hours or less and 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 above dosing regimen cycle 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.

As used herein, "inducing hypoglycemia" means lowering blood glucose levels in a subject than would otherwise have been indicated. 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 make the above fasting. Fasting causes the subject to hypoglycemia. The fasting period is determined by a doctor or the like in view 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, a period of time greater than increased expression of GLUT1 on the inner surface of blood vessels of cerebrovascular endothelial cells or reaching 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 target blood glucose level 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 object of maintaining hypoglycemia in the subject is achieved. Inducing hypoglycemia may be accompanied by other treatments that have no substantial effect on blood glucose.

As used herein, "inducing an increase in blood glucose level" means increasing a blood glucose level in a subject in which hypoglycemia is induced or a subject in which a hypoglycemic state is maintained. Blood glucose levels can be elevated by a variety of methods well known to those of skill in the art, eg, administration of one that induces an increase in blood glucose levels, eg, induces an increase in blood glucose levels such as glucose, fructose (fructose), galactose, etc. It can be increased by administration of monosaccharides, administration of polysaccharides that induce an increase in blood glucose level such as maltose, intake of carbohydrates that induce an increase in blood glucose level such as starch, or diet.

According to the present invention, there is provided a pharmaceutical composition comprising the composition of the present invention and a pharmaceutically acceptable excipient. The composition of the present invention may further contain a pharmaceutically acceptable excipient in addition to the antibody of the present invention. The compositions of the present invention can be in various forms, such as liquids (eg, injections), dispersants, suspensions, tablets, pills, powders, suppositories and the like. In a preferred embodiment, the pharmaceutical composition of the invention is an injection and can be administered parenterally (eg, intravenously, transdermally, intraperitoneally, and intramuscularly).

According to the present invention, there is provided a method of administering an antibody to a subject, which comprises administering the composition of the present invention to the subject. INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for delivering an antibody to a subject's brain parenchyma, which comprises administering the composition of the present invention to the subject. Administration can be intravenous administration. In these methods, the antibodies of the invention can be administered according to the dosing regimen according to the invention.

In some embodiments, in the composition comprising the polyion complex polymersome of the invention, either or both of the first polymer and the second polymer are linked to a non-charged hydrophilic polymer modified with a GLUT1 ligand. Thereby, the polyion complex type polymersome may recognizablely express the GLUT1 ligand by GLUT1. By doing so, the polyion complex type polymersome of the present invention is easily transferred from the blood vessel to the brain parenchyma across the blood-brain barrier. In this embodiment, the composition comprising the polyion complex type polymersome of the present invention can be administered according to the dosing regimen of the present invention.

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 surfaced on polyion complex polymersomes as recognizable 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-position, 3-position, or 6-position (and preferably O atoms bonded to the carbon atom). For example, a polyion complex type polymersome can be coated by covalently linking it to polyethylene glycol (O atom), and the coated polyion complex type polymersome can be preferably recognized by GLUT1. The coating of polyion complex polymersomes with GLUT1 increases the tendency of polyion complex polymersomes to stay in vascular endothelial cells when the amount increases, and when the amount decreases, polyion complex polymersomes pass through vascular endothelial cells (transcytosis) to the brain. The tendency to reach the substance of is increased. From this point of view, the degree of coverage of the polyion complex polymersome with the GLUT1 ligand can be adjusted. When the polyion complex type polymersome is coated with glucose, for example, 10 to 40 mol% (for example, 15 to 35 mol%, for example, 20 to 30 mol%) of the polymer constituting the polyion complex type polymersome is glucose. Can be linked (introduced).

In some embodiments, in the composition comprising the polyion complex polymersome of the invention, either or both of the first polymer and the second polymer are linked to the uncharged hydrophilic polymer and the uncharged hydrophilic polymer. Is not modified with GLUT1 ligand. In this embodiment, the composition containing the polyion complex type polymersome of the present invention can be administered into cerebrospinal fluid.

According to the present invention, there is a method for treating a brain disease in a subject in need thereof, which comprises administering the composition containing the polyion complex type polymersome of the present invention to the subject, and the polyion complex type of the present invention. Polymersomes are provided with a method comprising an aqueous solution containing an antibody that binds to a causative factor of the disease and neutralizes its activity. The combination of brain disease and the antibodies that can be administered to it can be as described above.

According to the present invention, it is a method for improving the permeability of a polyion complex type polymersome having an average particle size of 80 nm or more measured by a dynamic light scattering method to a glia limit film, and the polyion complex type polymer. Methods are provided that include cross-linking the somes by cross-linking with a linker that can be cleaved in a reducing environment.

