WO2012124688A1 - 肺送達のためのベクター、導入剤及び使用 - Google Patents
肺送達のためのベクター、導入剤及び使用 Download PDFInfo
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- WO2012124688A1 WO2012124688A1 PCT/JP2012/056406 JP2012056406W WO2012124688A1 WO 2012124688 A1 WO2012124688 A1 WO 2012124688A1 JP 2012056406 W JP2012056406 W JP 2012056406W WO 2012124688 A1 WO2012124688 A1 WO 2012124688A1
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- gala
- lipid
- chol
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- C12N2320/00—Applications; Uses
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Definitions
- the present invention relates to vectors, transduction agents and uses for pulmonary delivery.
- This application claims the benefit of priority of Japanese Patent Application No. 2011-55765 filed on March 14, 2011, the entirety of which is incorporated herein by reference. is there.
- nucleic acid drugs As a vector for delivering target substances such as low molecular weight drugs, nucleic acid drugs, antibody drugs, peptides, proteins, and sugars to target sites, liposomes with functional molecules introduced on the outer surface of the liposome membrane have been actively developed. Has been done.
- nucleic acid drugs include antisense, ribozyme, aptamer, decoy oligo, and siRNA.
- siRNA is particularly attracting attention, and clinical trials have been conducted by several pharmaceutical companies and venture companies in the United States and Europe.
- a liposome having a hydrophilic polymer for example, polyalkylene glycol such as polyethylene glycol
- a hydrophilic polymer for example, polyalkylene glycol such as polyethylene glycol
- the directivity of the liposome with respect to tumor cells can be improved by improving the blood retention of the liposome.
- a multifunctional envelope-type nanostructure MEND: Multifunctional envelope-type nano device; hereinafter, may be abbreviated as “MEND” in the present specification
- MEND Multifunctional envelope-type nano device
- Liposomes in which GALA is introduced on the outer surface of the liposome membrane using cholesterol bound to GALA have been developed (see Non-Patent Document 1). Liposomes become encapsulated in endosomes when endocytosed, and the liposomes in endosomes are degraded by the fusion of endosomes with lysosomes. According to these liposomes, the liposome-encapsulated substance escapes from endosomes, resulting in cytoplasm. Can be released into.
- GALA is an oligopeptide composed of 30 amino acid residues based on a repeating sequence of glutamic acid, alanine, leucine, and alanine (EALA), and is synthesized by a group such as Szoka (see Non-Patent Document 2).
- EALA glutamic acid
- Szoka a group such as Szoka
- GALA has a random coil structure due to the electric repulsion of glutamic acid under neutral pH conditions, but the ⁇ -helix has a high affinity for lipid membranes by eliminating the electric repulsion under acidic conditions. It is known to take a structure (see Non-Patent Document 2).
- GALA has so far caused membrane fusion between endosomal membranes and MEND lipid membranes under acidic conditions in endosomes by modifying GALA (Chol-GALA) bound to cholesterol (Chol) on the MEND surface. It has been used as a pH-responsive endosome escape promoting element that improves the activity of MEND by releasing an encapsulated substance into the cytoplasm (see Patent Document 3).
- GALA has been used as a functional element for the purpose of improving intracellular kinetics, but has not been known as a lung migratory element.
- lung is mentioned as one of the target organs of the liposome modified with a certain kind of endosome-soluble peptide (see Patent Document 4)
- the composition of the peptide described here is glutamic acid, alanine, Based on the repeating sequence of leucine and alanine (EALA), the composition of the GALA peptide according to the present application is greatly different, and further, the description that the liposome modified with the peptide specifically migrates to the lung is There is no description suggesting this.
- An object of the present invention is to provide a technique for specifically delivering a target substance to the lung.
- Another object of the present invention is to provide a vector which does not aggregate or hardly aggregates after storage for a certain period of time and / or has excellent storage stability.
- the present inventors have found that the GALA peptide exhibits a high ability to transfer to the lung and have completed the present invention. It was.
- the present invention provides the following uses, substance introduction agents and vectors.
- Item 1. Use of the GALA peptide represented by SEQ ID NO: 1 as a lung migratory element for delivering a target substance to the lung.
- Item 2. The use according to Item 1, wherein the GALA peptide is bound to a component of the vector.
- Item 3. Item 3.
- Item 1 or 2 wherein the vector contains a lipid and / or cholesterol, and the GALA peptide is bound to a cationic lipid and / or cholesterol.
- Item 4. Item 3. The use according to Item 1 or 2, wherein the vector contains a cationic lipid and / or cholesterol, and the GALA peptide is bound to the cationic lipid and / or cholesterol.
- Item 5. A substance-introducing agent that targets a lung that contains a target substance in a vector and the vector contains a GALA peptide represented by SEQ ID NO: 1.
- Item 6. Item 6.
- Item 7. Item 7.
- Item 7. The substance introduction agent according to Item 5 or 6, wherein the vector contains a cationic lipid and / or cholesterol, and the GALA peptide is bound to the cationic lipid and / or cholesterol.
- the vector is polyalkylene glycol, dextran, pullulan, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, Item 9.
- the substance introducing agent according to any one of Items 5 to 8 which is modified with a hydrophilic polymer selected from the group consisting of carrageenan.
- Item 10 The substance introduction agent according to any one of Items 5 to 9, wherein the target substance is a physiologically active substance that acts in the lung.
- Item 11. Item 11.
- the substance introduction agent according to any one of Items 5 to 10, wherein the target substance is selected from the group consisting of drugs, nucleic acids, peptides, proteins, sugars, and complexes thereof.
- the target substance is partial double-stranded RNA (mdRNA), nicked dsRNA (ndsRNA), dsRNA with gap (gdsRNA), short interfering nucleic acid (siNA), siRNA, microRNA (miRNA), short hairpin RNA (shRNA) ), Short interfering oligonucleotides, substituted short interfering oligonucleotides, modified short interfering oligonucleotides, chemically modified dsRNA, post-transcriptional gene silencing RNA (ptgsRNA), any duplex Item 12.
- mdRNA partial double-stranded RNA
- ndsRNA nicked dsRNA
- gdsRNA with gap gdsRNA
- siNA short interfering nucleic acid
- Item 13 Item 13.
- PEI polyethyleneimine
- Item 14 A vector for delivering a target substance to the lung, comprising the GALA peptide represented by SEQ ID NO: 1 as an element for selectively delivering the substance to the lung.
- Item 15. Item 15.
- the vector according to Item 14 wherein the vector comprises a cationic lipid and / or cholesterol, and the GALA peptide is bound to the cationic lipid and / or cholesterol.
- Item 17. The vector according to Item 17.
- the vector is polyalkylene glycol, dextran, pullulan, ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, Item 18.
- Item 19 The vector according to Item 18, wherein the polyalkylene glycol is PEG (preferably, PEG having a molecular weight of 2000).
- Item 20 is PEG (preferably, PEG having a molecular weight of 2000).
- auxiliary lipid Helper Lipid.
- Item 21 The vector according to item 20, wherein the auxiliary lipid is EPC, DOPC, DOPE, or SOPE.
- Item 22 The vector according to any one of Items 14 to 19, further comprising an auxiliary lipid (Helper Lipid).
- the vector is a liposome
- the liposome composition is selected from cationic lipid / Chol / auxiliary lipid / STR-PEG / Chol-GALA or auxiliary lipid / Chol / STR-PEG / Chol-GALA (more preferably selected from DOTMA, DODAP or DSTAP) Cationic lipid / Chol / EPC, auxiliary lipid selected from DOPE or SOPE / STR-PEG / Chol-GALA or EPC / Chol / STR-PEG / Chol-GALA, more preferably DOTMA / Chol / EPC / STR -PEG / Chol-GALA, DODAP / Chol / EPC / STR-PEG / Chol-GALA, DSTAP / Chol / EPC / STR-PEG / Chol-GALA, DOTMA / Chol / DOPE / STR- EG / Chol-
- the vector is a liposome containing DOTMA, Chol, and EPC as components of a lipid membrane, and the lipid composition (molar ratio) of the liposome is 10 to 50/20 to 50/20 to 70 for DOTMA / Chol / EPC.
- Item 15 The vector according to Item 14, wherein the liposome further comprises 1 to 15 mol% of STR-PEG2000 and 0.1 to 5 mol% of Chol-GALA with respect to the total lipid amount of DOTMA / Chol / EPC.
- Item 24 A method of introducing a target substance into a lung by administering to a mammal a vector encapsulating a target substance and binding a GALA peptide represented by SEQ ID NO: 1.
- Item 25 A method for treating lung cancer, comprising a step of administering a vector encapsulating an anticancer agent and binding a GALA peptide represented by SEQ ID NO: 1 to a mammal having lung cancer.
- Item 26. Item 25. The treatment method according to Item 24, wherein the lung cancer is a cancer that has metastasized to the lung.
- Item 27. A therapeutic agent for lung cancer, wherein an anticancer agent is encapsulated in a vector bound with the GALA peptide represented by SEQ ID NO: 1.
- Item 29. Item 16. The use according to Item 3, the substance introduction agent according to Item 7, and the vector according to Item 15, wherein the GALA peptide is bound to a lipid component in the range of 1 to 4 mol% with respect to the total amount of lipid.
- a vector or substance introduction agent for specifically delivering a target substance to the lung.
- the vector or the substance introduction agent of the present invention can be transferred to the lung, and at the same time, can be inhibited from transferring to the liver, which is a major accumulation organ.
- the vector or substance introduction agent of the present invention particularly the liposome, has a knockdown effect that surpasses existing siRNA delivery carriers, particularly in the lung, and it has been demonstrated that the introduction effect of the target substance is very excellent. Since the vector and the substance introduction agent containing the GALA peptide used in the present invention have no aggregation problem, they do not clog blood vessels.
- the result of having evaluated the pulmonary transfer property of a GALA modification liposome over time is shown.
- An enlarged view of the vertical axis of FIG. The result of having evaluated the liver transfer property of a GALA modification liposome over time is shown.
- the result of having mixed blood and GALA modification MEND in arbitrary ratios and evaluating the interaction with a blood cell component is shown.
- GALA-modified MEND labeled with siRNA [ 32 P] and lipid membrane [ 3 H] was administered via the tail vein, and [ 32 P] and [ 3 H] contained in the lungs for 1 hour were measured.
- the result of having evaluated the transfer property to the lung of the modified MEND is shown.
- FIG. 2 shows the results of tail vein administration of GALA-modified MEND encapsulating fluorescence-labeled siRNA, and the evaluation of the localization of GALA-modified MEND in the lung 1 hour later using a confocal laser scanning microscope.
- FIG. 3 shows the results of evaluation of the knockdown effect of GALA-modified MEND and GALA-unmodified MEND in the lung 24 hours after the tail vein administration of GALA-modified MEND and GALA-unmodified MEND by qRT-PCR method. The results of evaluating the knockdown effect of GALA-modified MEND in the lung 24 hours later by qRT-PCR after GALA-modified MEND administration in the tail vein are shown.
- the results of evaluating the knockdown effect of GALA-modified MEND in the liver 24 hours after administration of GALA-modified MEND by the qRT-PCR method are shown.
- the knockdown effect of MEND at the time of using DOTMA, DSTAP, and DODAP as a cationic lipid is shown.
- the knockdown effect of each MEND when EPC, DOPE, and SOPE are used as the helper lipid is shown.
- the change in body weight at the time of continuous administration of GALA-modified MEND for 4 days is shown.
- the measurement result of AST and ALT at the time of MEND 4 day continuous administration is shown.
- the result of the pharmacokinetics evaluation of PEG modification MEND is shown.
- the knockdown effect of PEG modification MEND is shown.
- the knockdown effect of a MEND preservation product is shown.
- the result of storage stability after storing GALA-modified MEND at room temperature for 1 month is shown.
- the results show that MENDs with different GALA modification amounts were administered into the tail vein, and the knockdown effect of GALA-modified MEND in the lung 24 hours later was evaluated by the qRT-PCR method.
- the lung metastasis inhibitory effect by GALA modification MEND administration in a lung metastasis model is shown.
- the knockdown effect by GALA modification MEND administration in a lung metastasis model is shown.
- the vector of the present invention can realize selective migration to the lung by containing the GALA peptide.
- the “GALA peptide represented by SEQ ID NO: 1” is the following 30 amino acid peptide. Ala Ala Leu Ala Glu Leu Ala Glu Ala Leu Ala Glu Ala Leu His Glu Ala Leu Ala Glu Ala Leu Ala Glu Trp
- the peptide described in SEQ ID NO: 1 has 4 units of a partial structure of glutamic acid (E) -alanine (A) -leucine (L) -alanine (A) in the sequence, and the partial structure is preferably 3 units or It is preferable that amino acid deletion, substitution or addition is performed in the other amino acid sequence while retaining 4 units, more preferably 4 units.
- These modified GALA peptides are also included in the “GALA peptide represented by SEQ ID NO: 1” of the present invention.
- the number and position of amino acids to be deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 1 are one or more, preferably one or several, and the specific range is deletion. Is usually 1 to 4, preferably 1 to 3, more preferably 1 to 2, and substitution is usually 1 to 6, preferably 1 to 4 and more preferably 1 to 2 The addition is usually 1 to 12, preferably 1 to 6, and more preferably 1 to 4.
