Polyion complex and pharmaceutical composition

Abstract

The main purpose of the present invention is to develop a DDS useful for treating metastatic cancer. A polyion complex according to an embodiment of the present invention comprises: a block copolymer having a hydrophilic polymer segment and a poly(amino acid) segment; and a nucleic acid, wherein the poly(amino acid) segment includes two or more cationic amino acid residues and two or more hydrophobic amino acid residues, the hydrophobic amino acid residues are arranged alternately and / or randomly, and the hydrophobic amino acid residues include hydrophobic amino acid residues having a side chain CLogP value of 3 or more. The polyion complex has an average particle diameter of 100 nm or more.

Description

Polyion complexes and pharmaceutical compositions

[0001] The present invention relates to a polyion complex comprising a nucleic acid and a cationic polymer, and a pharmaceutical composition comprising the polyion complex.

[0002] In recent years, nucleic acid drugs such as antisense nucleic acids, siRNA, miRNA, and aptamers, as well as gene therapies consisting of nucleic acids encoding therapeutic proteins such as plasmid DNA and mRNA, have been rapidly developing.

[0003] As carriers for drug delivery systems (DDS) for delivering nucleic acids to target cells, tissues, etc., cationic polymers capable of forming polyion complexes (PICs) with nucleic acids using electrostatic interactions as a driving force have been proposed. Examples of such cationic polymers include cationic block copolymers having hydrophilic polymer segments and cationic polymer segments, and micelle-like PICs formed by the assembly of cationic block copolymers so as to encapsulate nucleic acids have been proposed (for example, Patent Document 1).

[0004] WO2019 / 240223

[0005] Activated platelets are necessary for the spread and establishment of metastatic cancer and are known to be present in large quantities in the metastatic niche, which is an environment that promotes tumor formation. Platelets in the metastatic niche can cause thrombosis. Thrombosis acts as an obstacle to the EPR effect (Enhanced Permeability and Retention effect) and active targeting using ligands such as cyclic RGDs, making it difficult to adequately deliver drugs to target tissues. Therefore, there is a need for a drug delivery system (DDS) that is effective against metastatic cancer. The primary objective of this invention is to develop a DDS that is effective against metastatic cancer.

[0006] 1. The polyion complex of the embodiment of the present invention comprises a block copolymer having a hydrophilic polymer segment and a poly(amino acid) segment; a nucleic acid; wherein the poly(amino acid) segment comprises two or more cationic amino acid residues and two or more hydrophobic amino acid residues, wherein the hydrophobic amino acid residues are arranged alternately and / or randomly, and the hydrophobic amino acid residues include hydrophobic amino acid residues having a CLogP value of 3 or more in the (A) side chain, and the average particle size is 100 nm or more. 2. In the polyion complex described in 1 above, the hydrophilic polymer segment may include at least one hydrophilic polymer selected from the group consisting of poly(ethylene glycol), poly(saccharide), poly(vinylpyrrolidone), poly(vinyl alcohol), poly(acrylamide), poly(acrylic acid), poly(methacrylamide), poly(methacrylic acid), poly(methacrylic acid ester), poly(acrylic acid ester), poly(amino acid), poly(malic acid), and derivatives thereof. 3. 1. In the polyion complex described in 1 or 2 above, the molecular weight of the hydrophilic polymer may be 80 kDa or less. 4. In the polyion complex described in any of 1 to 3 above, the difference between the proportion of cationic amino acid residues located at odd positions of the poly(amino acid) segment and the proportion of cationic amino acid residues located at even positions of the poly(amino acid) segment may be 40% or more. 5. In the polyion complex described in any of 1 to 4 above, 80% or more of the cationic amino acid residues may be located at every other amino acid residue. 6. In the polyion complex described in any of 1 to 5 above, the cationic amino acid residue may be at least one selected from the group consisting of lysine residues, arginine residues, ornithine residues, histidine residues, homolysine residues, 2,4-diaminobutyric acid residues, 2,3-diaminopropionic acid residues, 2-aminoglycine residues, homoarginine residues, 2-amino-3-guanidinopropionic acid residues, and 2-amino-4-guanidinobutyric acid residues. 7. In the polyion complex described in any of the above 1 to 6, the hydrophobic amino acid residue (A) may have an alicyclic hydrocarbon group in its side chain.8. In the polyion complex described in any of 1 to 7 above, the hydrophobic amino acid residue (A) may be at least one selected from the group consisting of cyclohexylalanine residue, cyclopentylalanine residue, cyclohexylglycine residue, cyclopropylalanine residue, homonorleucine residue, 2-aminooctanoic acid residue, and 2-aminoundecanoic acid residue. 9. In the polyion complex described in any of 1 to 8 above, the nucleic acid may be a single-stranded nucleic acid. 10. In the polyion complex described in any of 1 to 9 above, the hydrodynamic diameter of the nucleic acid may be 10 nm or less. 11. In the polyion complex described in any of 1 to 10 above, the base length of the nucleic acid may be 50 bases or less. 12. In another aspect of the present invention, a pharmaceutical composition is provided. This pharmaceutical composition comprises the polyion complex described in any of 1 to 11 above. 13. The pharmaceutical composition described in 12 above may be used for the treatment of metastatic cancer. 14. 12 or 13 above may be used for the treatment of metastatic lung cancer. 15. In yet another aspect of the present invention, a method for treating metastatic cancer is provided, comprising administering an ion complex according to any one of 1 to 11 above to a target. 16. In the treatment method according to 15 above, the metastatic cancer may be metastatic lung cancer. 17. In yet another aspect of the present invention, an ion complex according to any one of 1 to 11 above is provided for use in the treatment of metastatic cancer. 18. In the ion complex according to 17 above, the metastatic cancer may be metastatic lung cancer. 19. In yet another aspect of the present invention, the use of an ion complex according to any one of 1 to 11 above is provided for manufacturing a pharmaceutical composition for the treatment of metastatic cancer. 20. In the use of the ion complex according to 19 above, the metastatic cancer may be metastatic lung cancer.

[0007] The polyion complex of the embodiment of the present invention may provide a useful DDS for the treatment of metastatic cancer.

[0008] These are the MALDI-TOF-MS spectral data of the block copolymers synthesized in the examples. This graph shows the size distribution of PICs prepared using (GlyLys) 10⁻⁰k, (ChaLys) 10⁻⁰k, or (NleLys) 10⁻⁰k. This graph shows the size distribution of PICs prepared using (GlyLys) 10⁻³k, (ChaLys) 10⁻³k, or (NleLys) 10⁻³k. This graph shows the size distribution of PICs prepared using (GlyLys) 10⁻¹⁰k, (ChaLys) 10⁻¹⁰k, or (NleLys) 10⁻¹⁰k. These are transmission electron microscope images of PIC prepared using (GlyLys) 10⁻³k, (ChaLys) 10⁻³k, or (NleLys) 10⁻³k. These are in vivo confocal laser scanning microscope images of PIC prepared using (GlyLys) 10⁻³k at 0 minutes, approximately 60 minutes, approximately 100 minutes, and approximately 160 minutes after injection. These are in vivo confocal laser scanning microscope images of PIC prepared using (ChaLys) 10⁻³k at 0 minutes, approximately 60 minutes, approximately 100 minutes, and approximately 160 minutes after injection. These are in vivo confocal laser scanning microscope images of PIC prepared using (NleLys) 10⁻³k at 0 minutes, approximately 60 minutes, approximately 100 minutes, and approximately 160 minutes after injection. These are in vivo confocal laser scanning microscope images of ASO at 0 minutes, approximately 60 minutes, approximately 100 minutes, and approximately 160 minutes after injection. This graph shows the relative intensity over time of PIC and ASO prepared using (GlyLys) 10⁻³k, (ChaLys) 10⁻³k, or (NleLys) 10⁻³k. These are in vivo imaging system images of the lungs, spleen, kidneys, and liver (from left to right) of mice administered with PIC prepared using 0kDa-PEG, 3kDa-PEG, or 10kDa-PEG. These are in vivo imaging system images of the lungs, spleen, kidneys, and liver (from left to right) of mice administered with PIC prepared using 20kDa-PEG or 40kDa-PEG. This graph shows the radioactivity efficiency indicating the amount of nucleic acid accumulation in the lungs and spleen of mice. These are in vivo imaging system images of the lungs, spleen, kidneys, and liver (from left to right) of mice prophylactically administered warfarin.This is a graph of radioactivity showing the accumulation of nucleic acids in the lungs and spleen of mice prophylactically administered warfarin. This is a graph of TGF-β1 expression levels in the lungs and spleen of mice. These are in vivo imaging system images taken at 0, 2, 5, 10, 20, and 30 minutes after injection. This is a graph showing the relationship between the Pearson correlation coefficient between platelets (DyLight488) and PIC (Alexa647) and time. These are in vivo imaging system images of subcutaneous tumors, heart, lungs, spleen, kidneys, and liver (from left to right) of mice administered PIC prepared using 0 kDa-PEG, 3 kDa-PEG, or 10 kDa-PEG. These are in vivo imaging system images of subcutaneous tumors, heart, lungs, spleen, kidneys, and liver (from left to right) of mice administered with PIC prepared using 20 kDa-PEG or 40 kDa-PEG. The graphs show the radioefficiency of nucleic acid accumulation in the kidneys and liver of mice. The graphs also show the radioefficiency of nucleic acid accumulation in subcutaneous tumors of mice. Finally, the graphs show the radioefficiency of nucleic acid accumulation in the lungs and spleen of mice. The graph also shows the change in survival rate of mice administered with PIC containing doxorubicin and (GlyLys) 10-10k, TGF-β1 ASO, GL3 ASO, or STAT3 ASO. This graph shows the median survival time (days) of mice administered a PIC containing doxorubicin, (GlyLys) 10-10k, TGF-β1 ASO, GL3 ASO, or STAT3 ASO.