According to the present invention, it is a method for producing a polyion complex type polymersome having an average particle size of 80 nm or more measured by a dynamic light scattering method, and the polyion complex type polymersome can be cleaved in a reducing environment. Methods are provided that include cross-linking by cross-linking with a linker.

In one embodiment, a method for producing a polyion complex type polymersome having an average particle size of 80 nm or more as measured by a dynamic light scattering method is a method of mixing a first polymer, a second polymer, and an aqueous solution containing a drug. The polyion complex polymersome is formed by reacting the obtained polyion complex polymersome with a cross-linking agent containing a linker (for example, a disulfide bond) that can be cleaved in a reducing environment to crosslink between polycations. Methods are provided that include forming the polymer.

In one embodiment, the method for producing a polyion complex polymersome having an average particle size of 80 nm or more as measured by a dynamic light scattering method is a polycation having an amino group in a side chain and a linker capable of cleaving in a reducing environment. Reacting with a cross-linking agent containing (eg, a disulfide bond) to form a crosslink between the polycations, and then cleavage (eg, in a disulfide bond) of the cross-linked polycation formed in a reducing environment. After that, the obtained polycation, the polyanion, and the aqueous solution containing the drug were mixed to form a polyion complex type polymersome, and then the obtained polyion complex type polymersome was incubated under oxidizing conditions. A method is provided that comprises forming a linker (eg, a disulfide bond) that can be cleaved between the polycations in a reducing environment.

According to the present invention, a composition containing the polyion complex type polymersome obtained by the method of the present invention is provided.

Example 1: Preparation of reduced environment-responsive PICsome In this example, PICsome, which is a hollow particle having a particle size larger than about 80 nm, was prepared.

Preparation method A
(A-1) Gluc-PEG-poly (α, β-apartic acid) (Gluc-PEG-P (Asp); _Mn of PEG: 2000, DP of P (Asp): 75) and PEG-PAsp (M). _n of PEG: 2000, DP of P (Asp): 75) was dissolved in 10 mM phosphate buffer (pH 7.4, 0 mM NaCl (10 mM PB)) to give Gluc-PEG-PAsp: PEG-PAsp. Both were mixed so as to be 1: 3.
(A-2) IgG (solanezumab, anti-hAPP (solanezumab) purchased from Fujifilm Wako Pure Chemical Industries, Ltd.) was also dissolved in 10 mM PB.
(A-3) A-1 and A-2 were mixed at a ratio of 1: 1 and adjusted to have a polymer concentration of 1 mg / mL and an IgG concentration of 10 mg / mL.
(A-4) Homo-poly ([5-aminopentyl] -α, β-aspartamide ((Homo-DAP) DP of P (Asp-AP) 70) also has a polymer concentration of 1 mg / mL. Dissolved in 10 mM PB.
(A-5) The solutions obtained in (A-3) and (A-4) above were mixed and stirred so that the polymer charge ratios were balanced, and self-associated PICsome was prepared by electrostatic interaction.
(A-6) Di (N-succinimidyl) 3,3'-dithiodipropionate (NHS-SS-NHS, purchased from Tokyo Chemical Industry Co., Ltd.) was dissolved in DMSO at 10 mg / mL.
Against (A-7) above obtained in (A-5) PICsome, the the (A-6) NHS-SS -NHS solution prepared in, the NHS group with respect to NH ₂ groups of Homo-DAP The mixture was added in equal amounts of 0, 0.3, 0.5, 0.8, 1.0, 1.2, 1.5, 2.0, 2.2 and allowed to stand overnight.
(A-8) To remove unreacted cross-linking agent and polymer, use VIVASPIN (MWCO = 300k), centrifuge at 4 ° C. and 1000 rpm, and purify by ultrafiltration to obtain PICsome (A). rice field.

Preparation method B
(B-1) The NHS-SS-NHS solution prepared in (A-6) above was added to a Homo-DAP aqueous solution (1 mg / mL) with _{respect to two} NH groups of Homo-DAP at 0, 0.3, 0. 5, 0.8, 1.0, 2.0 were added in equal amounts, allowed to stand overnight, and reduced with 200 mM dithiothreitol (DTT) to prepare Homo-DAP / SH.
(B-2) The solution obtained in (B-1) is mixed with the solution obtained in (A-3) so that the polymer charge ratios are balanced, stirred, and self-associated by electrostatic interaction to form PICsome. Prepared.
(B-3) In order to remove the unreacted cross-linking agent and polymer, VIVASPIN (MWCO = 300k) was used to centrifuge at 4 ° C. and 1000 rpm and purify by ultrafiltration to obtain PICsome (B). rice field.