- Amino acid substitutions include hydrophobic amino acids (Leu, Val, Ile, Ala), aromatic amino acids (Phe, Tyr, Trp), basic amino acids (Arg, Lys, His), acidic amino acids (Glu, Asp), neutral Similar amino acid substitutions between amino acids (Gly, Ser, Thr, Cys, Met, Gln, Asn, Pro) are preferred.
- the GALA peptide may be simply referred to as “GALA”.
- the GALA peptide is preferably bound to a component constituting the vector.
- components constituting the vector include lipids, proteins or peptides, sugar chains, water-soluble or water-miscible polymers (neutral, cationic or anionic), surfactants, and the like.
- the bond includes any bond such as a covalent bond, an ionic bond, a hydrogen bond, and a coordination bond, and is preferably a covalent bond or a coordination bond, and most preferably a covalent bond.
- the GALA peptide is contained in a carrier (vector) that can introduce a substance into cells.
- Carriers (vectors) that can introduce substances into cells include lipid-based transfection reagents, virus-derived particles, liposomes, polyplexes, micelles, and the like. In a preferred embodiment, they are modified or bound to liposomes.
- the GALA peptide may be bound to any of the components of the liposome such as phospholipid, cholesterol, lipid (preferably cationic lipid), auxiliary lipid (Helper Lipid) and the like.
- Chol-GALA cholesterol-associated peptide
- —O (C ⁇ O) — is bonded to the N-terminal amino group of the GALA peptide
- —NH 2 is the amino group protecting the C-terminal carboxyl group of the GALA peptide.
- the bond between cholesterol and GALA peptide may be a urethane bond as described above, and may be either an ester bond or an ether bond.
- the cholesteryl group may be bound to either the N-terminus or C-terminus of the GALA peptide, or may be bound to any amino acid side chain of the GALA peptide.
- the cholesteryl group may be bonded to the GALA peptide via any linker such as alkylene, peptide, or polyether.
- the C terminal is an amide, but the C terminal may be another group such as a carboxylic acid (COOH), an ester, or a salt of a carboxylic acid.
- the GALA peptide When the GALA peptide is bound to a lipid component such as cholesterol or phospholipid and contained in the vector or substance introduction agent, the GALA peptide is 0.1 to 5 mol%, preferably 0.3 to 4 mol, based on the total amount of lipid. %, More preferably 0.5 to 4 mol%, still more preferably 1 to 4 mol%, particularly preferably about 1.5 to 2 mol%.
- the “total lipid amount” shown in this specification does not include the amount of the lipid component bound to the modifying component of the liposome. That is, the amount of lipid component bound to the GALA peptide is not included.
- the vector is PEG modified, the amount of lipid component attached to PEG is not included.
- the GALA modification amount (the ratio of the lipid bound with the GALA peptide to the total lipid amount) Is 5 mol%.
- the GALA peptide is 0.01 to 10% by weight, preferably 0.1 to 5% by weight, more preferably 0.5 to 4% by weight, in particular, of the total weight of the vector. Preferably, about 1 to 2.5% by weight is used.
- a GALA peptide is bound to cholesterol
- the GALA peptide can be bound to a component of a vector other than cholesterol, and such aspects will be apparent to those skilled in the art.
- the vector and the substance introduction agent of the present invention have a zeta potential of about ⁇ 100 to 100 mV, preferably about ⁇ 50 to 50 mV, more preferably about ⁇ 30 to 30 mV at a pH near neutrality (for example, pH 7 or 7.4). is there.
- the zeta potential can be measured using a zeta sizer.
- the average particle size of the vector and the substance introduction agent of the present invention is not particularly limited.
- the particle size is 30 to 1000 nm, preferably 50 to 300 nm, more preferably 50 to 200 nm, and particularly 50 to 150 nm.
- the average particle diameter can be measured by, for example, a dynamic light scattering method, a static light scattering method, an electron microscope observation method, an atomic force microscope observation method, or the like.
- the introduction agent of the present invention contains a target substance to be delivered into a cell together with a vector.
- the target substance may be covalently bound to the vector or may form a complex with the vector.
- the vector is a hollow particle, it can be enclosed or encapsulated inside the vector.
- the target substance of the present invention is a physiologically active substance that exhibits an effect when introduced into lung cells.
- the substance-introducing agent of the present invention can be used either in vitro or in vivo for delivery of a target substance into lung cells.
- the type of the target substance is not particularly limited.
- drugs for example, drugs, nucleic acids, peptides (bioactive peptides such as oxytocin, bradykinin, thyrotropin releasing factor, enkephalin, peptide hormones, etc.), proteins (enzymes, interleukins, etc.) Cytokines, cell transmission factors, cell growth factors, etc.), sugars or complexes thereof, and the like, and can be appropriately selected according to the type of lung disease, diagnosis, treatment and the like.
- the “nucleic acid” includes analogs or derivatives thereof (for example, siRNA, peptide nucleic acid (PNA), phosphorothioate DNA, etc.) in addition to DNA or RNA. Further, the nucleic acid may be either single-stranded or double-stranded, and may be either linear or circular.
- the target substance is a drug
- anticancer agents for example, anticancer agents, vasodilators, pulmonary vasculitis treatment agents, antibacterial agents, antiviral agents, anti-inflammatory agents, bronchodilators, antitussives, pulmonary fibrosis inhibitors, antituberculosis agents, etc. Is mentioned.
- anticancer agents doxorubicin, daunorubicin, cisplatin, oxaliplatin, carboplatin, paclitaxel, irinotecan, SN-38, actinomycin D, vincristine, vinblastine, methotrexate, azathioprine, fluorouracil, mitomycin C, docetaxel, Famide, capecitabine, epirubicin, gemcitabine, mitoxantrone, leucovorin, vinorelbine, trastuzumab, etoposide, estramustine, prednisone, interferon alpha, interleukin-2, bleomycin, ifosfamide, mesna, altretamine, topotecan, cytarazolone, cytarazolone Dexamethasone, mercaptopurine, thioguanine, fludarabine, gemts Mab, idarubicin
- vasodilator examples include bosentan, ambrisentan, beraprost sodium, sildenafil, epoprostenol and the like.
- pulmonary vasculitis therapeutic agents include corticosteroids, cyclophosphamide, azathioprine, methotrexate, aspirin and the like.
- antibacterial agents include amphotericin B.
- Antiviral agents include vidarabine, acyclovir, trifluorothymidine and the like.
- anti-inflammatory agent examples include phenylbutazone, acetaminophen, ibuprofen, indomethacin, sulindac, piroxicam, diclofenac, prednisone, beclomethasone, dexamethasone and the like.
- the target substance is a nucleic acid
- mdRNA partial double-stranded RNA
- ndsRNA nicked dsRNA
- gdsRNA gapped dsRNA
- siRNA siRNA
- miRNA microRNA
- shRNA hairpin RNA
- ptgsRNA post-transcriptional gene silencing RNA
- dsRNA double-stranded RNA
- the target substance can be used alone or in admixture of two or more.
- two or more siRNAs can be used in combination.
- the substance introduction agent contains a nucleic acid such as siRNA as a target substance, it is preferable that a cation such as polyethyleneimine (PEI) coexists.
- PEI polyethyleneimine
- the double stranded RNA is at the 3 ′ end of the double stranded RNA, such as an overhang containing deoxyribonucleotides or two deoxyribonucleotides (eg, thymidine, adenine). It may contain 1 to 4 nucleotide overhangs at one or both ends. Double stranded RNA may have blunt ends at one or both ends. In some embodiments, the 5 'ends of the first and second strands are phosphorylated.
- the 3 'terminal nucleotide overhang may comprise a ribonucleotide or deoxyribonucleotide that is chemically modified in the sugar, base, or backbone of the nucleic acid.
- the 3'-terminal nucleotide overhang may comprise one or more universal base ribonucleotides.
- the 3 'terminal nucleotide overhang may comprise one or more acyclic nucleotides.
- the dsRNA is 5 ′ phosphate (Martinez et al., Ell Cell .. 110: 563-574, 2002; and Schwarz et al., Molec. Cell. 10: 537. -568, 2002) or a terminal phosphate group such as 5 ', 3' diphosphate.
- Double-stranded RNA consists of 2′-deoxy, 2′-O-methyl, 2′-O-methoxyethyl, 2′-O-2-methoxyethyl, halogen, 2′-fluoro, 2′-O-allyl, Or it may further comprise 2 ′ sugar substitutions such as any combination thereof.
- the double stranded RNA is alkyl, abasic, deoxyabasic, glyceryl, dinucleotide, acyclic nucleotide, on one or both ends of the first strand or one or more second strands, It further includes an end cap substituent such as an inverted deoxynucleotide moiety, or any combination thereof.
- the double stranded RNA is independently phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphate triester, aminoalkylphosphate triester, methylphosphonate, alkylphosphonate, 3′- Alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphonoacetates, thiophosphonoacetates, phosphinates, phosphoramidates, 3'-aminophosphoramidates, aminoalkyl phosphoramidates, At least one modified internucleoside linkage, such as thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphate triester, selenophosphate, boranophosphate linkage, or any combination thereof.
- Double-stranded RNA is 5-methylcytosine; 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl, 2-propyl, or other alkyl derivatives of adenine and guanine; 8-substituted Adenine and guanine (8-aza, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, etc.); 7-methyl, 7-deaza, and 3-deazaadenine and guanine; 2-thiouracil; 2 2-thiocytosine; 5-methyl, 5-propynyl, 5-halo (such as 5-bromo or 5-fluoro), 5-trifluoromethyl, or other 5-substituted uracil and cytosine; and 6- By using nucleic acid analogs such as azouracil, substitution or modification (including chemical modification) Can)
- RNA such as double stranded RNA (dsRNA)
- dsRNA double stranded RNA
- Non-limiting examples of such chemical modifications include internucleotide phosphorothioate linkages, 2′-deoxyribonucleotides, 2′-O-methylribonucleotides, 2′-deoxy-2′-fluororibonucleotides, “acyclic” "Incorporation of nucleotides, 5'-C-methyl nucleotides, and glyceryl and / or reverse deoxyabasic residues at the termini. These chemical modifications can maintain RNAi activity in the cell.
- the liposome is a closed vesicle having a lipid bilayer structure
- it may be a multilamellar liposome (MLV), SUV (small unilamellar vesicle), LUV (large uniaxial molecular vesicle), GUV (giant unimolecular cellular), etc. It may be a single membrane liposome.
- the vector (carrier) of the present invention can be modified with a hydrophilic polymer.
- hydrophilic polymers examples include polyalkylene glycols (polyalkylene glycol copolymers such as polyethylene glycol, polypropylene glycol, polybutylene glycol, or block copolymers of polyethylene glycol and polypropylene glycol), dextran, pullulan, ficoll, and polyvinyl alcohol.
- Styrene-maleic anhydride alternating copolymer divinyl ether-maleic anhydride alternating copolymer, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan, etc.
- polyalkylene glycol polyethylene glycol, Polypropylene glycol, polybutylene glycol, or block copolymer of polyethylene glycol and polypropylene glycol Copolymers of polyalkylene glycols such as body
- PEG polyethylene glycol
- the length of PEG can be appropriately selected within a molecular weight range of about 500 to 10,000, preferably a molecular weight of 1,000 to 5,000, and more preferably a molecular weight of 2,000.
- lipids modified with PEG include DSPE (distearoyl phosphatidylethanolamine) -PEG2000, DMPE (dimyristol phosphatidylylamine) -PEG2000, DSG (distearoylyglycerol, PEG2000)
- Examples thereof include C8 ceramide-PEG2000, C16 ceramide-PEG2000, and among these, STR-PEG2000 or C8ceramide-PEG2000 is preferable.
- Those skilled in the art can select the molecular weight of other hydrophilic polymers as appropriate.
- a liposome is modified with PEG, stearylated PEG (STR-PEG), C8 ceramide-PEG, or cholesterylated PEG (Chol-PEG) is used for lung migration performance and target substance (eg, nucleic acid drug such as siRNA).
- target substance eg, nucleic acid drug such as siRNA.
- a vector for example, a liposome preparation
- DSPE-PEG, DSG-PEG, C16 ceramide-PEG, etc. in blood stability It is preferable for improving.
- the hydrophilic polymer is preferably modified at a ratio of about 1 to 15 mol% when the lipid constituting the liposome is 100 mol%.
- liposomes will be described as an example of vectors (carriers) for delivering a target substance in vitro or in vivo to the lung.
- the present invention is not limited to liposomes, and any vector capable of introducing a GALA peptide into cells. These vectors (carriers) are included in the present invention.
- the type of lipid constituting the lipid bilayer is not particularly limited, and specific examples thereof include phosphatidylcholine (for example, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine).
- phosphatidylcholine for example, dioleoylphosphatidylcholine, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine.
- phosphatidylglycerol eg, dioleoylphosphatidylglycerol, dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol
- phosphatidylethanolamine eg, dioleoyl Phosphatidylethanolamine, dilauroyl phospha Phospholipids such as diethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidiethanolamine
- phosphatidylserine phosphatidylinositol, phosphatidic acid, cardiolipin, etc .
- phosphatidic acid phosphatidic acid
- cardiolipin etc.
- sphingomyelin ganglioside, etc.
- glycolipids examples thereof include glycolipids, and one or more of these can be used.
- the phospholipid may be egg yolk, soybean or other natural lipid derived from animals and plants (eg, egg yolk lecithin, soybean lecithin, etc.), synthetic lipid or semi-synthetic lipid. These lipids can be used alone or in admixture of two or more.