[0009] Preferred embodiments of the present invention will be described below, but the present invention is not limited to these embodiments. Each embodiment can be combined as appropriate unless such an understanding is clearly inappropriate from the context. In this specification, “~” representing a numerical range includes its upper and lower numerical limits.

[0010] In this specification, "polyion complex" refers to a complex formed by intermolecular interactions, including electrostatic interactions, between a polymer compound having cationic groups and a polymer compound having anionic groups.

[0011] A. Polyion Complex (PIC) The PIC according to an embodiment of the present invention comprises a block copolymer having a hydrophilic polymer segment and a poly(amino acid) segment, and a nucleic acid. The poly(amino acid) segment comprises two or more cationic amino acid residues and two or more hydrophobic amino acid residues, wherein the hydrophobic amino acid residues are arranged alternately and / or randomly. In other words, the poly(amino acid) segment does not have a block structure in which all of the two or more hydrophobic amino acid residues are arranged consecutively. Furthermore, the poly(amino acid) segment includes, as hydrophobic amino acid residues, hydrophobic amino acid residues having a CLogP value of 3 or more in the (A) side chain (hereinafter also referred to as hydrophobic amino acid residue (A)). The average particle size of this PIC is 100 nm or more. Using the PIC according to an embodiment of the present invention, an effective DDS can be provided against metastatic cancer.

[0012] The reason why PIC according to the embodiment of the present invention exhibits the above effects can be presumed to be as follows, although this does not limit the present invention: that platelets are physically activated by contact with PIC (for example, by activation of the P-selectin pathway which can mediate the interaction between activated platelets and leukocytes), and PIC can be delivered to metastatic cancer (e.g., the metastatic niche) through interaction with activated platelets. In particular, PIC having the above average particle size can suitably interact with activated platelets, and as a result, it is presumed that an effective DDS for metastatic cancer (e.g., the metastatic niche) is realized through the interaction between PIC and platelets. At this time, if the poly(amino acid) segment contains a hydrophobic amino acid residue (A) with a CLogP value of 3 or more in the side chain as a hydrophobic amino acid residue, PIC having the above average particle size can be suitably obtained. Furthermore, if the block copolymer has a hydrophilic polymer segment in addition to the poly(amino acid) segment, excessive activation of platelets can be suppressed. Furthermore, if two or more hydrophobic amino acid residues are arranged alternately and / or randomly (in other words, if not all of the two or more hydrophobic amino acid residues are arranged as consecutive blocks), the surfactant properties of the block copolymer will be reduced, which may suppress membrane disruption caused by surfactant properties (e.g., hemolysis of red blood cells). Excessive activation and membrane disruption of platelets can cause fatal pulmonary embolism, etc., and according to the above PIC, it is hypothesized that an effective DDS for metastatic cancer (e.g., metastatic niche) can be realized by utilizing the interaction with activated platelets while suppressing these problems.

[0013] The average particle size of the above PIC is 100 nm or more, preferably 100 nm to 500 nm, more preferably 100 nm to 300 nm, and even more preferably 100 nm to 200 nm. If the average particle size is 100 nm or more, interaction with activated platelets may be improved, and efficient delivery to metastatic cancer (e.g., metastatic niches) may be possible. The average particle size of the above PIC is the hydrodynamic diameter and can be determined, for example, by fluorescence correlation spectroscopy (FCS) analysis, dynamic light scattering (DLS) analysis, and ultracentrifugation analysis by sedimentation equilibrium (SE-AUC) analysis.

[0014] A-1. Block Copolymer The block copolymer has a hydrophilic polymer segment and a poly(amino acid) segment. The block copolymer is, for example, a diblock represented by the formula: A-L-B (wherein A represents a hydrophilic polymer segment, B represents a poly(amino acid) segment, and L represents a single bond or a divalent linking group). If the block copolymer has a hydrophilic polymer segment, excessive activation of platelets can be suppressed.

[0015] A-1-1. Hydrophilic Polymer Segments The hydrophilic polymer segment includes hydrophilic polymers. Specific examples of hydrophilic polymers include poly(ethylene glycol), poly(saccharide), poly(vinylpyrrolidone), poly(vinyl alcohol), poly(acrylamide), poly(acrylic acid), poly(methacrylamide), poly(methacrylic acid), poly(methacrylic acid ester), poly(acrylic acid ester), poly(amino acid) (e.g., poly(sarcosine), poly(aspartic acid), poly(glutamic acid), poly(serine), etc.), poly(malic acid), or derivatives thereof. Among these, polymers that do not have an electric charge at physiological pH (pH 7.4) are preferred, nonionic polymers are more preferred, and poly(ethylene glycol) is even more preferred. Terminally reactive poly(ethylene glycol) of various molecular weights is commercially available and readily obtainable.

[0016] The hydrophilic polymer may be linear or branched. In the case of a branched polymer, the number of chains is, for example, 2 to 4, preferably 2 or 3, and more preferably 2.

[0017] The molecular weight of the hydrophilic polymer (in the case of a branched-chain, the sum of the molecular weights of each chain) is preferably 80 kDa or less, more preferably 1 kDa to 80 kDa or less, and still more preferably 3 kDa to 80 kDa. If the molecular weight of the hydrophilic polymer is within the above range, a PIC with excellent colloidal stability can be obtained, and as a result, platelets can be moderately activated. In one embodiment, the molecular weight of the hydrophilic polymer can be selected as any appropriate value according to the application. Specifically, in the case of a PIC encapsulating a nucleic acid pharmaceutical, when the purpose is to deliver it to the lung, it can be set to about 3 kDa (for example, 1 kDa to 5 kDa), and when the purpose is to deliver it to the spleen, it can be set to about 20 kDa (for example, 10 kDa to 40 kDa). Also, in the case of a polymer-drug conjugate without using a nucleic acid pharmaceutical, it can be set to about 40 kDa (for example, 20 kDa to 80 kDa). The above molecular weight can be the number average molecular weight.

[0018] A-1-2. Poly(amino acid) segment The poly(amino acid) segment contains poly(amino acid). The poly(amino acid) contains two or more cationic amino acid residues and two or more hydrophobic amino acid residues, and the two or more hydrophobic amino acid residues are arranged alternately and / or randomly. The above hydrophobic amino acid residues include hydrophobic amino acid residues (A) having a ClogP value of 3 or more in the side chain.

[0019] As long as the effects of the present invention can be obtained, the poly(amino acid) may contain other amino acid residues other than the cationic amino acid residues and the hydrophobic amino acid residues (A). In one embodiment, the amino acid residues contained in the poly(amino acid) can be α-amino acid residues.