Evaluation of Physical Properties of PICsome The particle size and particle size distributions of PICsome (A) and (B) were evaluated by dynamic light scattering (DLS). Measurements were taken using DLS (Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK)), small-capacity quartz batch cell (ZEN2121, Malvern Instruments Ltd. Ltd., 10 mM Worcestershire, 10m, Worcestershire, pH7, Worcestershire, UK). ) I went inside.

The results were as shown in Tables 1 and 2. In both PICsome (A) and (B) prepared by the above different methods, monodisperse particle size formation was confirmed at a particle size (Size) of around 100 nm. As for PICsome (A), monodisperse particle formation was not confirmed when NHS-SS-NHS in an amount of 2.2 equivalent or more was added.

Stability evaluation (salt tolerance evaluation)
The salt tolerance of PICsome (A) and (B) was evaluated. The PICsome (A) and (B) solutions dispersed in pH 7.4, 0 mM NaCl prepared above and 10 mM PB (pH 7.4, 300 mM NaCl) were mixed 1: 1 and then 1 After hours, the particle size was evaluated by DLS.

The results were as shown in FIG. As shown in FIG. 1, in both PICsome (A) and (B), PICsome to which NHS-SS-NHS was not added could not maintain monodispersity, whereas 0.3 equal or more was added. The sample size and high monodispersity were maintained even at the salt concentration under physiological conditions.

Stability evaluation (reduction environmental stability and responsiveness evaluation)
Reduction of PICsome (A) and (B) Environmental stability (glutathione (GSH) concentration in blood (0.1 mM) and responsiveness evaluation (glutathione concentration in brain 2 mM) were performed. pH 7.4, 150. PICsome (A) and (B) solutions dispersed in mM NaCl,
(1) Mix 1: 1 with 10 mM PB (pH 7.4, 150 mM NaCl, 0.2 mM GSH) or (2) 10 mM PB (pH 7.4, 150 mM NaCl, 4 mM GSH), respectively. After 30 minutes, the particle size was evaluated by DLS.

The results were as shown in FIG. As shown in FIG. 2, both PICsome (A) and (B) maintained high monodispersity at a diameter of about 100 nm in the GSH concentration of 0.1 mM in blood as before the addition. On the other hand, it was clarified that the structure cannot be maintained when the NHS-SS-NHS addition amount is 0.3-1.0 equal to the GSH concentration of 2 mM in the brain, that is, it responds to the reducing environment. .. It was also suggested that the dose above 1.3 is stable in the reducing environment in the brain.

Blood Circulation Assessment Balb c (female, 6 weeks old, n = 3) was treated with Alexa647-labeled IgG (Alexa647-IgG).
(1) Encapsulated in PICsome cross-linked by EDC (Alexa647-IgG @ PICsome (non-cleavable)), or (2) Encapsulated in PICsome (A) (Alexa647-IgG @ PICsome (cleavable), NHS -SS-NHS: 0.3, 0.5 1.0 equal dose) was administered to the tail vein, blood was collected 1 hour after administration, and the fluorescence intensity in the blood was measured with a multi-plate reader (Tecan, SPARK). ..

The results were as shown in FIG. As shown in FIG. 3, it was revealed that about 50% of Alexa647-IgG @ PICsome (cleaving type) remained in the blood regardless of the amount of NHS-SS-NHS added. On the other hand, 80% of Alexa647-IgG @ PICsome (cleavage type) remained in the blood after 1 hour. As described above, the Cleavage-type crosslinked PICsome produced in this example had lower blood retention than the non-cleavage-type cross-linked PICsome.

Intracerebral dynamics of antibody @PICsome (cleavage type) In Alzheimer's disease model mouse APP / PS1 mouse (female; 3 months old) with 24-hour feeding control, (1) Alexa647-IgG @ PICsome prepared by EDC cross-linking as described above (1) Non-cleaving type), (2) Alexa647-IgG @ PICsome (cleaving type) (NHS-SS-NHS: 0.3 equal dose, PICsome stained with Dylight488) prepared according to Preparation Method A was administered to the tail vein. As the antibody, solanetumab, which is an anti-Aβ antibody, was used. A blood glucose operation was performed in which the sample was fasted 24 hours before the administration of the tail vein, and a 20 v / v% glucose solution was intraperitoneally administered 30 minutes before the administration.