- lipid bilayers for example, cholesterol, cholesterol succinic acid, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol to physically or chemically stabilize the lipid bilayer and to regulate the fluidity of the membrane
- Sterols derived from animals such as stigmasterol, sitosterol, campesterol, sterols derived from plants such as brassicasterol; Sterols derived from microorganisms such as timosterol and ergosterol; Sugars such as glycerol and sucrose; Triolein 1 type, or 2 or more types can be contained among glycerol fatty acid esters, such as trioctanoin.
- the content is not particularly limited, but is preferably 5 to 40% (molar ratio) with respect to the total amount of lipid constituting the lipid bilayer, and is preferably 10 to 30% (molar ratio). Further preferred.
- the liposome of the present invention has a GALA peptide composed of 30 amino acids on its surface.
- the outer surface of the lipid bilayer is the surface of the liposome, and for the multilamellar liposome, the outer surface of the outermost lipid bilayer is the surface of the liposome.
- the liposome of the present invention may have the above peptide in a portion other than the surface (for example, the inner surface of the lipid bilayer).
- the vector of the present invention preferably contains a cationic lipid.
- the cationic lipid include DODAC (dioctadecyldimethylammonium chloride), DOTMA (N- (2,3-dioleoyloxy) propyl-N, N, N-trimethylaluminumium), DDAB (didodecylamyloidium, dododecylamyloidium).
- a preferred cationic lipid is DOTMA, DSTAP or DODAP, and particularly preferably DOTMA.
- Cationic lipids can be used alone or in admixture of two or more.
- DOTMA and DSTAP have quaternary amines and are always positively charged, while DODAP has tertiary amines and has no charge at physiological pH.
- the structure and characteristics of the cationic lipid can be widened by changing the type and blending amount of the cationic lipid.
- the vector of the present invention preferably contains an auxiliary lipid.
- the co-lipid for example, EPC (egg phosphatidylcholine), DLPC (dilinoleoylphosphatidylcholine), DMPC (dimyristoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine), DSPC (distearoylphosphatidylcholine), POPC (palmitoyloleoylphosphatidylcholine), DOPC (dioleoylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), SOPE ( stearyloylylphosphatidyl holine), and the like.
- EPC egg phosphatidylcholine
- DLPC diilinoleoylphosphatidylcholine
- DMPC diimyristoylphosphatidylcholine
- DPPC dipalmito
- MEND containing DOPC as an auxiliary lipid shows no aggregation even after storage at room temperature for 1 month, and DOPC is excellent in storage stability.
- the GALA peptide is modified with a hydrophobic group or a hydrophobic compound, the hydrophobic group or the hydrophobic compound is inserted into the lipid bilayer, and the peptide is exposed from the lipid bilayer.
- the liposome which can be illustrated can be illustrated.
- “the peptide is exposed from the lipid bilayer” means that the peptide is exposed from either the outer surface or the inner surface of the lipid bilayer, or is exposed from both Is included.
- the hydrophobic group or the hydrophobic compound is not particularly limited as long as it can be inserted into the lipid bilayer.
- the hydrophobic group include saturated or unsaturated fatty acid groups such as stearyl group, palmityl group, oleyl group, palmitooleyl group, linoleyl group, and linoleyl group, aliphatic alcohol, aliphatic amine, and other carbon atoms of 10 or more.
- hydrocarbon-containing groups for example, sterol-derived groups such as cholesteryl group (Chol)
- fatty acid groups having 10 to 20 carbon atoms for example, palmitoyl group, oleyl group) , Stearyl group, arachidoyl group, etc.
- hydrophobic compound examples include phospholipids, glycolipids or sterols exemplified above, long-chain aliphatic alcohols (eg, phosphatidylethanolamine, cholesterol, etc.), polyoxypropylene alkyls, glycerin fatty acid esters, and the like. .
- the liposome of the present invention can be prepared using a known method such as a hydration method, an ultrasonic treatment method, an ethanol injection method, an ether injection method, a reverse phase evaporation method, or a freezing / thawing method. Examples of liposome production by the hydration method are shown below.
- a lipid membrane is obtained by dissolving a lipid that is a component of the lipid bilayer and the peptide modified with a hydrophobic group or a hydrophobic compound in an organic solvent, and then evaporating and removing the organic solvent.
- examples of the organic solvent include hydrocarbons such as pentane, hexane, heptane, and cyclohexane; halogenated hydrocarbons such as methylene chloride and chloroform; aromatic hydrocarbons such as benzene and toluene; methanol, ethanol, and the like Lower alcohols; esters such as methyl acetate and ethyl acetate; ketones such as acetone and the like. These may be used alone or in combination of two or more. Subsequently, the lipid membrane can be hydrated and stirred or sonicated to produce a liposome having the peptide on its surface.
- hydrocarbons such as pentane, hexane, heptane, and cyclohexane
- halogenated hydrocarbons such as methylene chloride and chloroform
- aromatic hydrocarbons such as benzene and toluene
- methanol, ethanol, and the like Lower alcohols
- Lipids which are constituents of the lipid bilayer, are dissolved in an organic solvent, and then the organic solvent is removed by evaporation to obtain a lipid membrane.
- the lipid membrane is hydrated and stirred or sonicated to produce liposomes.
- the peptide can be introduced onto the surface of the liposome by adding the peptide modified with a hydrophobic group or a hydrophobic compound to the external solution of the liposome.
- the said peptide can be introduce
- the preparation of liposomes in the case of using a quaternary amine as the cationic lipid is carried out by the same method as in Example 3-1, which will be described later, a method according to this, or a combination thereof with a conventional method.
- the liposome can be prepared by a method similar to Example 3-2 described later, a method analogous thereto, or a combination of these with a conventional method.
- the ratio of cationic lipid / Chol / auxiliary lipid can be appropriately changed.
- the composition ratio is preferably 10 to 50/20 to 50/20 to 70, more preferably 20 to 40/30 to 50/20 to 40, and 30/40/30. It is particularly preferred.
- a preferred combination of cationic lipid / Chol / auxiliary lipid is a cationic lipid selected from DOTMA, DODAP or DSTAP, or an auxiliary lipid selected from DOPE or SOPE, more preferably DOTMA / Chol / EPC, DODAP / Chol / EPC, DSTAP / Chol / EPC, DOTMA / Chol / DOPE, and DOTMA / Chol / SOPE.
- the addition ratio of Chol-GALA can be changed as appropriate.
- the total lipid amount (cationic lipid / Chol / total lipid of auxiliary lipid) for example, 0.1 to 5 mol%, preferably 0.3 to 4 mol%, more preferably 0.5 to 4 mol%, still more preferably 1 to 4 mol%, and particularly preferably 1.5 to 4 mol%. 2 mol%.
- PEG can be appropriately modified to the liposome.
- the addition ratio of PEG can be appropriately changed.
- 0.1 to 15 mol% with respect to the total lipid amount (cationic lipid / Chol / total lipid of auxiliary lipid) Preferably, it is 1 to 5 mol%.
- the added PEG is preferably PEG bonded to lipid, more preferably STR-PEG or C8 ceramide-PEG, and particularly preferably STR-PEG2000 or C8 ceramide-PEG2000.
- neutral liposomes having the liposome composition as auxiliary lipid / Chol can be prepared using the above-mentioned auxiliary lipid, and the composition ratio (molar ratio) can be appropriately changed. It is preferably 10 to 60, more preferably 60 to 80/20 to 40, and particularly preferably 70/30.
- auxiliary lipid / Chol are EPC / Chol, DLPC / Chol, DMPC / Chol, DPPC / Chol, DSPC / Chol, POPC / Chol, DOPC / Chol, DOPE / Chol, and SOPE / Chol, and more preferably Is EPC / Chol, DOPC / Chol, DOPE / Chol, SOPE / Chol, and particularly preferably EPC / Chol.
- the addition ratio of Chol-GALA can be appropriately changed. For example, 0.1% of the total lipid amount (auxiliary lipid / total lipid of Chol) can be used.
- PEG can be modified as appropriate to the liposome, and when PEG is modified, the addition ratio of PEG can be changed as appropriate and is based on the total lipid amount (auxiliary lipid / total lipid of Chol). Examples thereof include 0.1 to 15 mol%, and preferably 1 to 5 mol%.
- the added PEG is preferably a PEG modified with lipid, more preferably STR-PEG or C8 ceramide-PEG, and particularly preferably STR-PEG2000 or C8 ceramide-PEG2000.
- the preferred liposome composition of the present invention is cationic lipid / Chol / auxiliary lipid / STR-PEG / Chol-GALA or auxiliary lipid / Chol / STR-PEG / Chol-GALA, more preferably from DOTMA, DODAP or DSTAP.
- the STR-PEG is particularly preferably STR-PEG2000 having a PEG molecular weight of 2000.
- the most preferred liposome of the present invention has a composition of DOTMA / Chol / EPC / STR-PEG2000 / Chol-GALA, and the composition ratio (molar ratio) of DOTMA / Chol / EPC is 10 to 50/20 to 50/20.
- STR-PEG2000 is preferably 1-15 mol% and Chol-GALA is 0.1-5 mol% with respect to the total lipid amount of DOTMA / Chol / EPC;
- the composition ratio (molar ratio) of DOTMA / Chol / EPC is 20 to 40/30 to 50/20 to 40, and STR-PEG2000 is 1 to 5 mol% with respect to the total lipid amount of DOTMA / Chol / EPC.
- Chol-GALA is more preferably 1 to 4 mol%;
- the composition ratio (molar ratio) of DOTMA / Chol / EPC is 30/40/30.
- STR-PEG2000 is 1 to 5 mol% and Chol-GALA is 1 with respect to the total lipid amount of DOTMA / Chol / EPC. Most preferred is 5 to 2 mol%. Since STR-PEG2000 and Chol-GALA are modifying components of liposomes, their added amount was expressed as a ratio relative to the total lipid amount of liposomes composed of three components of DOTMA / Chol / EPC as 100 mol%.
- the target substance to be delivered into lung cells can be encapsulated inside the liposome of the present invention.
- the target substance to be encapsulated in the substance introduction agent (especially liposome) of the present invention is the above-mentioned drug (anticancer agent, vasodilator, antibacterial agent, etc.), nucleic acid (DNA, RNA, analogs or derivatives thereof) depending on the type of lung disease (For example, siRNA, peptide nucleic acid (PNA), phosphorothioate DNA, etc.), peptides (physiologically active peptides such as oxytocin, bradykinin, thyrotropin releasing factor, enkephalin, peptide hormones, etc.) etc.
- lung disease By encapsulating a target substance, it is possible to treat or prevent lung disease, as used herein as “pulmonary disease”, lung cancer, pulmonary inflammatory disease, pulmonary fibrosis, pulmonary embolism, pulmonary hypertension, Pulmonary vasculitis, acute respiratory distress syndrome (ARDS), asbestosis / dust disease, asthma, bronchodilation , Bronchopulmonary dysplasia (BPD), chronic bronchitis, chronic cough, chronic obstructive pulmonary disease (COPD), cold, cystic fibrosis, emphysema, hantavirus, histoplasmosis, influenza, legionellosis, lymphatic vessel Leiomyosarcosis, pertussis, pleurisy, pneumothorax, primary alveolar hypoventilation syndrome, alveolar proteinosis, respiratory distress syndrome, RS virus, sarcoidosis, severe acute respiratory syndrome (SARS), tuberculosis, etc.
- pulmonary disease lung cancer,
- Pulmonary diseases that are preferable for treatment or prevention include pulmonary diseases related to pulmonary blood vessels, and include lung cancer, pulmonary hypertension, pulmonary vasculitis, etc.
- Lung cancer includes not only primary lung cancer but also metastatic lung cancer that has metastasized from organs other than lung, and other organs that originate from lung cancer.
- the substance introduction agent of the present invention in order to suppress cancer metastasis to (eg, adrenal gland, liver, brain, bone, etc.) Treatment of lung cancer, cancer from lung
- the target substance encapsulated in the substance-introducing agent of the present invention for inhibiting metastasis include the above-mentioned anticancer agents (for example, amrubicin hydrochloride, gefitinib, cisplatin, vinblastine, mitomycin C, vinorelbine, paclitaxel, docetaxel, gemcitabine, carboplatin, Irinotecan, tegafur, etoposide, vincristine, cyclophosphamide, doxorubicin, ifosfamide, vindesine, etc.), factors involved in angiogenesis (eg, CD31, ESAM, VEGF, VEGFR, EGF, EGFR, Dll, SFRP, CD151, bFGF, TGF ⁇ 1, PDGF, HGF
- Examples of the target substance to be encapsulated in the substance-introducing agent of the present invention for pulmonary hypertension include the aforementioned vasodilators (for example, bosentan, ambrisentan, beraprost sodium, sildenafil, epoprostenol, etc.), factors involved in vasodilation (e.g., endothelin receptor (ET a, ET B), PDE5, etc.) are preferred anti siRNA of.
- vasodilators for example, bosentan, ambrisentan, beraprost sodium, sildenafil, epoprostenol, etc.
- factors involved in vasodilation e.g., endothelin receptor (ET a, ET B), PDE5, etc.
- the target substance to be encapsulated in the substance introduction agent of the present invention for pulmonary vasculitis corticosteroids, cyclophosphamide, azathioprine, methotrexate, aspirin and the like are preferable.
- the substance introduction agent of the present invention can be used either alone or together with other treatments for lung diseases.