[0020] In the poly(amino acid) segment, the difference between the ratio of cationic amino acid residues arranged at odd positions and the ratio of cationic amino acid residues arranged at even positions is, for example, 40% or more, preferably 50% or more, more preferably 60% or more, and may be 80% or more, or 90% to 100%. A large difference may mean that the cationic side chains are preferentially arranged on one side of the poly(amino acid) segment. A poly(amino acid) segment having such a structure can promote electrostatic complexation with a nucleic acid (for example, a single-stranded nucleic acid), so that a PIC having a desired particle size can be more suitably obtained. The position numbers of the amino acid residues in the poly(amino acid) segment are not particularly limited, but the position number of the amino acid residue at the N-terminus can be set to 1.

[0021] In the poly(amino acid) segment, preferably 80% or more, more preferably 90% to 100% of the cationic amino acid residues can be arranged every other amino acid residue. The side chains of the cationic amino acid residues arranged every other amino acid residue can be arranged in one direction at a certain interval (about 7 nm). Also, the anionic groups (typically phosphate groups) of adjacent nucleotides in the nucleic acid can be arranged in one direction at a certain interval (about 6 nm). Therefore, according to a block copolymer in which 80% or more of the cationic amino acid residues are alternately arranged with other amino acid residues, the distribution of the positive charges of the cationic side chains and the distribution of the negative charges of the nucleic acid can be preferably matched, and electrostatic complexation can be further promoted. As a result, it may be easier to adjust the properties of the PIC, such as the particle size.

[0022] In the poly(amino acid) segment, it is preferable that at least one cationic amino acid residue and a hydrophobic amino acid residue are arranged adjacent to each other. If a cationic amino acid residue and a hydrophobic amino acid residue are adjacent to each other, the anion of the nucleic acid can be neutralized by the cation of the cationic amino acid residue, and the hydrophobic amino acid residue adjacent to the cationic amino acid residue can easily interact with the nucleobase. As a result, the formation of a polyion complex having an average particle size of 100 nm or more can be further promoted.

[0023] Any suitable amino acid residue can be used as the cationic amino acid residue. Preferably, an amino acid residue having one positive charge at physiological pH (pH 7.4) is used. Specifically, examples include lysine residues, arginine residues, ornithine residues, histidine residues, homolysine residues, 2,4-diaminobutyric acid residues, 2,3-diaminopropionic acid residues, 2-aminoglycine residues, homoarginine residues, 2-amino-3-guanidinopropionic acid residues, and 2-amino-4-guanidinobutyric acid residues. Preferably, lysine residues, arginine residues, and ornithine residues are used. Only one cationic amino acid residue may be used, or two or more may be used in combination.

[0024] As described above, the hydrophobic amino acid residue (A) used is an amino acid residue whose side chain CLogP value is 3 or higher. The particle size of the PIC can affect the degree of interaction with activated platelets, and by using a hydrophobic amino acid residue (A), PICs of a desired particle size can be suitably formed. The CLogP value of the side chain of the hydrophobic amino acid residue (A) is preferably 3 to 6, more preferably 3 to 5, and even more preferably 3.5 to 4. The CLogP value of the side chain of the hydrophobic amino acid residue can be calculated by any suitable method. For example, it can be calculated using any suitable software. In this specification, ChemDraw ver. 22.0.0 was used, and the calculation was performed using the algorithm provided by BioByte (http: / / www.biobyte.com / ).

[0025] The hydrophobic amino acid residue (A) preferably has an aliphatic hydrocarbon group in its side chain. The aliphatic hydrocarbon group may be linear, branched, or cyclic. If the hydrophobic amino acid residue (A) has an aliphatic hydrocarbon group in its side chain, interaction with the aromatic ring of nucleic acids is promoted, and the formation of polyion complexes with an average particle size of 100 nm or more can be further promoted. Furthermore, if the amino acid has an aliphatic hydrocarbon group in its side chain, the binding energy with nucleic acid bases becomes larger, and the self-assembly of block copolymers can be suppressed. More preferably, the hydrophobic amino acid residue (A) has an alicyclic hydrocarbon group in its side chain, and even more preferably, it has an alicyclic hydrocarbon group having 5 or more carbon atoms in its side chain. If the hydrophobic amino acid residue (A) has an alicyclic hydrocarbon group having 5 or more carbon atoms in its side chain, molecular compatibility with nucleic acids can be further improved.

[0026] Examples of hydrophobic amino acid residues (A) include cyclohexylalanine residues, cyclopentylalanine residues, cyclohexylglycine residues, cyclopropylalanine residues, homonorleucine residues, 2-aminooctanoic acid residues, and 2-aminoundecanoic acid residues. Preferably, cyclohexylalanine residues, cyclopentylalanine residues, and cyclohexylglycine residues are used. Only one hydrophobic amino acid residue may be used, or two or more may be used in combination.

[0027] As the other amino acid residues mentioned above, any suitable amino acid residue can be used, provided that the effects of the present invention are obtained. The other amino acid residues may be, for example, uncharged amino acid residues selected from hydrophobic amino acids other than the hydrophobic amino acid residue (A) mentioned above (e.g., Ala, Ile, Leu, Val, Met, Phe, Trp, Tyr, Nle (norleucine)), and polar neutral amino acids (e.g., Asn, Cys, Gln, Ser, Thr). Only one of these other amino acid residues may be used, or two or more may be used in combination.

[0028] The total number of amino acid residues contained in poly(amino acids) (in other words, the degree of polymerization of poly(amino acids)), the number of cationic amino acid residues, and the number of hydrophobic amino acid residues (A) can be appropriately set according to the base length or negative charge amount of the nucleic acid to be delivered, synthesis efficiency, cost, etc. The total number of amino acid residues contained in poly(amino acids) is, for example, 100 or less, preferably 60 or less, more preferably 10 to 50, and even more preferably 10 to 30.

[0029] The ratio of cationic amino acid residues to the total number of amino acid residues in poly(amino acids) is, for example, 20% to 80%, preferably 30% to 70%, and more preferably 40% to 60%. The number of cationic amino acid residues in poly(amino acids) is two or more, and may be 5 to 25 or 8 to 12.

[0030] The number of cationic amino acid residues in the poly(amino acid) can be adjusted so that the ratio of the positive charge amount derived from the cationic side chain of the poly(amino acid) to the negative charge amount of the nucleic acid to be delivered (former:latter) is preferably 10:1, more preferably 5:1. This further promotes electrostatic compounding between the nucleic acid and the block copolymer, and makes it easier to adjust the properties of the PIC, such as size.

[0031] The ratio of the number of hydrophobic amino acid residues (A) to the total number of amino acid residues contained in poly(amino acid) is, for example, 20% to 80%, preferably 30% to 70%, and more preferably 40% to 60%. The number of hydrophobic amino acid residues (A) contained in poly(amino acid) is 2 or more, and may be 5 to 25 or 8 to 12.

[0032] The ratio of the number of cationic amino acid residues to the number of hydrophobic amino acid residues (A) in poly(amino acids) (former:latter) is preferably 20:80 to 80:20, and can be, for example, 30:70 to 70:30 or 40:60 to 60:40.

[0033] When poly(amino acid) contains other amino acid residues (amino acid residues other than the cationic amino acid residue and the hydrophobic amino acid residue (A)), the ratio of the number of other amino acid residues to the total number of amino acid residues contained in poly(amino acid) is, for example, 25% or less, preferably 20% or less, and more preferably 10% or less. The number of other amino acid residues contained in poly(amino acid) may be, for example, 0, 1, 2, 3, 4, or 5.

[0034] If other hydrophobic amino acid residues other than hydrophobic amino acid residue (A) are included as other amino acid residues, the ratio of hydrophobic amino acid residue (A) to the total number of hydrophobic amino acids is preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, 80% or more, or 90% to 100%.

[0035] The above block copolymer can be obtained by linking a hydrophilic polymer segment and a poly(amino acid) segment via any suitable linking group. Any suitable linking group can be used as long as the effects of the present invention are obtained. The linking group may be, for example, a C1-C6 linear or branched alkylene group, an amide bond (-NHCO-), an ester bond (-COO-), an ether bond (-O-), an environmentally responsive bond (disulfide bond, boronic acid ester, etc.), a secondary or tertiary amino group, a carbonyl group, or any combination thereof.