Twenty-four hours after sample administration, the abdomen was opened under isoflurane anesthesia, perfused with a 4% paraformaldehyde (PFA) solution (solvent: D-PBS (-)) from the portal vein, and the craniotomy was performed to remove the brain. The removed brain is immersed in a 4% PFA solution (solvent: D-PBS (-)) overnight at 4 ° C, and then in a 20% sucrose solution (solvent: D-PBS (-)) overnight at 4 ° C. It was soaked and then soaked in a 30% sucrose solution (solvent: D-PBS (−)) at 4 ° C. overnight. O. C. T. After embedding in compound and freezing in low-temperature hexane, frozen sections having a thickness of 14 μm were prepared using a cryostat (CM1950 freezing microtome for infection prevention measures). Subsequently, immunostaining of amyloid β was performed. First, thioflavin was diluted with a PBS solution containing 0.1% tween20 (PBS-T). Subsequently, the tissue was washed with PBS-T for 5 minutes, and then blocking was performed at room temperature for 15 minutes using Blocking one (nacalai tesque) as a blocking buffer. After washing with PBS-T for 5 minutes, thioflavin was reacted at room temperature for 1 hour. After washing with PBS-T three times for 5 minutes, embedding was performed with Aqueous Mounting Medium. The obtained sections were observed with a confocal laser scanning microscope.

The results were as shown in FIG. Alexa647-IgG @ PICsome (non-cleaving type) did not cross the glia limit membrane and the antibody shown in red remained in the CSF, whereas Alexa647-IgG @ PICsome (cleaving type) showed in red. It was observed that the antibody recognized Aβ (antibody and Aβ were localized), no co-localization between the antibody and the polymer was observed, PICsome was cleaved in the brain, and the antibody was released into the brain parenchyma. Shown.

From this result, it was clarified that the crosslinked polyion complex polymersome in which the polymers were crosslinked via a linker that can be cleaved in the reducing environment in the brain enhances the permeability to the glia limit membrane.

Claims (12)

A composition comprising polyion complex polymersomes having an average particle size of 80 nm or more and a polydispersity index (PDI) of 0.2 or less as measured by a dynamic light scattering method.
Polyion complex polymersomes include a first polymer containing a cationic polymer and a second polymer containing an anionic polymer, and either or both of the first polymer and the second polymer are uncharged hydrophilic. A block copolymer that further contains polymer blocks,
A composition in which the polymers are crosslinked by a linker that can be cleaved in a reducing environment. The first polymer has a biocompatible main chain and a side chain with an amino group, and the cross-linking is N-hydroxysuccinimide (NHS) -L _1- S-S-L _2- NHS {in the formula, L. ₁ and L ₂ are independently substituted lower alkylene, optionally substituted lower alkynyl, optionally substituted lower alkenyl, and optionally substituted polyalkylene glycol. Here, one carbon atom of the lower alkylene, lower alkynyl and lower alkenyl may be replaced by a heteroatom selected from the group consisting of O, S, and N}.
The composition according to claim 1, wherein the composition is made between the first polymers according to the above. The composition of claim 1 or 2, wherein the uncharged hydrophilic polymer block is modified with a GLUT1 ligand at the terminal opposite the cationic and / or anionic polymer. The composition according to claim 3, wherein the GLUT1 ligand is glucose. The composition according to any one of claims 1 to 4, wherein the polycation in the first polymer is a polymer of a natural cationic amino acid or a polymer of a non-natural cationic amino acid. The composition according to any one of claims 1 to 5, wherein the first polymer comprises a non-charged hydrophilic polymer, and the non-charged hydrophilic polymer is a polyalkylene glycol and a polyoxazoline. The first polymer is
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 bound via a linker and may be linked.
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 , where p is 2, 3 or 4 and q. Is 2, 3, 4, or 5,
R ⁴ is a hydrogen, protecting group, hydrophobic group, or polymerizable group.
X is a cationic amino acid and
n is an integer of 2 to 5000,
n ₁ is an integer of 0 to 5000,
n ₃ is an integer of 0 to 5000,
n − n _{1 − n 3} is an integer of 0 or more, and each repeating unit in the equation is shown in a specific order for convenience of description, but each repeating unit can exist in any order, and each repeat unit. The composition according to any one of claims 1 to 6, wherein the repeating units may exist at random, and each repeating unit may be the same or different. The composition according to any one of claims 1 to 7, which comprises an aqueous solution containing a drug. The composition according to any one of claims 1 to 8, which is administered parenterally. The composition according to claim 9, which is administered to cerebrospinal fluid. The composition according to claim 9, which is administered intraperitoneally or intravenously. It is a method of administering a drug to a subject who needs it.
A method comprising administering to the subject the composition of claim 8.

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