- the target substance When the target substance is water-soluble, it is possible to encapsulate the target substance in the aqueous phase inside the liposome by adding the target substance to an aqueous solvent used when hydrating the lipid membrane in the production of liposomes. it can.
- the target substance When the target substance is fat-soluble, the target substance can be encapsulated in the lipid bilayer of the liposome by adding the target substance to an organic solvent used in the production of the liposome.
- “encapsulation” includes both a case where a target substance is contained inside a hollow particle such as a liposome and a case where the substance is held on a surface portion constituting a vector such as a lipid bilayer membrane.
- the species to which the target substance is delivered is not particularly limited as long as it is a vertebrate having a lung, but is preferably a mammal.
- mammals include humans, monkeys, cows, sheep, goats, horses, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and the like.
- the liposome of the present invention can be used in the state of a dispersion, for example.
- a dispersion solvent for example, buffer solutions such as physiological saline, phosphate buffer solution, citrate buffer solution, and acetate buffer solution can be used.
- additives such as sugars, polyhydric alcohols, water-soluble polymers, nonionic surfactants, antioxidants, pH regulators, hydration accelerators may be added to the dispersion.
- the liposome of the present invention can also be used in a state where the dispersion is dried (for example, freeze-dried, spray-dried, etc.).
- the dried liposome can be made into a dispersion by adding a buffer solution such as physiological saline, phosphate buffer, citrate buffer, or acetate buffer.
- Each liposome can be used both in vitro and in vivo.
- examples of the administration route include intravenous injection, infusion, etc., and the dosage and administration frequency depend on the type and amount of the target substance encapsulated in the liposome according to the present invention. It can be adjusted accordingly.
- the liposome of the present invention can be safely administered without any weight loss or liver damage.
- EPC was purchased from Nippon Oil & Fats (Tokyo, JAPAN).
- DOTAP, DSTAP, DODAP, Cholesterol, DOPE, SOPE, DOPC, C8 Ceramide-PEG2000 were purchased from Avanti Polar Lipids (Alabaster, AL, USA).
- DOTMA was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, JAPAN).
- STR-PEG2000 was purchased from Wako Pure Chemical Industries, Ltd.
- PEI branch type, Mw. Ave. 10,000
- Transaminase CII-Test Wako was purchased from Wako Pure Chemical Industries, Ltd.
- RNAlater was purchased from Ambion. High Capacity RNA-to-cDNA Kit and TaqMan Gene Expression Master Mix were purchased from Applied Biosystems.
- CD31 Forward CAGAGCGGATAATTGCCATCC (SEQ ID NO: 10) CD31 Reverse: ACAGGATGGAAATCACAACTTTCATC (SEQ ID NO: 11) CD31 Probe: [FAM] ACCCTCAGGATCTCGCTGAACACCGC [TAMRA] (SEQ ID NO: 12) CD34 Forward: TCTGCCTGGAACTAAGTGAAGC (SEQ ID NO: 13) CD34 Reverse: CCTCAGACTGGGCTAGAAGCA (SEQ ID NO: 14) CD34 Probe: [FAM] ACCAGCATCAGCCCTCAGCCTCCTCC [TAMRA] (SEQ ID NO: 15) ICR mouse male 5 weeks old and C57BL / 6 mouse male 6 weeks old were purchased from Japan SLC. In addition, unless otherwise specified, commercially available special grades or first grades or equivalents thereof were used.
- lipid thin film For the preparation of the lipid thin film, a pump DIVAC 1.2, Trap EVALA UNI TRAP UT-1000 (both Tokyo Science Instruments) were used.
- sonication a bath sonicator AU-25C (Aiwa Medical Industry) was used.
- ZETA SIZER Nano-ZS (Malvern Instruments Ltd.) was used for the measurement of particle diameter and ⁇ potential by dynamic light scattering (DLS).
- DLS dynamic light scattering
- ABI 7500 real-time system (Applied Biosystems) was used.
- the liquid scintillation counter TRI-CABB 1600TR (PACKARD) was used for ⁇ -ray measurement.
- CLSM confocal laser scanning microscope
- A1 Nikon
- laser Ar laser and He / Ne laser were used.
- PCR thermal cycler TP3000 (Takara Bio Inc.) was used.
- Docu-pHMeter (Sartorius) was used.
- the lipid used was adjusted to an arbitrary concentration (1 to 10 mM) by diluting with an appropriate amount of EtOH.
- Chol-GALA was purified by reverse phase HPLC using COSMOSIL 5C4-AR-300 (Size 10 ⁇ 250 mm). H 2 O / 0.1% TFA was Buffer A, and CH 3 CN / 0.1% TFA was Buffer B. After injecting Chol-GALA (Purity:> 71%) dissolved in DMF (4 mg / mL as Crude) at a flow rate of 2.0 mL / min and 25 ° C., 50% B ⁇ 95% B (20 min) A gradient was applied (details below). Chol-GALA was recovered by detecting the absorbance at 215 nm and lyophilized.
- the particle size and ⁇ (zeta) potential of Liposome and MEND were measured using Zetasizer Nano ZS (Malvern Instruments, UK).
- the experimental data is expressed as an average value ⁇ standard deviation of the experimental values of three times or more unless otherwise specified.
- the test was performed by One-way ANOVA, and further multiple comparison was performed by the Dunnett method, and the case where P ⁇ 0.05 was considered significant.
- lipid composition is EPC / Chol / STR-PEG2000 / Chol.
- a lipid thin film, which is GALA was prepared (abbreviated as “GALA-Liposome” in FIG. 1).
- GALA-Liposome lipid thin film represented by “Cationic-Liposome” in FIG. 1
- the molar ratio of DOTMA (EtOH solution), Chol, and EPC is set to 30/40/30, and the lipid composition is obtained by the same operation as above.
- a lipid thin film (Liposome b) was prepared in which DOTMA / Chol / EPC. 2) Preparation of liposome 10 mM HEPES buffer 5% Glucose (HBG) (in vivo experiment), 10 mM so that the lipid concentration prepared in 1) above is 2.64 mM (in vivo experiment) and 0.55 mM (in vitro experiment). After adding HEPES buffer (HB) (in vitro experiment), the mixture was hydrated at room temperature for 15 minutes or more. Then, the liposome was prepared by performing ultrasonic treatment for about 1 minute with a bathtub-type sonicator.
- Example 2 Evaluation of liposome pharmacokinetics 1
- Administration of liposome, recovery of organ, measurement of [ 3 H] The lipid membrane of the liposome prepared in 2) above is labeled with [ 3 H] did.
- Mice ICR, 5 weeks old, rabbit
- Mice were administered from the tail vein under the condition of 10 ⁇ L Liposome / g mouse.
- Mice were treated with ether anesthesia 1, 5, 15 minutes and 1, 6 hours after administration, and then laparotomized. After blood was collected from the inferior vena cava, the lungs and liver were removed.
- FIG. 1-A, FIG. 1-B, and FIG. 2 show the results of evaluating the migration of the GALA-modified liposome (GALA-Liposome) over time into the lungs and liver.
- GALA-Liposome GALA-modified liposome
- Example 3-1 Preparation of MEND1 1) Preparation of lipid thin film In the same manner as in Example 1, DOTMA (EtOH solution), Chol (EtOH solution), EPC (EtOH solution) were added to a glass test tube so that the molar ratio was 30/40/30, and an appropriate amount of EtOH was added.
- lipid thin film (liposome b) whose lipid composition is DOTMA / Chol / EPC was prepared by distilling a solvent off.
- DOTMA / Chol / EPC / STR-PEG2000 lipid thin film
- MEND1 lipid thin film
- DOTMA lipid thin film
- Chol-GALA lipid thin film
- siRNA core particle solution was added to the test tube prepared with the lipid thin film obtained in the above 1) so that the lipid concentration was 2.64 mM, and then hydrated at room temperature for 15 minutes or more. Then, MEND was prepared by performing ultrasonic treatment for about 1 minute with a bath sonicator, and MEND1 and GALA-MEND1 encapsulating siRNA core particles were prepared.
- Example 3-2 Preparation of MEND2
- DODAP EtOH solution
- Chol EtOH solution
- EPC EtOH solution
- STR-PEG2000 (EtOH solution) was added to the obtained lipid solution, and 5 mol% of the total lipid amount of liposome c was added to prepare a lipid thin film having a lipid composition of DODAP / Chol / EPC / STR-PEG2000 (hereinafter referred to as “lipid lipid”). , And may be abbreviated as “MEND2”).
- MEND2 lipid composition of DODAP / Chol / EPC / STR-PEG2000
- MEND2 lipid composition of DODAP / Chol / EPC / STR-PEG2000
- siRNA solution and the PEI solution were diluted with HBG (pH unadjusted (pH 5.0)) to prepare acidic solution core particles.
- HBG pH unadjusted (pH 5.0)
- MEND MEND is prepared by adding an equal amount of HBG (pH 8.1) to the siRNA core particle solution so that the prepared solution becomes neutral, and siRNA core particles are prepared.
- Encapsulated MEND2 and GALA-MEND2 were prepared.
- Example 4 Interaction with blood cells Mouse blood (containing heparin, 20 units / mL) and GALA-MEND1 solution were mixed at a volume ratio of 1: 1, and the condition was 37 ° C. Mix for 5 minutes using a shaker (Sample 1). Blood was further added to this mixed solution to obtain a volume ratio of 10: 1, and then mixed under the same conditions as before (Sample 2). 10 ⁇ L of Samples 1 and 2 were added on a slide glass and a cover glass was placed thereon, and then observed using a microscope.
- FIG. 3 shows the results of evaluating the interaction with blood cell components by mixing blood and the obtained GALA-MEND1 at an arbitrary ratio.
- MEND1 or GALA-MEND1 solution (2 mg siRNA / kg mouse) was administered from the tail vein of a mouse (ICR, 5 weeks old, sputum). One hour after administration, the mice were treated with ether anesthesia, then laparotomy was performed, blood was collected from the inferior vena cava, and the lungs were removed.
- FIG. 4 shows the results of evaluating the transferability of GALA-modified MEND to the lung.
- Example 6 Observation of localization of MEND in lung by CLSM MEND1 and GALA-MEND1 prepared using Cy5-labeled siRNA were used as administration samples.
- MEND1 or GALA-MEND1 solution (2 mg siRNA / kg mouse) obtained in Example 3-1 was administered from the tail vein of a mouse (ICR, 5 weeks old, sputum). One hour after tail vein administration, the lungs were removed from the mice under anesthesia, and lung tissue pieces of about several mm were prepared.
- the prepared lung tissue pieces were infiltrated into a GSL I-B4 Isolectin FITC conjugate (Funakoshi) solution (diluted to 20 ⁇ g / mL with physiological saline) for 30 minutes, and then observed using CLSM.
- GSL I-B4 Isolectin FITC conjugate (Funakoshi) solution (diluted to 20 ⁇ g / mL with physiological saline) for 30 minutes, and then observed using CLSM.
- FIG. 5 shows the results of evaluating the localization of GALA-modified MEND in the lung 1 hour later using a confocal laser scanning microscope after administering MEND1 or GALA-MEND1 encapsulating fluorescently labeled siRNA into the tail vein.
- Example 7-1 In vivo knockdown effect with or without GALA modification Using GALA-modified MEND and GALA unmodified MEND, the knockdown effect with or without GALA modification was examined. 1) Preparation of MEND solution A GALA-MEND1 solution and a GALA-unmodified MEND1 solution were prepared in the same manner as in Example 3-1.
- RNA extraction was performed using RNA mini kit and QIA cube (manufactured by QIAGEN) according to the attached protocol. 4) Reverse transcription reaction CDNA was prepared using 1 ⁇ g of Total RNA according to the attached protocol using High Capacity RNA-to-cDNA Kit.
- Denture and reverse transcription reaction were performed using a thermal cycler under the conditions of Denatur (65 ° C., 5 min ⁇ 4 ° C., hold) and reverse transcription reaction (42 ° C., 60 min ⁇ 95 ° C., 5 min ⁇ 4 ° C., hold). . 5) mRNA quantification by Real-time PCR method MRNA (CD31) was quantified using a relative quantification method ( ⁇ Ct method) based on the TaqMan method. 100 ⁇ M Upper Primer 0.25 ⁇ L, 100 ⁇ M Lower Primer 0.25 ⁇ L, 100 ⁇ M probe 0.0625 ⁇ L, filtered DDW 6.9375 ⁇ L, TaqMan M.I. M.M.
- initial denaturation was performed at 95 ° C. for 10 min, followed by 40 cycles of PCR reaction with 95 ° C. for 15 sec PCR denaturation reaction and 60 ° C. for 1 min annealing / extension reaction. .
- CD34 as an internal standard gene, the amount of CD31 mRNA was calculated using relative quantification by the ⁇ Ct method.
- FIG. 6 shows the results of evaluating the knockdown effects of GALA-MEND1 and MEND1 in the lung 24 hours later by qRT-PCR method after administering GALA-MEND1 and MEND1 into the tail vein.
- GALA (+) indicates the result of GALA-MEND1
- GALA ( ⁇ ) indicates the result of MEND1.
- Example 7-2 In vivo knockdown evaluation 1
- in vivo transcription, mRNA extraction, reverse transcription reaction, and mRNA quantification by Real-time PCR method were used.
- GALA modification amount is 2 mol% with respect to the total lipid amount.
- FIG. 7 shows the result of evaluating the knockdown effect of GALA-modified MEND in the lung 24 hours later by qRT-PCR method after administering GALA-modified MEND into the tail vein.