[0036] A specific example of the above block copolymer is shown in formula (1) below. (In the formula, X independently represents a hydrophobic amino acid residue, Y independently represents a cationic amino acid residue, L represents a divalent linking group, R １ R represents a hydrogen atom or a linear or branched alkyl group having 1 to 12 carbon atoms, which may be substituted. ２When it is bonded to the N-terminus (—NH—) of the poly(amino acid) segment, it represents a hydrogen atom, a linear or branched alkyl group with 1 to 12 carbon atoms which may be substituted, or a linear or branched alkylcarbonyl group with 1 to 24 carbon atoms which may be substituted. When it is bonded to the C-terminus (—C(O)—) of the poly(amino acid) segment, it represents a hydroxyl group, an oxybenzyl group, an —O—R ２ａ group or an —NH—R ２ｂ group, where R ２ａ and R ２ｂ each independently represent a linear or branched alkyl group with 1 to 12 carbon atoms which may be substituted, l is an integer from 10 to 230, n is an integer from 1 to 60, m1 is 0 or 1, and m2 is 0 or 1).

[0037] In the above formula (1), X represents a hydrophobic amino acid residue (A). Two or more Xs may be the same amino acid residue (A) or different amino acid residues (A).

[0038] In the above formula (1), R １ , R ２ , R ２ａ , and R ２ｂ Examples of the linear or branched alkyl group with 1 to 12 carbon atoms defined by include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, a tert-butyl group, an n-hexyl group, a decyl group, and an undecyl group. Examples of the alkyl moiety with 13 or more carbon atoms include a tridecyl group, a tetradecyl group, a pentadecyl group, a nonadecyl group, a docosanyl group, and a tetracosyl group.

[0039] Regarding R １ , R ２ , R ２ａ , and R ２ｂ , examples of the substituent when substituted include a C1-6 alkoxy group (e.g., a methoxy group, an ethoxy group), an aryloxy group (e.g., a phenyloxy group, a tolyloxy group), an aryl C1-3 oxy group, a hydroxyl group, a C1-6 haloalkyl group (e.g., a trifluoromethyl group), and a halogen atom (e.g., chlorine and fluorine).

[0040] L is the linking portion between the hydrophilic polymer segment, the poly(ethylene glycol) segment, and the poly(amino acid) segment, and may be any suitable linking group. When L is bonded to the C-terminus (-C(O)-) of the poly(amino acid) segment, it may be, for example, -NH-, -O-, or -O-L. ａ -NH-, -CO-, -CH ２ -, and -OL-L ａ -S-L ａ -NH- (Here, L ａ Each of these can be a linking group selected independently from alkylene groups having 1 to 6 carbon atoms. Furthermore, when L is bonded to the N-terminus (-NH-) of a poly(amino acid) segment, it can be, for example, -OCO-L. ｂ -CO-, and -NHCO-L ｂ -CO- (here, L ｂ Each of these can be a linking group selected independently from alkylene groups having 1 to 6 carbon atoms.

[0041] l represents the number of repeating ethylene glycol units. l is preferably an integer between 10 and 230, more preferably between 20 and 170, and even more preferably between 45 and 90.

[0042] m1, m2, and n relate to the number of repeating amino acid residues, and 2×n + m1 + m2 represents the degree of polymerization of poly(amino acids). 2×n + m1 + m2 can be set according to the amount of negative charge of the nucleic acid to be delivered, for example, an integer from 4 to 60, preferably an integer from 10 to 40, and more preferably an integer from 10 to 30.

[0043] The above block copolymer can be prepared by any suitable method. For example, the block copolymer can be obtained by extending poly(amino acids) from the C-terminus to the N-terminus by peptide solid-phase synthesis using a carrier (e.g., polystyrene beads) having polyethylene glycol (PEG) chains linked via a linker and having amino groups at their free ends, followed by deprotection of the side chains and cleavage from the resin. Alternatively, the block copolymer can be obtained by synthesizing poly(amino acids) by peptide solid-phase synthesis and reacting the N-terminus or C-terminus of the obtained poly(amino acids) with terminally functionalized PEG.

[0044] In embodiments of the present invention, "block copolymer" includes those in the form of salts. Thus, in this specification, the term "block copolymer" includes pharmaceutically acceptable salts of block copolymers, such as hydrochloride, hydrobromide, maleate, mesylate, phosphate, sulfate, tartrate, acetate, lactate, trifluoroacetate, etc., unless explicitly stated otherwise, unless otherwise clearly inappropriate.

[0045] A-2. Nucleic acids Nucleic acids are typically oligonucleotides or polynucleotides, with nucleotides consisting of a purine or pyrimidine base, a pentose, and a phosphate group as the basic unit. Examples include oligo- or poly-double-stranded RNA, oligo- or poly-double-stranded DNA, oligo- or poly-single-stranded DNA, and oligo- or poly-single-stranded RNA. Oligo- or poly-double-stranded nucleic acids and oligo- or poly-single-stranded nucleic acids, in which RNA and DNA are mixed on a single strand, may also be used. The nucleotides contained in the nucleic acid may be of the natural type or chemically modified non-natural type. Preferably, groups or molecules such as amino groups, thiol groups, or fluorescent compounds may be attached to the nucleotides.

[0046] The base length of the nucleic acid (the length of the portion constituting the double helix in the case of double-stranded nucleic acid) is typically 50 bases or less, preferably 8 to 50 bases, more preferably 10 to 30 bases, and even more preferably 13 to 25 bases. If the base length of the nucleic acid is within the above range, the formation of PICs with an average particle size of 100 nm or more can be further promoted.

[0047] The hydrodynamic diameter of the nucleic acid is preferably 10 nm or less, more preferably 6 nm or less, and even more preferably 5 nm or less. If the hydrodynamic diameter of the nucleic acid is within the above range, the formation of PICs with an average particle size of 100 nm or more can be further promoted. The hydrodynamic diameter of the nucleic acid can be measured by the method described above.

[0048] Preferred examples of the above nucleic acids include antisense oligonucleotides (ASOs), CpG oligonucleotides, aptamers, siRNA, miRNA, and decoys. These nucleic acids can be designed with reference to a gene or protein that may be the target of treatment and used as nucleic acid drugs.

[0049] Preferably, single-stranded nucleic acids are used as the nucleic acids. The distance between charges in single-stranded nucleic acids approximates the distance between charges in the block copolymer, and the charge distribution can be matched. As a result, electrostatic compounding between the block copolymer and nucleic acids is promoted, and the particle size of the PIC can be adjusted more easily.

[0050] A-3. Method for preparing PIC The above PIC can be prepared by mixing the block copolymer and the nucleic acid in a buffered aqueous solution as needed to achieve a desired N / P ratio.

[0051] The N / P ratio is preferably 1 or higher, more preferably 2 or higher, and even more preferably 3 or higher. The upper limit of the N / P ratio can be, for example, 200 or less. The N / P ratio refers to the ratio of the molar concentration (N) of cationic groups (typically amino groups) derived from the side chains of the protonable block copolymer to the molar concentration (P) of anionic groups (typically phosphate groups) derived from nucleic acids at the pH of the above aqueous solution (for example, 7.4).

[0052] B. Pharmaceutical Compositions The pharmaceutical compositions according to embodiments of the present invention include the polyion complex described in Section A, and may further include any suitable additives as needed. The type and amount of additives can be appropriately determined by those skilled in the art depending on the purpose. Specific examples of additives include excipients, isotonic agents, pH adjusters, buffers, stabilizers, and the like.

[0053] In one embodiment, the pharmaceutical composition is an injectable preparation. The pharmaceutical composition as an injectable preparation may be a powder formulation, a lyophilized formulation, or a liquid formulation. The liquid formulation contains an aqueous medium, and typically, the block copolymer and nucleic acid form a PIC in the aqueous medium. On the other hand, the powder formulation and the lyophilized formulation are dissolved in an aqueous medium such as water for injection before use. The block copolymer and nucleic acid can form a PIC in the aqueous solution obtained by dissolution.