- FIG. 8 shows the results of evaluating the knockdown effect of GALA-modified MEND in the liver 24 hours later by qRT-PCR method after administering GALA-modified MEND in the tail vein.
- FIG. 9 shows the results of evaluating the knockdown effect of GALA-modified MEND in the spleen 24 hours later by qRT-PCR method after administering GALA-modified MEND into the tail vein.
- GALA-modified MEND has a significantly stronger knockdown effect in the lung than AtuPLEX, which is an existing carrier.
- GALA-modified MEND was shown to have a knockdown effect in the liver when administered at 1.0-4.0 mg / kg, as compared to the existing carrier AtuPLEX. Compared with FIG. 7, it was shown that the knockdown effect in the lung was much stronger than in the liver.
- GALA-modified MEND did not have a significant knockdown effect in the spleen.
- Example 7-3 Knockdown effect of MEND with different cationic lipids
- GALA-MENDs with different cationic lipids were prepared, and the knockdown effects of each GALA-MEND were compared.
- 1) Preparation of MEND GALA-MEND1 (lipid composition is DOTMA / Chol / EPC / STR-PEG2000 / Chol-GALA) using DOTMA as the cationic lipid was prepared by the same method as in Example 3-1, and the cation was prepared.
- GALA-MEND3 (lipid composition is DSTAP / Chol / EPC / STR-PEG2000 / Chol-GALA) using DSTAP as a functional lipid is the same as the preparation method described in Example 3-1 except that DOTMA is changed to DSTAP. I went.
- GALA-MEND2 (lipid composition is DODAP / Chol / EPC / STR-PEG2000 / Chol-GALA) using DODAP as the cationic lipid was prepared in the same manner as in Example 3-2. 2)
- In vivo knockdown evaluation The knockdown effect of each GALA-MEND containing the different cationic lipids obtained in 1) above was evaluated. The evaluation method was the same as in Example 7-1.
- FIG. 10 shows the knockdown effect of GALA-MEND1 to 3 when DOTMA, DODAP, and DSTAP are used as cationic lipids.
- the dose was 0.5 mg siRNA / kg.
- MEND according to the present invention has a high knockdown effect regardless of whether the cationic lipid is a tertiary amine or a quaternary amine.
- MEND containing DOTMA as a cationic lipid has the highest knockdown effect.
- Example 7-4 MEND Knockdown Effect with Different Helper Lipids MENDs with different helper lipids were prepared, and the knockdown effects of each MEND were compared.
- MEND containing Helper lipid was performed by the same method as in Example 3-1. That is, a liposome having a DOTMA / Chol / Helper lipid ratio of 30/40/30 was prepared, and 5 mol% of STR-PEG2000 and 2 mol% of Chol-GALA were modified with respect to the total lipid amount of the liposome. GALA modified MEND was prepared.
- FIG. 11 shows the knockdown effect of each GALA-modified MEND when EPC, DOPE, and SOPE are used as the helper lipid.
- EPC is a lipid composition of DOTMA / Chol / EPC / STR-PEG2000 / Chol-GALA
- DOPE is a lipid composition of DOTMA / Chol / DOPE / STR-PEG2000 / Chol-GALA
- SOPE “" Indicates GALA modified MEND having a lipid composition of DOTMA / Chol / SOPE / STR-PEG2000 / Chol-GALA. The dose was 0.5 mg siRNA / kg.
- the GALA-modified MEND solution prepared in 1) above was administered continuously for 4 days from the tail vein of a mouse (C57BL / 6, 6w, sputum) (using a 27G injection needle).
- the body weight of the mice was measured each day from the start date of administration to 1 day after the final administration, and the change in body weight during continuous administration was calculated.
- the dose was 1 or 2 mg siRNA / kg / day.
- FIG. 12 shows changes in body weight during continuous administration of GALA-modified MEND for 4 days.
- Example 8-2 MEND AST and ALT at the time of continuous administration for 4 days
- the GALA-modified MEND used in Example 8-1 was administered to the mice via the tail vein for 4 consecutive days, and the mice were treated with ether anesthesia 24 hours after the final administration, followed by laparotomy, using a 23G injection needle and a 1 mL syringe. Blood was collected from the posterior vena cava. After leaving still at 4 degreeC for 4 hours, it centrifuged (4 degreeC, 12000 rpm, 2 minutes), and collect
- the GALA-modified MEND according to the present invention was not observed to change in AST and ALT, and it was considered that liver damage did not occur the next day after continuous administration for 4 days.
- Example 9 Evaluation of pharmacokinetics of PEG-modified MEND The pharmacokinetics of GALA-modified MEND with and without PEG modification were compared using PEG-unmodified GALA-modified MEND and PEG-modified GALA-modified MEND.
- MEND produced by the method of Example 3-1 was used.
- the amount of PEG modification was 1 mol% or 5 mol% with respect to the total lipid amount.
- the pharmacokinetic evaluation of MEND was performed according to Example 5.
- PEG unmodified MEND lipid composition: DOTMA / Chol / EPC
- 1% STR-PEG2000 modified MEND lipid composition: DOTMA / Chol / EPC / STR-PEG2000
- PEG-unmodified GALA-modified MEND lipid composition: DOTMA / Chol / EPC / Chol-GALA
- PEG-modified GALA-modified MEND lipid composition: DOTMA / Chol / EPC / STR-PEG2000 / Chol-GALA
- DOTMA / Chol / FIG. 14 shows a comparison of the lung migration rate of EPC / C8 Ceramide-PEG2000 / Chol-GALA).
- Example 10 Knockdown effect of PEG-modified MEND Using PEG-unmodified GALA-modified MEND and PEG-modified GALA-modified MEND, the knockdown effect of GALA-modified MEND with and without PEG modification was compared.
- the MEND used was the PEG-unmodified GALA-modified MEND and PEG-modified GALA-modified MEND used in Example 9. That is, STR-PEG2000 and C8ceramide-PEG2000 were used as PEG, and the amount of PEG modification was 1 mol% and 5 mol% with respect to the total lipid amount.
- the same method as in Example 7-1 was used for the MEND knockdown evaluation. The dose was 1 mg siRNA / kg.
- FIG. 15 shows a comparison of the knockdown effect between PEG-unmodified GALA-modified MEND and PEG-modified GALA-modified MEND.
- the GALA-modified MEND used was the PEG-unmodified GALA-modified MEND and PEG-modified GALA-modified MEND used in Example 9.
- STR-PEG2000 was used as the PEG, and the modification amount of the PEG was 5 mol% with respect to the total lipid amount.
- MEND was stored for 16 days at room temperature in HBG solution (pH 7.4).
- HBG solution pH 7.4
- FIG. 16 shows the knockdown effect of PEG-unmodified GALA-modified MEND and PEG-modified GALA-modified MEND stored for 16 days at room temperature.
- the GALA-modified MEND used was prepared by the method of Example 3-1, a method according to this, or a combination of these with conventional methods. MEND was stored in HBG solution for 1 month at room temperature. The properties of MEND after one month at room temperature are shown in FIG. In FIG.
- EPC is DOTMA / Chol / EPC / STR-PEG2000 / Chol-GALA
- DLPC is DOTMA / Chol / DLPC / STR-PEG2000 / Chol-GALA
- DMPC is DOTMA / Chol / DMPC / STR-PEG2000 / Chol-GALA
- DPPC is DOTMA / Chol / DPPC / STR-PEG2000 / Chol-GALA
- DSPC is DOTMA / Chol / DSPC / STR-PEG2000 / Chol-GALA
- POPC is DOTMA / Chol / POPC / STR-PEG2000 / Chol-GALA
- DOPC is DOTMA / Chol / DOPC / STR-PEG2000 / Chol-GALA
- DOPE is DO Shows the MA / Chol / DOPE / STR-PEG2000 / DOPE
- FIG. 18 shows the results of the tail vein administration of MENDs with different GALA modification amounts, and evaluation of the knockdown effects of MENDs with different GALA modification amounts in the lung 24 hours later by the qRT-PCR method.
- Example 14 Antitumor Effect in Melanoma Lung Metastasis Cancer Model by MEND Administration 1) Preparation of MEND Solution
- a GALA-modified MEND solution and a GALA-unmodified MEND solution were prepared in the same manner as in Example 3-1.
- GALA-modified MEND (1 mg) encapsulating control siRNA (anti-Luc (GL3)) or anti-CD31 siRNA every 3 days from the next day after transplantation of M16-administered B16-F10-luc2 cells to a melanoma lung metastasis cancer model siRNA / KG mouse) was administered to model mice, and the mice were laparotomized 17 days after tumor implantation, and the lungs were removed. The extracted lung was divided into two for evaluation of tumor lung metastasis and CD31 mRNA expression level. Note that the body weight of the mice was monitored during the MEND administration period.
- the administration of GALA-modified MEND encapsulating anti-CD31 siRNA significantly suppressed the progression of lung metastasis compared to the untreated group and the control siRNA administration group.
- administration of GALA-modified MEND encapsulating anti-CD31 siRNA significantly suppressed the expression level of CD31 mRNA compared to the untreated group and the control siRNA administration group. So far, it has been reported that knockout of CD31 or administration of anti-CD31 antibody inhibits angiogenesis in tumor tissue, and as a result, progression of melanoma lung metastasis is suppressed (Proc Natl Acad Sci US A. 2010 Oct 26; 107 (43): 18616-21).
- the vector modified with the GALA peptide of the present invention specifically delivers a carrier for delivering a target substance specifically to the lung, preferably a medicine, more preferably a nucleic acid medicine such as siRNA to the lung. It became clear that it was useful as a career.
- Liposomes (MEND) as substance introduction agents obtained in the examples had an average size of about 90 to 170 nm and a zeta potential in the range of ⁇ 50 to 50 mV.
- the sizes and zeta potentials of the liposomes prepared in Examples 1 and 3 are shown in Table 2 and Table 3, respectively.
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Abstract
Description
関連出願の相互参照
本願は、2011年3月14日に出願した特願2011-55765号明細書(その全体が参照により本明細書中に援用される)の優先権の利益を主張するものである。
本発明は、また、一定期間保存後も凝集しないか、もしくはほとんど凝集しないか、及び/又は保存安定性にも優れたベクターを提供することを目的とする。
すなわち、本発明は、以下の使用、物質導入剤及びベクターを提供するものである。
項1. 配列番号1で表されるGALAペプチドの、目的物質を肺に送達させるためのベクターの肺移行性素子としての使用。
項2. 前記GALAペプチドがベクターの構成成分に結合されている、項1に記載の使用。
項3. 前記ベクターが脂質及び/又はコレステロールを含み、前記GALAペプチドがカチオン性脂質及び/又はコレステロールに結合されている、項1または2に記載の使用。
項4. 前記ベクターがカチオン性脂質および/又はコレステロールを含み、前記GALAペプチドがカチオン性脂質及び/又はコレステロールに結合されている、項1または2に記載の使用。
項5. 目的物質をベクターに内包し、かつ、前記ベクターが配列番号1で表されるGALAペプチドを含む肺を標的とする物質導入剤。
項6. GALAペプチドがベクターの構成成分に結合されている、項5に記載の物質導入剤。
項7. 前記ベクターが脂質及び/又はコレステロールを含み、前記GALAペプチドがカチオン性脂質及び/又はコレステロールに結合されている、項5または6に記載の物質導入剤。
項8. 前記ベクターがカチオン性脂質及び/又はコレステロールを含み、前記GALAペプチドがカチオン性脂質及び/又はコレステロールに結合されている、項5または6に記載の物質導入剤。
項9. 前記ベクターがポリアルキレングリコール、デキストラン、プルラン、フィコール、ポリビニルアルコール、スチレン-無水マレイン酸交互共重合体、ジビニルエーテル-無水マレイン酸交互共重合体、アミロース、アミロペクチン、キトサン、マンナン、シクロデキストリン、ペクチン、カラギーナンからなる群から選ばれる親水性ポリマーにより修飾されている、項5~8のいずれか1項に記載の物質導入剤。
項10. 前記目的物質が肺で作用する生理活性物質である、項5~9のいずれか1項に記載の物質導入剤。
項11. 前記目的物質が薬物、核酸、ペプチド、タンパク質、糖及びこれらの複合体からなる群から選ばれる、項5~10のいずれか1項に記載の物質導入剤。
項12. 前記目的物質が部分二重鎖RNA(mdRNA)、ニックの入ったdsRNA(ndsRNA)、ギャップのあるdsRNA(gdsRNA)、短干渉核酸(siNA)、siRNA、マイクロRNA(miRNA)、短ヘアピンRNA(shRNA)、短干渉オリゴヌクレオチド、置換された短干渉オリゴヌクレオチド、修飾された短干渉オリゴヌクレオチド、化学修飾されたdsRNA、転写後遺伝子サイレンシングRNA(ptgsRNA)からなる群から選ばれるいずれかの二重鎖RNA(dsRNA)である、項5~11のいずれか1項に記載の物質導入剤。
項13. 前記目的物質が核酸であり、ポリエチレンイミン(PEI)を核酸とともに含む、項11又は12に記載の物質導入剤。
項14. 配列番号1で表されるGALAペプチドを肺に選択的に物質を送達させるための素子として含有する、目的物質を肺に送達させるためのベクター。
項15. 前記ベクターが脂質及び/又はコレステロールを含み、前記GALAペプチドがカチオン性脂質及び/又はコレステロールに結合されている、項14に記載のベクター。
項16. 前記ベクターがカチオン性脂質及び/又はコレステロールを含み、前記GALAペプチドがカチオン性脂質及び/又はコレステロールに結合されている、項14に記載のベクター。
項17. 前記ベクターがカチオン性脂質を含み、カチオン性脂質がDOTMA、DSTAP及びDODAPからなる群から選ばれる少なくとも1種を含む、項15又は16に記載のベクター。
項18. 前記ベクターがポリアルキレングリコール、デキストラン、プルラン、フィコール、ポリビニルアルコール、スチレン-無水マレイン酸交互共重合体、ジビニルエーテル-無水マレイン酸交互共重合体、アミロース、アミロペクチン、キトサン、マンナン、シクロデキストリン、ペクチン、カラギーナンからなる群から選ばれる親水性ポリマーにより修飾されている、項14~17のいずれか1項に記載のベクター。
項19. ポリアルキレングリコールがPEG(好ましくは、分子量が2000のPEG)である項18記載のベクター。
項20. さらに、補助脂質(Helper Lipid)を含むことを特徴とする、項14~19のいずれか1項に記載のベクター。
項21. 補助脂質(Helper Lipid)がEPC、DOPC、DOPE又はSOPEであることを特徴とする項20記載のベクター。
項22. 前記ベクターがリポソームであり、リポソーム組成がカチオン性脂質/Chol/補助脂質/STR-PEG/Chol-GALA又は補助脂質/Chol/STR-PEG/Chol-GALA(より好ましくはDOTMA、DODAP又はDSTAPから選ばれるカチオン性脂質/Chol/EPC、DOPE又はSOPEから選ばれる補助脂質/STR-PEG/Chol-GALA又はEPC/Chol/STR-PEG/Chol-GALAであり、更に好ましくはDOTMA/Chol/EPC/STR-PEG/Chol-GALA、DODAP/Chol/EPC/STR-PEG/Chol-GALA、DSTAP/Chol/EPC/STR-PEG/Chol-GALA、DOTMA/Chol/DOPE/STR-PEG/Chol-GALA、DOTMA/Chol/SOPE/STR-PEG/Chol-GALA又はEPC/Chol/STR-PEG/Chol-GALA)である項14記載のベクター。
項23. 前記ベクターがDOTMA、Chol及びEPCを脂質膜の構成要素として含むリポソームであり、前記リポソームの脂質組成(モル比)は、DOTMA/Chol/EPCが10~50/20~50/20~70であり、前記リポソームは、DOTMA/Chol/EPCの総脂質量に対しSTR-PEG2000を1~15モル%、Chol-GALAを0.1~5モル%をさらに含む、項14記載のベクター。
項24. 目的物質を内包し、かつ、配列番号1で表されるGALAペプチドを結合したベクターを哺乳動物に投与し、前記目的物質を肺に導入する方法。
項25. 抗癌剤を内包し、かつ、配列番号1で表されるGALAペプチドを結合したベクターを肺癌を有する哺乳動物に投与する工程を含む、肺癌の治療方法。
項26. 前記肺癌が肺転移した癌である項24に記載の治療方法。
項27. 配列番号1で表されるGALAペプチドを結合したベクターに抗癌剤を内包することを特徴とする肺癌治療剤。
項28. 前記肺癌が肺転移した癌である項27に記載の肺癌治療剤。
項29. 前記GALAペプチドが、総脂質量に対し1~4モル%の範囲で脂質成分に結合されている、項3に記載の使用、項7に記載の物質導入剤、項15に記載のベクター。
本発明で使用するGALAペプチドを含むベクター及び物質導入剤は、凝集の問題がないため、血管を詰まらせることがない。
Ala Ala Leu Ala Glu Leu Ala Glu Ala Leu Ala Glu Ala Leu His Glu Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Ala Glu Trp (配列番号1)
配列番号1記載のペプチドは、その配列中に、グルタミン酸(E)-アラニン(A)-ロイシン(L)-アラニン(A)の部分構造を4単位有するが、当該部分構造を好ましくは3単位又は4単位、より好ましくは、4単位を保持したまま、それ以外のアミノ酸配列において、アミノ酸の欠失、置換又は付加が行われることが好ましい。これらの改変型GALAペプチドも本発明の「配列番号1で表されるGALAペプチド」に含まれる。
(コレステリル)-O(C=O)-(WEAALAEALAEALAEHLAEALAEALEALAA)-NH2
上記において、「-O(C=O)-」はGALAペプチドのN末端のアミノ基と結合しており、「-NH2」はGALAペプチドのC末端のカルボキシル基がアミノ基で保護されていることを意味する。コレステロールとGALAペプチドの結合は、上記のようにウレタン結合でもよく、エステル結合、エーテル結合のいずれでもよい。コレステリル基は、GALAペプチドのN末端、C末端のいずれに結合してもよく、或いはGALAペプチドの任意のアミノ酸の側鎖に結合してもよい。また、コレステリル基はアルキレン、ペプチド、ポリエーテルなどの任意のリンカーを介してGALAペプチドに結合してもよい。さらに上記ではC末端がアミドになっているが、C末端はカルボン酸(COOH)、エステル、カルボン酸の塩などの他の基であってもよい。
上記に、GALAペプチドをコレステロールに結合させる実施形態について記載したが、コレステロール以外のベクターの構成要素にGALAペプチドを結合させることができ、その態様は当業者には明らかである。
目的物質が核酸の場合、例えば部分二重鎖RNA(mdRNA)、ニックの入ったdsRNA(ndsRNA)、ギャップのあるdsRNA(gdsRNA)、短干渉核酸(siNA)、siRNA、マイクロRNA(miRNA)、短ヘアピンRNA(shRNA)、短干渉オリゴヌクレオチド、置換された短干渉オリゴヌクレオチド、修飾された短干渉オリゴヌクレオチド、化学修飾されたdsRNA、転写後遺伝子サイレンシングRNA(ptgsRNA)からなる群から選ばれるいずれかの二重鎖RNA(dsRNA)が好ましく例示される。目的物質は単独で或いは2種以上を混合して用いることができる。例えば2種以上のsiRNAを併用することも可能である。
物質導入剤がsiRNAなどの核酸を目的物質として含む場合、ポリエチレンイミン(PEI)のようなカチオンを共存させるのが好ましい。
二重鎖RNAは、5‐メチルシトシン;5-ヒドロキシメチルシトシン;キサンチン;ヒポキサンチン;2-アミノアデニン;6-メチル,2-プロピル、またはアデニンおよびグアニンのその他のアルキル誘導体;8-置換されたアデニンおよびグアニン(8-アザ、8-ハロ、8-アミノ、8-チオール、8-チオアルキル、8-ヒドロキシル等);7-メチル、7-デアザ、および3-デアザアデニンならびにグアニン;2-チオウラシル;2-チオチミン;2-チオシトシン;5-メチル、5-プロピニル、5-ハロ(5-ブロモまたは5-フルオロ等)、5-トリフルオロメチル、または他の5-置換されたウラシルおよびシトシン;ならびに6-アゾウラシルなどの核酸アナログを使用することで、置換ないし修飾(化学修飾を含む)することができる。
リポソームは、脂質二重層構造を有する閉鎖小胞である限り、多重膜リポソーム(MLV)であってもよいし、SUV(small unilamellar vesicle) 、LUV(large unilamellar vesicle) 、GUV(giant unilamellar vesicle)等の一枚膜リポソームであってもよい。
本発明のベクター(キャリア)は、親水性ポリマーにより修飾することができる。
親水性ポリマーとしては、ポリアルキレングリコール(ポリエチレングリコール、ポリプロピレングリコール、ポリブチレングリコール、或いはポリエチレングリコールとポリプロピレングリコールのブロック共重合体などのポリアルキレングリコールの共重合体)、デキストラン、プルラン、フィコール、ポリビニルアルコール、スチレン-無水マレイン酸交互共重合体、ジビニルエーテル-無水マレイン酸交互共重合体、アミロース、アミロペクチン、キトサン、マンナン、シクロデキストリン、ペクチン、カラギーナンなどが挙げられ、好ましくはポリアルキレングリコール(ポリエチレングリコール、ポリプロピレングリコール、ポリブチレングリコール、或いはポリエチレングリコールとポリプロピレングリコールのブロック共重合体などのポリアルキレングリコールの共重合体)、特に好ましくはポリエチレングリコール(PEG)が挙げられ、これらの親水性ポリマーによりベクター(キャリア)を修飾するのが好ましい。PEGの長さは、500~10000程度の分子量の範囲で適宜選択することができ、好ましくは分子量が1000~5000であり、より好ましくは分子量が2000である。PEGにより修飾された脂質としては、DSPE(distearoyl phosphatidylethanolamine)-PEG2000、DMPE(dimyristoyl phosphatidylethanolamine)-PEG2000、DSG(distearoylglycerol)-PEG2000、DMG(dimyristoylglycerol)-PEG2000、コレステリル化PEG2000、STR(Stearyl)-PEG2000又はC8セラミド-PEG2000、C16セラミド-PEG2000などが挙げられ、これらのうち、STR-PEG2000又はC8セラミド-PEG2000が好ましい。他の親水性ポリマーの分子量も同様に当業者は適宜選択できる。
本発明のリポソームは、30アミノ酸から構成されるGALAペプチドを表面に有する。なお、一枚膜リポソームについては脂質二重層の外表面がリポソームの表面であり、多重膜リポソームについては最外層の脂質二重層の外表面がリポソームの表面である。また、本発明のリポソームは、表面以外の部分(例えば、脂質二重層の内表面)に上記ペプチドを有していてもよい。
カチオン性脂質において、DOTMA及びDSTAPは4級アミンを有しており,常に正電荷を帯びているが,DODAPは3級アミンを有しており,生理的pHにおいて電荷を持たないものである。このようにカチオン性脂質の種類、配合量を変更することで、カチオン性脂質の構造,特徴に幅を持たせることができる。
本発明のリポソームの好ましい態様として、上記GALAペプチドが疎水性基又は疎水性化合物で修飾されており、疎水性基又は疎水性化合物が脂質二重層に挿入され、上記ペプチドが脂質二重層から露出しているリポソームを例示することができる。なお、本態様において、「ペプチドが脂質二重層から露出している」には、ペプチドが脂質二重層の外表面又は内表面のいずれか一方から露出している場合、両方から露出している場合が含まれる。
水和法によるリポソームの製造例を以下に示す。
脂質二重層の構成成分である脂質と、疎水性基又は疎水性化合物で修飾された上記ペプチドとを有機溶剤に溶解した後、有機溶剤を蒸発除去することにより脂質膜を得る。この際、有機溶剤としては、例えば、ペンタン、ヘキサン、ヘプタン、シクロヘキサン等の炭化水素類;塩化メチレン、クロロホルム等のハロゲン化炭化水素類;ベンゼン、トルエン等の芳香族炭化水素類;メタノール、エタノール等の低級アルコール類;酢酸メチル、酢酸エチル等のエステル類;アセトン等のケトン類等が挙げられ、これらを単独で又は2種以上を組み合わせて使用することができる。次いで、脂質膜を水和させ、攪拌又は超音波処理することにより、上記ペプチドを表面に有するリポソームを製造することができる。
そのDOTMA/Chol/EPCの組成比率(モル比)は20~40/30~50/20~40であり、DOTMA/Chol/EPCの総脂質量に対して、STR-PEG2000は1~5モル%、Chol-GALAは1~4モル%であるのがより好ましく;