[0054] Diseases that can be treated with the above-mentioned pharmaceutical composition include, for example, cancer. The cancer may be metastatic cancer (e.g., metastatic lung cancer, metastatic breast cancer, metastatic melanoma, metastatic colon cancer, etc.). According to the pharmaceutical composition of the embodiment of the present invention, since PIC can also be delivered to the spleen where platelets are stored, a synergistic effect with antitumor immunity via the activation of splenic immune cells can be expected. Furthermore, since platelets can be therapeutically regulated, it can also be applied to fibrous diseases (e.g., hepatic fibrosis, cardiac fibrosis, pulmonary fibrosis, etc.), atherosclerosis, rheumatoid arthritis, etc.

[0055] The above-mentioned pharmaceutical composition can be administered via vein, abdominal cavity, intradermal, subcutaneous, or intramuscular routes. Intravenous injection is preferred. The target population is typically humans or other mammals (mice, rats, rabbits, monkeys, dogs, horses, etc.).

[0056] C. Method of Use The PIC of the embodiments of the present invention can be used for any suitable application. For example, the PIC can be administered with any suitable drug to treat a disease. Examples of diseases include those listed above. The PIC of the embodiments of the present invention can also be used in the preparation of pharmaceutical compositions. Examples of pharmaceutical compositions include those listed above.

[0057] The present invention will be specifically described below with reference to examples, but the present invention is not limited in any way to these examples.

[0058] <Materials> TentaGel® PAP resin (number average molecular weight (Mn): 3kDa and 10kDa) and TentaGel® RAM resin were purchased from Rapp Polymere GmbH (Tübingen, Germany). Benzotriazole-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole hydrate (HOBt), and 9-fluorenylmethyloxycarbonyl (Fmoc) protected amino acids were purchased from Watanabe Chemical Industry Co., Ltd. N-methyl-2-pyrrolidone (NMP), trifluoroacetic acid (TFA), piperidine, dichloromethane (DCM), acetic anhydride, triisopropylsilane (TIPS), sucrose, triethylamine (TEA), succinic anhydride, pyridine, 4-dimethylaminopyridine (DMAP), di-tert-butyl decarbonate (Boc ２0), and warfarin were purchased from Fujifilm Wako Pure Chemical Corporation. Doxorubicin hydrochloride (Dox HCl) was purchased from BLDpharm Inc. Sterilized HEPES (1M, pH 7.3) was purchased from Amresco. Chemically modified antisense nucleic acids (ASOs) were synthesized by Gene Design, Inc. All nucleotides in the ASOs were synthesized to have a phosphorothioate backbone. The base sequences of the ASOs are as follows: TGF-β1 targeted ASO: 5'-TCGatg5mcgcttCCG-3' (SEQ ID NO: 1). STAT-3 (signal transduction and transcription activator 3) targeted ASO: 5'-GAAAattcattcttCCA-3' (SEQ ID NO: 2). GL3-targeted ASO: 5'-TCGAagtactcagcgtaaGTT-3' (SEQ ID NO: 3). Uppercase and lowercase letters represent locked nuclear acid (LNA) (C indicates LNA 5-methylcytosine) and DNA (5mc indicates 5-methylcytosine), respectively. Fluorescently labeled ASO was obtained by conjugating Alexa Fluor® 647 dye to the 5' end of the TGF-β1-targeted ASO. Luciferase-expressing mouse epithelial breast cancer cells "4T1-Luc" were purchased from the Japan Bioresource Research Collection Cell Bank and cultured in RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. BALB / c mice (female, 6 weeks old) were purchased from Oriental Yeast Co., Ltd. All animal experiments were conducted in accordance with the guidelines for the breeding and use of experimental animals established by the University of Tokyo and the Nano Medical Innovation Center of the Kawasaki City Industrial Promotion Foundation.

[0059] <Statistical Analysis> Statistical analysis was performed using the Microsoft Excel add-in "Real Statistics Resource Pack" with either a one-way ANOVA using Tukey's post-hoc test or a two-way ANOVA. Unless otherwise specified, data are presented as mean ± standard deviation. Significant statistical differences are indicated by *p < 0.05.

[0060] [Experimental Example 1] Synthesis of Block Copolymers Having 3kDa PEG Segments or 10kDa PEG Segments Block copolymers having 3kDa PEG segments or 10kDa PEG segments were synthesized by standard Fmoc solid-phase peptide synthesis using a multi-solid-phase synthesizer (Kokusan Kagaku Co., Ltd., "KMS-3"). Specifically, the procedure is as follows. Throughout the entire synthesis process, NMP was used as the coupling and washing solvent. The first amino acid was introduced to the PEG chain with terminal amino groups on the TentaGel PAP resin by deprotecting the Fmoc group by two 10-minute treatments with 20% piperidine in NMP. Five equivalents of Fmoc-Lys(Boc)-OH were activated with five equivalents of PyBOP / HOBt and ten equivalents of TEA, and reacted overnight with the free N-terminal amino acid in the resin. Next, at each coupling step, 5 equivalents of Fmoc amino acids were activated with 5 equivalents of PyBOP / HOBt and 10 equivalents of TEA, and reacted with the free N-terminal amino acids in the resin for 1 hour. After each coupling step, the Fmoc groups were removed by two 10-minute treatments with 20% piperidine. After the final deprotection step, the free N-terminus was acetylated by applying acetic anhydride / NMP / TEA (1 / 3 / 2) for 35 minutes. The PEG polymer was cleaved from the resin using TFA / water / TIPS (95 / 2.5 / 2.5) to remove the side-chain protecting groups. The polymer was purified six times by dialysis with deionized water, followed by lyophilization to recover the product as a white powder. In this manner, block copolymers were obtained in which Lys and Gly were arranged in this order 10 times in a 3k or 10k PEG from the PEG terminal side ((GlyLys)10-3k and (GlyLys)10-10k), block copolymers in which Lys and Cha were arranged in this order 10 times in a 3k or 10k PEG from the PEG terminal side ((ChaLys)10-3k and (ChaLys)10-10k), and block copolymers in which Lys and Nle were arranged in this order 10 times in a 3k or 10k PEG from the PEG terminal side ((NleLys)10-3k and (NleLys)10-10k). The degree of polymerization of the poly(amino acid) in each block copolymer is 20.The characteristics of the product were determined by MALDI-TOF-MS (ultraflextreme; Bruker Daltonics) in linear positive ion mode (range of 2 kDa to 20 kDa) using an α-cyano-4-hydroxycinnamic acid matrix. Figure 1 shows the MALDI-TOF-MS spectral data for (GlyLys) 10⁻³k, (GlyLys) 10⁻¹⁰k, (ChaLys) 10⁻³k, (ChaLys) 10⁻¹⁰k, (NleLys) 10⁻³k, and (NleLys) 10⁻¹⁰k.

[0061] [Experimental Example 2] Synthesis of poly(amino acids) Poly(amino acids) were synthesized by standard Fmoc solid-phase peptide synthesis using a multi-solid-phase synthesizer (Kokusan Kagaku Co., Ltd., "KMS-3"). Specifically, the procedure was as follows. Throughout the entire synthesis process, NMP was used as the coupling and washing solvent. The first amino acid was introduced onto the Tentagel RAM resin by deprotecting the Fmoc group with two 10-minute treatments of 20% piperidine in NMP. Five equivalents of Fmoc-Lys(Boc)-OH were activated with five equivalents of PyBOP / HOBt and ten equivalents of TEA, and reacted with the free N-terminal amino acid in the resin overnight. Next, at each coupling step, five equivalents of Fmoc amino acids were activated with five equivalents of PyBOP / HOBt and ten equivalents of TEA, and reacted with the free N-terminal amino acid in the resin for one hour. After each coupling step, the Fmoc group was removed by two 10-minute treatments with 20% piperidine. After the final deprotection step, the free N-terminus was acetylated by applying acetic anhydride / NMP / TEA (1 / 3 / 2) for 35 minutes. Poly(amino acids) was cleaved from the resin using TFA / water / TIPS (95 / 2.5 / 2.5) to remove the side chain protecting groups. Poly(amino acids) was purified six times by dialysis with deionized water, followed by lyophilization to recover the product as a white powder. The product was characterized by ESI-MS (micro-TOF-Q (micrOTOF-Q); Bruker Daltonics). In this manner, poly(amino acids) ((GlyLys)10-0k) was obtained in which Lys and Gly were arranged in this order 10 times, poly(amino acids) ((ChaLys)10-0k) in which Lys and Cha were arranged in this order 10 times, and poly(amino acids) ((NleLys)10-0k) in which Lys and Nle were arranged in this order 10 times. The degree of polymerization of each poly(amino acid) was 20, the N-terminus was acetylated, and the C-terminus was amidated.

[0062] [Experimental Example 3] Synthetic fragment condensation method for block copolymers having 20 kDa PEG segments or 40 kDa PEG segments was used to synthesize 20 kDa and 40 kDa PEG polymers. Cationic amino acid sequences were synthesized on preloaded H-Lys(Boc)-2-ClTrt resin using the standard Fmoc solid-phase synthesis method described above. Side-chain protected cationic fragments were treated with hexafluoro-2-propanol (HFIP) / DCM(1 / 3) for 40 minutes and cleaved from the resin. Next, 5 equivalents of the side-chain protected cationic fragments were activated with 5 equivalents of HBTU / HOBt and 10 equivalents of TEA, and reacted overnight with 1 equivalent of adenosine 5-methyl phosphate (MEPA). Next, the side-chain protecting groups were removed using TFA / water / TIPS(95 / 2.5 / 2.5). Next, the polymer was dialyzed six times with deionized water, and then freeze-dried to recover the product as a white powder. In this manner, block copolymers were obtained in which Lys and Gly are arranged in this order 10 times from the PEG terminal side of a 20 kDa or 40 kDa PEG ((GlyLys) 10-20 k, and (GlyLys) 10-40 k); in which Lys and Cha are arranged in this order 10 times from the PEG terminal side of a 20 kDa or 40 kDa PEG ((ChaLys) 10-20 k, and (ChaLys) 10-40 k); and in which Lys and Nle are arranged in this order 10 times from the PEG terminal side of a 20 kDa or 40 kDa PEG ((NleLys) 10-20 k, and (NleLys) 10-40 k). The degree of polymerization of the poly(amino acid) in each block copolymer is 20.

[0063] [Experimental Example 4] Preparation of PIC The ASO described above and the polymers (block copolymers or poly(amino acids)) obtained in Experimental Examples 1, 2, or 3 were separately dissolved in 10 mM HEPES / 10% sucrose buffer (pH 7.4) at concentrations of 25 μM and 175 μM, respectively. These solutions were mixed by pipetting so that the amines of the polymers and the thiophosphate in the ASO had an N / P ratio of 5 (final ASO concentration: 12.5 μM) to obtain the PIC solution. The PIC solution was then allowed to stand at room temperature for 20 minutes, freeze-dried, and stored at 4°C. Before use, the freeze-dried powder was mixed with Milli-Q water to prepare the PIC solution again, allowed to stand at room temperature for 10 minutes, and used in the following experiments.

[0064] <Fluorescence Correlation Spectroscopy (FCS)> The size distribution of PIC was measured using FCS with the "MF20" intermolecular interaction analysis system (Olympus Corporation) equipped with a 633 nm He-Ne laser. The PIC solution was diluted to 25 nM with 10 mM HEPES / 5% glucose. Diffusion time was measured at room temperature with a sampling time of 120 seconds. The autocorrelation curve was fitted using QuickFit 3.0 with triplet three-dimensional diffusion, as shown below. (In the formula, τ represents relaxation time, τ Ｄｉ α represents the diffusion time of the sample component, γ represents the aspect ratio of the focal volume, N represents the number of discretization steps to extract the maximum entropy distribution (MEM), and α i (where represents the relative amplitude of the components). The MEM method aimed to maximize the Shannon-Janes entropy (S), which is defined as follows: (In the formula, ρ ｊ (This represents the probability of detecting a component in the confocal region.) The diffusion coefficient D can be obtained from the following equation. (In the formula, W ｘｙ (This represents the width of the focal region.) ｘｙ This is an Atto-655 carboxylic acid solution (D = 426 μm at 25°C). ２ s －１ Calibrated with W xyThe size was less than 500 nm. The size distribution (hydrodynamic diameter, dh) was calculated using the Stokes-Einstein equations, as shown in the following formula. (In the formula, k Ｂ (where θ represents the Boltzmann constant, T represents temperature, and η represents solvent viscosity).

[0065] Figure 2 shows the size distribution of PICs prepared using (GlyLys) 10-0k, (ChaLys) 10-0k, or (NleLys) 10-0k. Figure 3 shows the size distribution of PICs prepared using (GlyLys) 10-3k, (ChaLys) 10-3k, or (NleLys) 10-3k. Figure 4 shows the size distribution of PICs prepared using (GlyLys) 10-10k, (ChaLys) 10-10k, or (NleLys) 10-10k. Table 1 shows the average particle size of each PIC. The average particle size of PICs using (ChaLys) 10-10k and (ChaLys) 10-3k, which have Cha residues with a CLogP value of 3 or higher, was 100 nm or larger. Note: Chemdraw ver. Using version 22.0.0 and the algorithm provided by BioByte (http: / / www.biobyte.com / ), the CLogP values ​​for the side chain of the hydrophobic amino acid residue Cha were calculated to be 3.9, and the CLogP value for the side chain of Nle was 2.8.

[0066] <Negative Staining Transmission Electron Microscopy Observation> The re-prepared PIC solution was diluted 10-fold with 10 mM HEPES / 10% sucrose buffer (pH 7.4). The 400-mesh copper grid was glow-discharged for 3 minutes using a PIB-10 ion bombarder (Vacuum Device Inc.) before use. After standing for 20 seconds, the sample solution was removed from the grid and completely dried. 2 μL of 2% uranylacetic acid was pipetteed onto the grid and stood for 1 minute. The staining solution was then wiped off with filter paper and imaged using a transmission electron microscope (JEOL, "JEM-1400", accelerating voltage 120 kV).

[0067] Figure 5 shows transmission electron microscope images of PICs prepared using (GlyLys) 10⁻³k, (ChaLys) 10⁻³k, or (NleLys) 10⁻³k. It was confirmed that the PICs prepared using (ChaLys) 10⁻³k had larger particle sizes than those prepared using (GlyLys) 10⁻³k or (NleLys) 10⁻³k.

[0068] <In Vivo Confocal Laser Scanning Microscopy> A mouse (n=1) was intravenously injected with 200 μL of a PIC or ASO sample in 10 mM HEPES / 10% sucrose (26 μg Alexa647-ASO per mouse). The skin of the mouse's earlobe was continuously observed for 6 hours using an in vivo confocal laser scanning microscope (IVCLSM) (A1R confocal LSM, Nikon). To analyze the IVCLSM results, an ROI was placed in the interstitial region, and the average fluorescence intensity within the ROI was measured 6 hours after injection. The intensity values ​​were normalized to the maximum intensity in the vascular system immediately after injection.

[0069] Figures 6-9 show in vivo confocal laser scanning microscope images of PIC and ASO prepared with (GlyLys) 10⁻³k, (ChaLys) 10⁻³k, or (NleLys) 10⁻³k at 0 minutes, approximately 60 minutes, approximately 100 minutes, and approximately 150 minutes after injection. Figure 10 shows a graph of the relative fluorescence intensity of PIC and ASO prepared with (GlyLys) 10⁻³k, (ChaLys) 10⁻³k, or (NleLys) 10⁻³k over time. PIC prepared with (ChaLys) 10⁻³k showed similar fluorescence intensity to ASO from 0 to 100 minutes, after which the fluorescence intensity decreased significantly. This was presumed to be because PIC moved from the bloodstream to the extravascular space due to interaction with platelets, as will be discussed later.

[0070] [Experimental Example 5] 4T1-Luc lung metastasis model BALB / c mice (female, 6 weeks old) were given 2 x 10⁻¹⁴ ５4T1-Luc cells / 100 μL of PBS were administered intravenously. On day 7 post-transplant, the establishment of lung metastatic breast cancer was confirmed using an in vivo imaging system (IVIS, PerkinElmer) that acquired bioluminescence images 12 minutes after intraperitoneal injection of 200 μL of D-luciferin. The bioluminescence signal intensity in the lungs was 10 ５ Photons / second ~10 ６ When the photon / second level reached a certain level, the PIC solution was administered intravenously.