そのDOTMA/Chol/EPCの組成比率(モル比)は30/40/30であり、DOTMA/Chol/EPCの総脂質量に対して、STR-PEG2000は1~5モル%、Chol-GALAは1.5~2モル%であるのが最も好ましい。STR-PEG2000とChol-GALAはリポソームの修飾成分であるので、それらの添加量はDOTMA/Chol/EPCの3成分からなるリポソームの総脂質量を100モル%とし、それに対する比率として表した。
EPCは日本油脂(Tokyo,JAPAN)から購入した。DOTAP、DSTAP、DODAP、Cholesterol、DOPE、SOPE、DOPC、C8 セラミド-PEG2000はAvanti Polar Lipids(Alabaster,AL,USA)から購入した。DOTMAは東京化成工業株式会社(Tokyo,JAPAN)から購入した。STR-PEG2000は和光純薬株式会社より購入した。
Chol-GALA (コレステリル-O(C=O)-WEAALAEALAEALAEHLAEALAEALEALAA-NH2,Mw.3444.0, >71% putity)は、前記特許文献3に記載の方法、これに準じた方法又はこれらと常法とを組み合わせることにより合成した。
PEI(branch type,Mw.ave.10,000)は和光純薬株式会社より購入した。
トランスアミナーゼCII-テストワコーは和光純薬株式会社より購入した。
RNAlaterはAmbionより購入した。High Capacity RNA-to-cDNA Kit、TaqMan Gene Expression Master MixはApplied Biosystemsより購入した。siRNA及びCy5標識siRNAの合成は北海道システムサイエンスに委託した。Primer及びProbeの合成は常法に従い合成した。使用したsiRNA、Primer、Probeの配列を以下に示す。
[siRNA]
CD31-1 sense : GCACAGUGAUGCUGAACAATT (配列番号2)
CD31-1 antisense : UUGUUCAGCAUCACUGUGCTT (配列番号3)
CD31-2 sense : GUGCAUAGUUCAAGUGACATT (配列番号4)
CD31-2 antisense : UGUCACUUGAACUAUGCACTT (配列番号5)
CD31-3 sense : GCAAGAAGCAGGAAGGACATT (配列番号6)
CD31-3 antisense : UGUCCUUCCUGCUUCUUGCTT (配列番号7)
Luciferase sense : GCGCUGCUGGUGCCAACCCTT (配列番号8)
Luciferase antisense : GGGUUGGCACCAGCAGCGCTT (配列番号9)
CD31 siRNAは上記3種類を等量混合して使用した。
[Primer、Probe]
CD31 Forward : CAGAGCGGATAATTGCCATTCC (配列番号10)
CD31 Reverse : ACAGGATGGAAATCACAACTTCATC (配列番号11)
CD31 Probe : [FAM] ACCCTCAGGATCTCGCTGAACACCGC [TAMRA](配列番号12)
CD34 Forward : TCTGCCTGGAACTAAGTGAAGC (配列番号13)
CD34 Reverse : CCTCAGACTGGGCTAGAAGCA (配列番号14)
CD34 Probe : [FAM] ACCAGCATCAGCCTCAGCCTCCTCC [TAMRA](配列番号15)
ICRマウス雄5週齢、C57BL/6マウス雄6週齢は日本SLCより購入した。
その他、特に記述がない限り、市販の特級または一級のもの、あるいはそれに準ずるものを用いた。
Real-time PCRには、ABI 7500 real-time system(Applied Biosystems)を用いた。
pH測定には、Docu-pHMeter(Sartorius)を用いた。
<条件>
インジェクション量:250μL(4mg/mL in DMF)
流速:2mL/min
カラム温度:25℃
プロトコル:
1) 脂質薄膜の調製
ガラス試験管にEPC(EtOH溶液)、Chol(EtOH溶液)をモル比70/30で調製し(リポソームa)、リポソームaの総脂質量に対して、STR-PEG2000(EtOH溶液)を5モル%になるように添加し、EtOHを適当量添加後、デシケーターで減圧乾燥し、溶媒を留去することで、脂質組成がEPC/Chol/STR-PEG2000である脂質薄膜を調製した(図1では“Liposome”と略す)。また、EPC/Chol/STR-PEG2000にChol-GALAを修飾する際には、リポソームaの総脂質量の2モル%を脂質溶液中に添加し、脂質組成がEPC/Chol/STR-PEG2000/Chol-GALAである脂質薄膜を調製した(図1では“GALA-Liposome”と略す)。また、図1で“Cationic-Liposome”で表される脂質薄膜を調製する際には、DOTMA(EtOH溶液)、Chol、EPCをモル比30/40/30とし、上記と同様の操作で脂質組成がDOTMA/Chol/EPCである脂質薄膜(リポソームb)を調製した。
2) リポソームの調製
上記1)で調製した脂質薄膜に脂質濃度が2.64mM(in vivo実験)、0.55 mM(in vitro実験)となるように10mM HEPES buffer 5% Glucose(HBG)(in vivo実験)、10mM HEPES buffer(HB)(in vitro実験)を添加後、室温で15分以上水和した。その後、浴槽型ソニケーターで約1分間超音波処理を行うことで、リポソームを調製した。
(実施例2)リポソームの体内動態評価
1) リポソームの投与、臓器の回収、[3H]の測定
上記2)で調製されたリポソームの脂質膜を[3H]で標識することで投与サンプルとした。
10μL Liposome/g mouseの条件で、マウス(ICR、5週齢、♂)尾静脈より投与した。投与1、5、15分および1、6時間後にエーテル麻酔によりマウスを処置した後、開腹し、下大静脈より採血した後、肺、肝臓を摘出した。生理食塩水で臓器をよく洗浄し、重量を測定した後(肝臓に関しては、肝臓を細断後よく混合し、肝臓0.2gを使用した。)、プラスチックバイアルに入れ、2mLのSoluene-350を添加し、50℃で一晩インキュベーションすることで組織を溶解させた。この溶液に対して、Hionic fluor 10mLを添加しよく混合させた後、4℃で一晩静置し、液体シンチレーションカウンターにて[3H]のカウントを測定した。また、[3H]の投与量を評価するため、投与したLiposomeサンプル10μLに対してHionic fluor 10mLを添加し、よく混合させた後、4℃で一晩静置し、同様に[3H]のカウントを測定した。
各臓器サンプルの[3H]カウント(測定値)を測定に供した臓器重量で除することにより、各臓器1gに含まれる[3H]を算出した後、投与したリポソームに含まれる[3H]で除することで、投与量に対する臓器移行量を割合として算出した。
(実施例3-1)MEND1の調製
1) 脂質薄膜の調製
実施例1と同様の方法により、ガラス試験管にDOTMA(EtOH溶液)、Chol(EtOH溶液)、EPC(EtOH溶液)をモル比30/40/30になるように添加し、EtOHを適当量添加後、デシケーターで減圧乾燥し、溶媒を留去することで、脂質組成がDOTMA/Chol/EPCである脂質薄膜(リポソームb)を調製した。STR-PEG2000(EtOH溶液)を修飾する際には、リポソームbの総脂質量の5モル%を脂質溶液中に添加し、脂質組成がDOTMA/Chol/EPC/STR-PEG2000である脂質薄膜(以下、“MEND1”と略す場合がある)を調製した。Chol-GALAを修飾する際には、リポソームbの総脂質量の2モル%を脂質溶液中に添加し、脂質組成がDOTMA/Chol/EPC/STR-PEG2000/Chol-GALAである脂質薄膜(以下、“GALA-MEND1”と略す場合がある)を調製した。
2) siRNA complex(siRNAコア粒子)の調製
Charge ratio(+/-)=1.8となるようにPEI/siRNA complexを調製した。ボルテックス中、0.333 mg/mLのsiRNA溶液に対して、0.125mg/mLのPEI溶液を滴下(volume比 siRNA溶液:PEI溶液=6:4)し、室温で15分間以上インキュベーションすることで調製した。siRNA溶液およびPEI溶液は、HBGにより希釈したものを用いた。
3)MENDの調製
上記1)で得られた脂質薄膜を調製した試験管に、siRNAコア粒子溶液を、脂質濃度が2.64mMになるように添加後、室温で15分以上水和させた。その後、浴槽型ソニケーターで約1分間超音波処理を行うことでMENDを調製し、siRNAコア粒子を封入したMEND1及びGALA-MEND1を作製した。
(実施例3-2)MEND2の調製
1) 脂質薄膜の調製
実施例1記載の調製法に準じて、ガラス試験管にDODAP(EtOH溶液)、Chol(EtOH溶液)、EPC(EtOH溶液)をモル比30/40/30になるように添加し、成分がDODAP/Chol/EPCである脂質溶液(リポソームc)を調製した。得られた脂質溶液にSTR-PEG2000(EtOH溶液)を、リポソームcの総脂質量の5モル%を添加し、脂質組成がDODAP/Chol/EPC/STR-PEG2000である脂質薄膜を調製した(以下、“MEND2”と略す場合がある)。Chol-GALAを修飾する際には、リポソームcの総脂質量の2モル%をMEND2の脂質溶液中に添加し、EtOHを適当量添加後、デシケーターで減圧乾燥し、溶媒を留去することで、脂質組成がDODAP/Chol/EPC/STR-PEG2000/Chol-GALAである脂質薄膜を調製した(以下、“GALA-MEND2”と略す場合がある)。
2) siRNA complex(siRNAコア粒子)の調製
Charge ratio(+/-)=1.8となるようにPEI/siRNA complexを調製した。ボルテックス中、0.333mg/mLのsiRNA溶液に対して、0.125mg/mLのPEI溶液を滴下(volume比 siRNA溶液:PEI溶液=6:4)し、室温で15分間以上インキュベーションすることで調製した。siRNA溶液およびPEI溶液は、HBG(pH未調整 (pH 5.0))により希釈したものを用いることで酸性溶液コア粒子を調製した。
3)MENDの調製
上記1)で得られた脂質薄膜を調製した試験管に、siRNAコア粒子溶液を、脂質濃度が2.64mMになるように添加後、室温で15分以上水和させた。その後、浴槽型ソニケーターで約1分間超音波処理後、調製溶液が中性となるようにHBG(pH8.1)をsiRNAコア粒子溶液と等量添加することでMENDを調製し、siRNAコア粒子を封入したMEND2及びGALA-MEND2を作製した。
(実施例4)血球との相互作用
マウス血液(へパリン含、20 units/mL)とGALA-MEND1溶液を1:1のvolume比になるように混合し、37℃条件下でシェイカーを用いて5分間混合した(サンプル1)。この混合液に、血液をさらに添加しvolume比10:1とした後、先ほどと同様の条件で混合した(サンプル2)。サンプル1、2をスライドガラス上に10μL添加しカバーガラスを載せた後、顕微鏡を用いて観察した。
(実施例5)MENDの体内動態評価
MENDの脂質膜を[3H]、siRNAを[32P]で標識することで投与サンプルとした。
マウス(ICR、5週齢、♂)の尾静脈よりMEND1あるいはGALA-MEND1溶液(2mg siRNA/kg mouse)を投与した。投与1時間後にエーテル麻酔によりマウスを処置した後、開腹し、下大静脈より血液を採取した後、肺を摘出した。重量を測定した後、0.1mg程度をプラスチックバイアルに入れ、2mLのSoluene-350を添加し、50℃で一晩インキュベーションすることで組織を溶解させた。
この溶液に対して、H2O2 200μL(100μL×2)を添加することで脱色処理をした後、Hionic fluor 10mLを添加しよく混合させた。4℃で一晩静置し、液体シンチレーションカウンターにて[3H]、[32P]のカウントを測定した。また、未投与マウスから摘出した肺に既知量のMEND1溶液を添加し同様の操作を行うことで検量線を作製し、[3H]、[32P]の各臓器への移行量を算出した。
siRNAを[32P]で、脂質膜を[3H]で標識したMEND1あるいはGALA-MEND1を尾静脈投与し、1時間の肺に含まれる[32P]、[3H]を測定することにより、GALA修飾MENDの肺への移行性を評価した結果を図4に示す。
(実施例6)CLSMによるMENDの肺での局在観察
Cy5標識siRNAを用いて調製したMEND1およびGALA-MEND1を投与サンプルとした。
マウス(ICR、5週齢、♂)の尾静脈より実施例3-1で得られたMEND1あるいはGALA-MEND1溶液(2mg siRNA/kg mouse)を投与した。尾静脈投与1時間後に、麻酔下でマウスより肺を摘出し、数mm程度の肺組織片を作製した。作製した肺組織片をGSL I-B4 Isolectin FITC conjugate(フナコシ社製)溶液中(生理食塩水を用いて20μg/mLに希釈したもの)に30分間浸透させた後、CLSMを用いて観察した。
(実施例7-1)GALA修飾の有無によるin vivo ノックダウン効果
GALA修飾MEND及びGALA未修飾MENDを用いて、GALA修飾の有無によるノックダウン効果を調べた。
1)MEND溶液の調製
実施例3-1と同様の方法により、GALA-MEND1溶液及びGALA未修飾のMEND1溶液を調製した。即ち、脂質組成がDOTMA/Chol/EPC=30/40/30で調製したリポソームの総脂質量に対しSTR-PEG2000を5モル%修飾したものがMEND1であり、更にChol-GALAを総脂質量に対して2モル%修飾したものがGALA-MEND1である。
2) in vivo transfection
マウス(C57BL/6、6w、♂)の尾静脈より上記1で得られたMEND溶液(0.5-4mg siRNA/kg mouse)を投与した。尾静脈投与24時間後に、エーテル麻酔によりマウスを処置した後、開腹し、各臓器(肺、肝臓、脾臓)を摘出した。摘出した臓器は、RNAlaterに浸し、4℃で一晩置いたのち、-20℃で保存した。
3) mRNA抽出
-20℃で保存した臓器サンプルを室温に戻し、20-30mgを評量したのち、RNA mini kitおよびQIA cube(QUIAGEN社製)を用いて添付のプロトコルに従いRNA抽出を行った。
4) 逆転写反応
Total RNA 1μgをHigh Capacity RNA-to-cDNA Kitを用いて添付プロトコルに従い、cDNAを調製した。Denatureおよび逆転写反応は、サーマルサイクラーを用いて、Denature (65℃,5min→4℃,hold)、逆転写反応(42℃,60min→95℃,5min→4℃,hold)の条件で行った。
5) Real-time PCR法によるmRNA定量