[0071] <Intravenous Injection and In vivo Distribution> Three mice (n=3) were intravenously injected with 20 μL of a PIC or ASO sample in 10 mM HEPES / 10% sucrose (13 μg, Alexa647-ASO / mouse). For polymer samples in PBS, the fluorescence signal was quantified based on the absorbance at 680 nm (bandwidth 10 nm, absorbance = 2.0) using a NanoDrop One microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific Life Technologies Japan). 200 μL of each sample was injected and quantified. Mice were sacrificed 24 hours after administration, and individual organs, including the lungs, spleen, kidneys, and liver, as well as subcutaneous tumors for PIC solution, were excised. The fluorescence signals of Alexa 647-ASO or Alexa 680-polymer within the tissue were measured using an IVIS Spectrum optical imager.

[0072] Figures 11 and 12 show in vivo imaging system images of the mouse lungs, spleen, kidneys, and liver (from left to right), and Figure 13 shows graphs of radioactivity efficiency indicating the accumulation of nucleic acids in the mouse lungs and spleen. Statistical analysis was performed using one-way analysis of variance (ANOVA). In Figure 13, ***** represents p < 0.00001, **** represents p < 0.0001, *** represents p < 0.001, ** represents p < 0.01, and * represents p < 0.05. PIC prepared using a polymer containing (ChaLys)10 was confirmed to accumulate in large quantities in the lungs and spleen where breast cancer had metastasized (Figures 11 and 12). This trend was even more pronounced in 3k and 10k PEG chains. Table 2 compares the accumulation levels in the lungs and spleen of (GlyLys)10, (ChaLys)10, and (NleLys)10 for each PEG chain of 0k, 3k, and 10k. In the lungs and spleen, (ChaLys)10 showed significantly higher accumulation than (GlyLys)10 and (ChaLys)10 in all PEG chains. Furthermore, the accumulation levels in the lungs and spleen of (ChaLys)10-0k, (ChaLys)10-3k, and (ChaLys)10-10k and ASO were compared (Table 3). (ChaLys)10-3k and (ChaLys)10-10k showed significantly higher accumulation levels in the lungs and spleen than ASO. The accumulation levels in the lungs and spleen of (ChaLys) 10-0k, (ChaLys) 10-3k, (ChaLys) 10-10k, (ChaLys) 10-20k, and (ChaLys) 10-40k were compared with those of ASO (Table 4). (ChaLys) 10-3k and (ChaLys) 10-10k showed significantly higher accumulation levels in the lungs and spleen than (ChaLys) 10-20k, (ChaLys) 10-40k, and ASO.

[0073] The procedure was the same as described above, except that 200 μg of intraperitoneal warfarin prophylactically was administered to each mouse one hour before intravenous administration of the sample. Samples of PIC or ASO prepared with (ChaLys) 10-3k were intravenously injected into mice (n=4), and their in vivo distribution was examined. Figure 14 shows in vivo imaging system images of the lungs, spleen, kidneys, and liver (from left to right) of mice that received prophylactic warfarin, and Figure 15 shows graphs of radioactivity showing the amount of nucleic acid accumulation in the lungs and spleen of mice that received prophylactic warfarin. As a result, it was confirmed that the amount of nucleic acid accumulation in the lungs and spleen with PIC administration was significantly higher than with ASO administration alone (Figure 15). This indicates that prophylactic warfarin administration, which is performed on patients undergoing chemotherapy or after tumor resection surgery, does not significantly affect the delivery effect of PIC.

[0074] <In vivo gene knockdown assay> Mice (PIC and ASO administered: n=4, control: n=3) were intravenously injected with 200 μL of PIC or ASO sample in 10 mM HEPES / 10% sucrose (single dose of 12 μg TGF-β1 ASO per mouse). Mice were sacrificed 48 hours after administration, and their lungs and spleens were removed and immersed overnight in 500 μL of RNAeasy solution (Qiagen) at 4°C. The tissues were then homogenized, and RNA was extracted using Maxwell® RSC simply RNA Tissue Kit (Promega). cDNA was prepared from RNA from which genomic DNA had been removed using ReverTra Ace (Toyobo), and qRT-PCR was performed using FastStart Universal SYBR-Green Master (Roche) and QuantStudio 7 Flex System (Applied Biosystems). The relative expression level of TGF-β1 was determined using the endogenous housekeeping gene GAPDH. Primers were purchased from Hokkaido System Science. The mouse TGF-β1 primer sequences are as follows: 5'-CTCCCCGTGGCTTCTAGTGC-3' (forward primer) (SEQ ID NO: 4) 5'-GCCTTAGTTTGGGACAGGATCTG-3' (reverse primer) (SEQ ID NO: 5) The results are shown in Figure 15.

[0075] As shown in Figure 16, TGF-β1 expression in the lungs and spleen was significantly suppressed in both cases: when PIC using (ChaLys)10-3k was administered, and when ASO was administered alone. Furthermore, TGF-β1 expression was significantly suppressed when PIC was administered compared to when ASO was administered alone.

[0076] [Experimental Example 6] A mouse (n=1) administered diblock copolymer was intravenously injected with 200 μL of a PIC sample prepared using diblock copolymer of Cha10Lys10PEG69 in 10 mM HEPES / 10% sucrose (NP=5, 26 μg Alexa647-ASO per mouse). The skin of the mouse's earlobe was continuously observed for 3 hours using an in vivo confocal laser scanning microscope (IVCLSM) (A1R confocal LSM, Nikon). The mouse developed respiratory distress and died 3 hours after administration. Lethality was thought to have been induced by the surfactant properties of the diblock copolymer.

[0077] [Experimental Example 7] Evaluation of interaction between platelets and PIC Ten minutes before PIC administration, a mouse (n=1) was intravenously injected with DyLight 488-conjugated anti-GPIbβ antibody (X488, EMFRET Analytics) to label platelets in vivo. Next, the mouse was intravenously injected with 200 μL of a PIC sample (NP=5, 13 μg Alexa647-ASO per mouse) prepared using (ChaLys) 10⁻³ kD in 10 mM HEPES / 10% sucrose. The skin of the mouse's earlobe was continuously observed for 30 minutes using an in vivo confocal laser scanning microscope (IVCLSM) (A1R confocal LSM, Nikon). To analyze the interaction between platelets and polyplexes, the colocalization of DyLight488 and Alexa647 was evaluated using Pearson's correlation coefficient with respect to time. In vivo imaging system images taken at 0, 2, 5, 10, 20, and 30 minutes after injection are shown in Figure 17. A graph showing the relationship between the Pearson correlation coefficient between platelets (DyLight488) and PIC (Alexa647) and time is also shown in Figure 17. PIC began to accumulate from 2 minutes after administration, and the fluorescence of both platelets and PIC decreased after 20 minutes (Figure 17). Furthermore, the Pearson correlation coefficient showed high values ​​from 3 to 10 minutes after administration, suggesting that platelets and PIC colocalized. After 10 minutes after administration, the Pearson correlation coefficient decreased. These results suggest that platelets and PIC interact and exhibit similar behavior in the blood.

[0078] [Experimental Example 8] Double breast cancer 4T1 cell model Six-week-old female BALB / c mice were anesthetized with isoflurane. Subcutaneous transplantation (3 x 10) ６ (100 μL of cells suspended in PBS), and intravenous administration (2 x 10⁶ 4T1-Luc cells) ５ The procedure (suspending each individual in 100 μL of PBS) was performed simultaneously. Subcutaneous transplantation was performed in the right lower abdomen of mice. The tumor volume was 30 mm. ３ 100mm ３ When the tumor volume reached a certain point, the polymer sample was administered intravenously. Tumor volume (mm) ３ The in vivo distribution was calculated using the following formula. In the formula, L represents the longest diameter of the tumor (mm), and W represents the shortest diameter perpendicular to L (mm). This formula was used to approximate the tumor as an ellipsoid, ensuring consistency in volume estimation across all groups.