TaqMan法に基づいた相対定量法(ΔΔCt法)を用いてmRNA(CD31)の定量を行った。目的の濃度に希釈したcDNA 5μLに対して、100μM Upper Primer 0.25μL、100μM Lower Primer 0.25μL、100μM probe 0.0625μL、filtrated DDW 6.9375μL、TaqMan M.M.2×12.5μL添加した後、95℃、10minで初期変性を行った後、95℃、15secのPCR変性反応、60℃、1minのアニーリング/伸長反応を1サイクルとして40サイクルPCR反応を行った。CD34を内部標準遺伝子として、ΔΔCt法による相対定量を用いてCD31のmRNA量を算出した。
(実施例7-2)In vivoノックダウン評価
1)実施例7-1と同様の方法により、in vivo transfection、mRNA抽出、逆転写反応及びReal-time PCR法によるmRNA定量を用いて、実施例3-1と同様の方法により作製したGALA修飾MEND(脂質組成は、モル比でDOTMA/Chol/EPC=30/40/30であり、GALA修飾量は総脂質量に対して2モル%である)を尾静脈投与し、24時間後の肺、肝臓及び脾臓におけるGALA修飾MENDのノックダウン効果をqRT-PCR法により評価した。
(実施例7-3)カチオン性脂質の異なるMENDのノックダウン効果
カチオン性脂質の異なるGALA-MENDを調製して、各GALA-MENDのノックダウン効果を比較した。
1)MENDの調製
カチオン性脂質としてDOTMAを用いたGALA-MEND1(脂質組成はDOTMA/Chol/EPC/STR-PEG2000/Chol-GALA)の調製は実施例3-1と同様の方法により行い、カチオン性脂質としてDSTAPを用いたGALA-MEND3(脂質組成はDSTAP/Chol/EPC/STR-PEG2000/Chol-GALA)の調製はDOTMAをDSTAPに変更する以外は実施例3-1記載の調製法に準じて行った。また、カチオン性脂質としてDODAPを用いたGALA-MEND2(脂質組成はDODAP/Chol/EPC/STR-PEG2000/Chol-GALA)の調製は、実施例3-2と同様の方法により行った。
2)in vivoノックダウン評価
上記1)で得られた異なるカチオン性脂質を含有する各GALA-MENDにおけるノックダウン効果を評価した。評価方法は、前記実施例7-1と同様の方法により行った。
Helper lipidの異なるMENDを調製して、各MENDのノックダウン効果を比較した。
Helper lipidを含むMENDの調製は、前記実施例3-1と同様の方法により行った。即ち、DOTMA/Chol/Helper lipidをモル比30/40/30とするリポソームを調製し、当該リポソームの総脂質量に対して、STR-PEG2000を5モル%、Chol-GALAを2モル%修飾したGALA修飾MENDを調製した。
異なるHelper lipidを含有する各GALA修飾MENDにおけるノックダウン効果を評価した。評価方法は、前記実施例7-1と同様の方法により行った。
(実施例8-1)MEND4日連続投与時における体重変化
1)MEND溶液の調製
実施例3-1の方法に準じてGALA修飾MEND溶液を調製した。即ち、脂質組成は、DOTMA/Chol/EPC=30/40/30(モル比)であり、GALA修飾量は、総脂質量に対して2モル%とした。
上記1)で作製したGALA修飾MEND溶液をマウス(C57BL/6、6w、♂)の尾静脈より4日間連続で投与した(27Gの注射針を使用)。投与開始日から最終投与後1日まで各日マウスの体重を測定し、連続投与時における体重変化を算出した。なお、投与量は1あるいは2mg siRNA/kg/dayとした。
(実施例8-2)MEND 4日間連続投与時におけるAST及びALT
実施例8-1で使用したGALA修飾MENDを4日間連続でマウスに尾静脈投与し、最終投与後24時間にエーテル麻酔によりマウスを処置した後、開腹し、23Gの注射針及び1mLシリンジを用いて後大静脈より血液を採取した。4℃で4時間静置した後、遠心(4℃、12000rpm、2min)し、上清を血清として回収した。血清5μLに対してAST及びALT測定キット(トランスアミナーゼCII-テストワコー)を用いて添付の方法に従い、血清中AST及びALTを測定した。その結果を図13に示す。
(実施例9)PEG修飾MENDの体内動態評価
PEG未修飾のGALA修飾MENDとPEG修飾GALA修飾MENDを用いて、PEG修飾の有無によるGALA修飾MENDの体内動態を比較した。
MENDの体内動態評価は、実施例5に準じて行った。なお、PEG未修飾MEND(脂質組成:DOTMA/Chol/EPC)、1%STR-PEG2000修飾MEND(脂質組成:DOTMA/Chol/EPC/STR-PEG2000)、1%C8セラミド-PEG2000修飾MEND(脂質組成:DOTMA/Chol/EPC/C8セラミド-PEG2000)の体内動態評価はN=2で行った。
PEG未修飾のGALA修飾MEND(脂質組成:DOTMA/Chol/EPC/Chol-GALA)とPEG修飾GALA修飾MEND(脂質組成:DOTMA/Chol/EPC/STR-PEG2000/Chol-GALA、及びDOTMA/Chol/EPC/C8セラミド-PEG2000/Chol-GALA)の肺移行率の比較を図14に示す。
PEG未修飾のGALA修飾MENDとPEG修飾GALA修飾MENDを用いて、PEG修飾の有無によるGALA修飾MENDのノックダウン効果を比較した。
使用したMENDは、実施例9で使用したPEG未修飾のGALA修飾MENDとPEG修飾GALA修飾MENDを用いた。即ち、PEGはSTR-PEG2000及びC8セラミド-PEG2000を使用し、PEG修飾量は総脂質量に対して1モル%及び5モル%とした。MENDのノックダウン評価は、実施例7-1と同様の方法を用いた。なお、投与量は1mg siRNA/kgとした。PEG未修飾のGALA修飾MENDとPEG修飾GALA修飾MENDのノックダウン効果の比較を図15に示す。
次に、一定期間保存後のPEG未修飾のGALA修飾MENDとPEG修飾GALA修飾MENDのノックダウン効果を比較した。
(実施例12)GALA修飾MENDの保存安定性
次に、Helper lipidの変更によるGALA修飾MENDの物性への影響を、室温で1ヶ月後のGALA修飾MENDの凝集の有無により確認した。
(実施例13) in vivo ノックダウンに及ぼすGALA修飾量の影響
GALA修飾量が異なるMENDを用いて、GALA修飾量がノックダウン効果に及ぼす影響を評価した。即ち、実施例7-1と同様の方法により、in vivo transfection、mRNA抽出、逆転写反応及びReal-time PCR法によるmRNA定量を用いて、実施例3-1と同様の方法により作製したGALA修飾MEND(脂質組成は、モル比でDOTMA/Chol/EPC/=30/40/30であり、当該リポソームの総脂質量に対するSTR-PEG2000修飾量は5モル%であり、GALA修飾量は1~4モル%である)を尾静脈投与し、24時間後の肺におけるGALA修飾MENDのノックダウン効果をqRT-PCR法により評価した。GALA修飾量が異なるMENDを尾静脈投与し、24時間後の肺でのGALA修飾量が異なるMENDの各ノックダウン効果をqRT-PCR法により評価した結果を図18に示す。
(実施例14)MEND投与によるメラノーマ肺転移がんモデルにおける抗腫瘍効果
1)MEND溶液の調製
実施例3-1と同様の方法により、GALA修飾MEND溶液及びGALA未修飾MEND溶液を調製した。脂質組成は、脂質組成は、モル比でDOTMA/Chol/EPC/=30/40/30であり、当該リポソームの総脂質量に対するSTR-PEG2000修飾量は5モル%であり、GALA修飾量は2モル%である。
2)メラノーマ肺転移がんモデルの作製
ルシフェラーゼ(GL4)安定発現B16-F10マウスメラノーマ細胞(B16-F10-luc2; Caliper Life Sciences, MA, USA)を10%FBSを含むRPMI-1640メディウム中で48時間培養した。マウス(C57BL/6、6w、♂)の尾静脈より、B16-F10-luc2細胞(2x105cells/100μL)を投与することによりメラノーマ肺転移がんモデルを作製した(day0)。
3)メラノーマ肺転移がんモデルへのMEND投与
B16-F10-luc2細胞移植後翌日より3日おきに、コントロールsiRNA(anti-Luc(GL3))あるいはanti-CD31 siRNAを封入したGALA修飾MEND(1mg siRNA/KG mouse)をモデルマウスに投与し、腫瘍移植後17日にマウスを開腹し、肺を摘出した。摘出した肺は腫瘍肺転移評価とCD31 mRNA発現量評価用として2つに分割した。なお、MEND投与期間中、マウスの体重をモニタリングした。
4)腫瘍肺転移の定量評価
アシストチューブ中で摘出した肺サンプルに対してin vivo lysis buffer 1 mLを添加し、POLYTRONホモジナイザー(KINEMATICA社製)を用いてホモジナイズした。ホモジェネートをサンプルチューブに回収した後、遠心(4℃、13,000rpm、10分間)し、その上清20μLをLuciferase assay substrate 50μLと混合させた後、ルミノメーター(Luminescencer-PSN, ATTO社製)でルシフェラーゼ活性を測定した。ルシフェラーゼ活性(RLU/lung)は肺全体におけるrelative light units(RLU)として算出した。結果を図19に示す。
5)CD31 mRNA発現量評価
実施例7-1と同様の方法により、摘出した肺におけるCD31 mRNA量を算出した。結果を図20に示す。
これまでにCD31のノックアウトあるいは抗CD31抗体投与は腫瘍組織における血管新生を阻害し、その結果メラノーマ肺転移の進行が抑制されることが報告されている(Proc Natl Acad Sci U S A. 2010 Oct 26;107(43):18616-21)。今回の得られたGALA修飾MEND投与により得られた肺転移の進行抑制効果は、CD31 mRNAのノックダウンにより肺転移腫瘍組織における血管新生が阻害されたことに起因していると考えられた。
なお、肺転移モデルマウスにおいてGALA修飾MEND投与による体重減少は見られなかったことから、GALA修飾MENDは毒性を示すことなく、肺転移の治療が可能であることが示唆された。
さらに、siRNA封入GALA修飾リポソームが、肺において効率的なノックダウン効果を有することが認められた。
また、本発明のGALA修飾MENDを投与しても、体重減少や肝障害が見られず、安全に投与することができることが確認された。
以上から、本発明のGALAペプチドで修飾されたベクターは、目的物質を肺に特異的に送達させるためのキャリア、好ましくは医薬、より好ましくはsiRNA等の核酸医薬を肺に特異的に送達させるためのキャリアとして有用であることが明らかになった。
実施例1及び3で調製したリポソームのサイズ及びゼータ電位をそれぞれ表2及び表3に示す。
Claims (15)
- 配列番号1で表されるGALAペプチドの、目的物質を肺に送達させるためのベクターの肺移行性素子としての使用。
- 前記GALAペプチドがベクターの構成成分に結合されている、請求項1に記載の使用。
- 前記ベクターが脂質及び/又はコレステロールを含み、前記GALAペプチドがカチオン性脂質及び/又はコレステロールに結合されている、請求項1または2に記載の使用。
- 前記ベクターがカチオン性脂質および/又はコレステロールを含み、前記GALAペプチドがカチオン性脂質及び/又はコレステロールに結合されている、請求項1または2に記載の使用。
- 目的物質をベクターに内包し、かつ、前記ベクターが配列番号1で表されるGALAペプチドを含む肺を標的とする物質導入剤。
- GALAペプチドがベクターの構成成分に結合されている、請求項5に記載の物質導入剤。
- 前記ベクターが脂質及び/又はコレステロールを含み、前記GALAペプチドがカチオン性脂質及び/又はコレステロールに結合されている、請求項5または6に記載の物質導入剤。
- 前記目的物質が薬物、核酸、ペプチド、タンパク質、糖及びこれらの複合体からなる群から選ばれる、請求項5~7のいずれか1項に記載の物質導入剤。
- 配列番号1で表されるGALAペプチドを肺に選択的に物質を送達させるための素子として含有する、目的物質を肺に送達させるためのベクター。
- 前記ベクターが脂質及び/又はコレステロールを含み、前記GALAペプチドがカチオン性脂質及び/又はコレステロールに結合されている、請求項9に記載のベクター。
- 前記ベクターがカチオン性脂質を含み、カチオン性脂質がDOTMA、DSTAP及びDODAPからなる群から選ばれる少なくとも1種を含む、請求項9又は10に記載のベクター。
- 前記ベクターがポリアルキレングリコール、デキストラン、プルラン、フィコール、ポリビニルアルコール、スチレン-無水マレイン酸交互共重合体、ジビニルエーテル-無水マレイン酸交互共重合体、アミロース、アミロペクチン、キトサン、マンナン、シクロデキストリン、ペクチン、カラギーナンからなる群から選ばれる親水性ポリマーにより修飾されている、請求項9~11のいずれか1項に記載のベクター。
- さらに、補助脂質(Helper Lipid)を含み,補助脂質(Helper Lipid)がEPC、DOPC、DOPE又はSOPEであることを特徴とする請求項9~12のいずれか1項に記載のベクター。
- 前記ベクターがDOTMA、Chol及びEPCを脂質膜の構成要素として含むリポソームであり、前記リポソームの脂質組成(モル比)は、DOTMA/Chol/EPCが10~50/20~50/20~70であり、前記リポソームは、DOTMA/Chol/EPCの総脂質量に対しSTR-PEG2000を1~15モル%、Chol-GALAを0.1~5モル%をさらに含む、項9記載のベクター。
- 目的物質を内包し、かつ、配列番号1で表されるGALAペプチドを結合したベクターを哺乳動物に投与し、前記目的物質を肺に導入する方法。
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Also Published As
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EP2687204A4 (en) | 2014-09-03 |
US9532950B2 (en) | 2017-01-03 |
CN103561725A (zh) | 2014-02-05 |
JPWO2012124688A1 (ja) | 2014-07-24 |
EP2687204B1 (en) | 2020-10-14 |
ES2832330T3 (es) | 2021-06-10 |
EP2687204A1 (en) | 2014-01-22 |
US20140079770A1 (en) | 2014-03-20 |
CN103561725B (zh) | 2018-08-31 |
JP5850915B2 (ja) | 2016-02-03 |
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