[0079] <Intravenous Injection and Biodistribution> The polymer used in PIC was labeled overnight with Alexa 680 NHS ester (InvivoGen, USA) in a 100 mM sodium bicarbonate solution. For the polymer samples in PBS, the fluorescence signal was quantified based on the absorbance at 680 nm (bandwidth 10 nm, absorbance = 2.0) using a NanoDrop One microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific Life Technologies Japan). 200 μL of each sample was injected into mice (n=3) and quantified. The mice were sacrificed 8 days after administration, and individual organs, including the heart, lungs, spleen, kidneys, and liver, as well as subcutaneous tumors in the PIC solution, were excised. The fluorescence signal of Alexa 680 polymer in the tissue was measured using an IVIS Spectrum optical imager. Figures 19 and 20 show in vivo imaging system images of mouse subcutaneous tumors, heart, lungs, spleen, kidneys, and liver (from left to right). Figure 21 shows the radioactivity efficiency graphs indicating nucleic acid accumulation in mouse kidneys and livers, Figure 22 shows the radioactivity efficiency graphs indicating nucleic acid accumulation in mouse subcutaneous tumors, and Figure 23 shows the radioactivity efficiency graphs indicating nucleic acid accumulation in mouse lungs and spleen. In Figures 21 to 23, ***** represents p < 0.00001, **** represents p < 0.0001, *** represents p < 0.001, ** represents p < 0.01, and * represents p < 0.05. The radioactivity efficiency in the kidneys and liver, hydrophobicity (CLogP value of hydrophobic amino acid residues), and PEG chain length were analyzed using two-way ANOVA. In the kidneys, hydrophobic η ２ 0.97, η of the PEG chain length ２ The effect size was 0.94, and both showed large effect sizes. Furthermore, a comparison was made using one-way ANOVA for hydrophobic (polyamino acid residues) or PEG chains that showed large effect sizes (Table 5). In the kidney, the accumulation of 0 kDaPEG- (ChaLys) and 40 kDaPEG- (ChaLys) was the lowest. In the liver, hydrophobic η ２ 0.76, η of the PEG chain length ２The value was 0.80. Furthermore, using one-way ANOVA, the accumulation levels were compared based on hydrophobicity (polyamino acid residues) or PEG chains, which had large effect sizes (Table 6). In the liver, the accumulation level of 40 kDaPEG-(ChaLys) was the lowest.

[0080] In tumors, hydrophobic η ２ 0.18, η of the PEG chain length ２ The value was 0.59. In addition, one-way analysis of variance (ANOVA) was used to compare the accumulation levels for PEG chain lengths that showed large effect sizes (Table 7). In tumors, the accumulation level of 40 kDaPEG-(ChaLys) was the highest.

[0081] In the lungs, hydrophobic η ２ 0.82, η of the PEG chain length ２ The value was 0.86. In addition, one-way analysis of variance (ANOVA) was used to compare the accumulation amounts based on polyamino acid residues or PEG chains (Table 8). In the spleen, hydrophobic η ２ 0.86, η of the PEG chain length ２ The value was 0.77. In addition, one-way analysis of variance (ANOVA) was used to compare the accumulation amounts based on polyamino acid residues or PEG chains (Table 9).

[0082] <Evaluation of therapeutic effect> 4T1-Luc cells (2 × 10⁻¹⁶) were administered to 6-week-old female BALB / c mice (n=4). ５ The drug was suspended in 100 μL of PBS and administered intravenously. Seven days after transplantation, the establishment of metastatic breast cancer in the lungs was confirmed using an in vivo imaging system (IVIS, PerkinElmer). Specifically, 200 μL of D-luciferin was administered intraperitoneally, and the presence of metastatic breast cancer was confirmed by acquiring bioluminescence images 12 minutes later. The bioluminescence intensity of the lungs was 10 ５ Photons / second ~10 ６When the photon count reached a certain level (photons / second), the PIC solution was administered intravenously. The PIC solution was prepared using the following procedure. First, doxorubicin hydrochloride, (GlyLys) 10-10k, and TGF-β1 ASO, GL3 ASO, or STAT3 ASO were added and mixed to obtain the PIC solution. Each PIC solution was prepared to contain approximately 10 μg of doxorubicin (Dox), approximately 12 μg of TGF-β1 ASO, approximately 13 μg of GL3 ASO, and approximately 13 μg of STAT3 ASO. Specifically, the ASO and doxorubicin (Dox) polymer were dissolved in 10 mM HEPES / 10% sucrose buffer (pH 7.4) to a concentration of 25 μM for ASO and 175 μM for Dox polymer. The solution of Dox and (GlyLys) 10-10k was reacted at room temperature for 20 minutes, then lyophilized and stored at 4°C. Before use, the lyophilized powder was resuspended in Milli-Q water and reacted at room temperature for 10 minutes. Each PIC solution was administered intravenously on days 7, 9, and 11 after transplantation. For comparison, mice administered a buffer (10 mM HEPES / 10% sucrose buffer (pH 7.4)) instead of the PIC solution were also evaluated in the same manner. Mice were euthanized when a 20% decrease in body weight from the start of treatment was observed. Survival rates (survival rate (%) = total number of euthanized mice / total number of mice used in the experiment × 100) were calculated from the mice used in the experiment and the mice that were euthanized. A graph of the change in survival rate is shown in Figure 24, and a graph of the median survival time is shown in Figure 25. The survival rates of mice administered with the buffer and mice administered with Dox-TGF-β1 were the same (Figure 24). Furthermore, mice administered with Dox-STAT3 had the highest survival rate, and there was a significant difference in median survival time compared to mice administered with the buffer or Dox-TGF-β1 (p < 0.05).

[0083] The PIC of the present invention can be suitably used in the fields of pharmaceutical compositions utilizing nucleic acid drugs and pharmaceutical compositions utilizing polymer-drug conjugates that do not use nucleic acid drugs.

Claims

1. A polyion complex comprising: a block copolymer having a hydrophilic polymer segment and a poly(amino acid) segment; a nucleic acid; wherein the poly(amino acid) segment comprises two or more cationic amino acid residues and two or more hydrophobic amino acid residues, the hydrophobic amino acid residues are arranged alternately and / or randomly, the hydrophobic amino acid residues include hydrophobic amino acid residues having a CLogP value of 3 or more in the (A) side chain, and the average particle size is 100 nm or more.

2. The polyion complex according to claim 1, wherein the hydrophilic polymer segment comprises at least one hydrophilic polymer selected from the group consisting of poly(ethylene glycol), poly(saccharide), poly(vinylpyrrolidone), poly(vinyl alcohol), poly(acrylamide), poly(acrylic acid), poly(methacrylamide), poly(methacrylic acid), poly(methacrylic acid ester), poly(acrylic acid ester), poly(amino acid), poly(malic acid), and derivatives thereof.

3. The polyion complex according to claim 1, wherein the molecular weight of the hydrophilic polymer is 80 kDa or less.

4. The polyion complex according to claim 1, wherein the difference between the proportion of cationic amino acid residues located at odd positions of the poly(amino acid) segment and the proportion of cationic amino acid residues located at even positions of the poly(amino acid) segment is 40% or more.

5. The polyion complex according to claim 4, wherein 80% or more of the cationic amino acid residues are arranged every other amino acid residue.

6. The polyion complex according to claim 1, wherein the cationic amino acid residue is at least one selected from the group consisting of lysine residue, arginine residue, ornithine residue, histidine residue, homolysine residue, 2,4-diaminobutyric acid residue, 2,3-diaminopropionic acid residue, 2-aminoglycine residue, homoarginine residue, 2-amino-3-guanidinopropionic acid residue, and 2-amino-4-guanidinobutyric acid residue.

7. The polyion complex according to claim 1, wherein the hydrophobic amino acid residue (A) has an alicyclic hydrocarbon group in its side chain.

8. The polyion complex according to claim 1, wherein the hydrophobic amino acid residue (A) is at least one selected from the group consisting of cyclohexylalanine residue, cyclopentylalanine residue, cyclohexylglycine residue, cyclopropylalanine residue, homonorleucine residue, 2-aminooctanoic acid residue, and 2-aminoundecanoic acid residue.

9. The polyion complex according to claim 1, wherein the nucleic acid is a single-stranded nucleic acid.

10. The polyion complex according to claim 1, wherein the hydrodynamic diameter of the nucleic acid is 10 nm or less.

11. The polyion complex according to claim 1, wherein the base length of the nucleic acid is 50 bases or less.

12. A pharmaceutical composition comprising the polyion complex described in any one of claims 1 to 11.

13. The pharmaceutical composition according to claim 12, used for the treatment of metastatic cancer.

14. The pharmaceutical composition according to claim 13, used for the treatment of metastatic lung cancer.

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