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LIPID MEMBRANE STRUCTURE FOR DELIVERY INTO SIRNA CELL meetings

Foreign code F190009750
File No. (S2017-0830-N0)
Posted date May 7, 2019
Country WIPO
International application number 2018JP022940
International publication number WO 2018230710
Date of international filing Jun 15, 2018
Date of international publication Dec 20, 2018
Priority data
  • P2017-117708 (Jun 15, 2017) JP
Title LIPID MEMBRANE STRUCTURE FOR DELIVERY INTO SIRNA CELL meetings
Abstract A lipid membrane structure contains, as a lipid component, a lipid compound represented by formula (I): (R1)(R2)C(OH)-(CH2)a-(O-CO)b-X [wherein a represents an integer of 3 to 5; b represents an integer of 0 or 1; R1 and R2 independently represent a linear hydrocarbon group that may have -CO-O-; and X represents a 5- to 7-membered non-aromatic heterocyclic group or a group represented by formula (B) (wherein d represents an integer of 0 to 3; and R3 and R4 independently represent a C1-4 alkyl group or a C2-4 alkenyl group, wherein R3 and R4 may be bonded together to form a 5- to 7-membered non-aromatic hetero ring (wherein one or two C1-4 alkyl groups or C2-4 alkenyl groups may be present as substituents on the ring))].
Outline of related art and contending technology BACKGROUND ART
Agent specific to the diseased part of the lipid membrane as a means of transporting an agent is a liposome structure has been proposed a method of encapsulating. In particular, the treatment of malignant tumors filled with anti-tumor agents in the art that the effectiveness of the liposomes are many reported. In addition, the available gene expression as a multi-functional lipid membrane structure enveloped nanostructures (MEND: Multifunctional envelope-type nano device; hereinafter, abbreviated as' MEND ' is used herein. For example see Non-Patent Document 1 or the like) is proposed. This structure is, in a particular cell such as a gene for the selective delivery can be used as a drug, for example, such as gene therapy of a tumor is known to be useful.
Lipid drug using the film structure, nucleic acid, peptide, polypeptide, a sugar such as the target organ or tumor tissue such as the target substance to a specific site as a means for delivering, to the surface of the lipid membrane structure is modified with functional molecules have been proposed many methods. Anti-tumor agents of an agent such as a lipid containing membrane structure may be, reaches to a target cell within the cell by endocytosis incorporated in the state and are encompassed within the endosome, thereafter, a lysosomal enzyme hydrolysis action or the like included in the receiving agent has been released into the cytoplasm. Endosome incorporated into the liposome in order to enhance the release of drug from, the surface of the liposome (GALA: Non-Patent Document 2) the peptide in the liposome (Non-Patent Document 3) or (Patent Document 4) the MEND has been proposed.
In addition, the nucleic acid containing a target substance such as a lipid film structure is transferred into the nucleus of the target cell as a means for, for example, the external surface of the liposome in the liposome octaarginine (Patent Document 1, Non-Patent Document 4), nuclear translocation peptides modified with a lipid membrane (Patent Document 2) has a membrane of two 2, galactose or mannose surface of monosaccharides such as liposome (Patent Document 3) has been proposed. Modified saccharide multiple lipid membrane structure is a lipid membrane and nucleus (T-MEND) of the fuzed film, in the in vitro test results and thereby improving the efficiency of gene expression have been reported. Further, (non-patent document 5) by modifying the KALA peptide lipid film structure of a material such as nucleic acid into the nucleus of the cell efficiently deliver (Patent Document 5) have been reported.
On the other hand, dendritic cells, the center of the immune response from the responsible for antigen presenting cell, a cancer immunotherapy is one of significant target cell 1. Dendritic cells from a cancer patient collected, outside the activation is conducted after introduction of an antigen, an immune cell therapy is administered to the patient again (dendritic cell therapy) have also been carried out. In recent years, in a dendritic cell has been found from immunosuppressive factors, dendritic cells are also attracting attention as a target of the siRNA and pharmaceutical, dendritic cells can be combined with therapies therapy, cancer immunity inducing the more powerful and can be expected.
Conventional, dendritic cells for introducing RNA into the nucleus, a lentiviral vector expressing the shRNA used immunosuppressive factors knocked down are reported (Non-Patent Document 6, Non-Patent Document 7) is present. However, artificial delivery systems using siRNA introduction to the dendritic cells is hardly reported. The use of viral vectors of the target gene knockdown be achieved in a high efficiency, there is a problem in safety.
SiRNA delivery system is introduced as an artificial R8/GALA-D-MEND(D-MEND) have been reported (Non-Patent Document 8). D-MEND is, the cell affinity (R8) peptide octaarginine element in the endosome escape element GALA MEND modified peptide, the number of the envelope film nano MEND control carrier. D-MEND is, commonly used in cancer cells are HeLa cells at concentrations as low as about 12 nm 70% the concentration of the siRNA knockdown of shown, its activity is introduced into the general purpose as a reagent (LFN2000) compared to 2 2000 Appropriate times more activity.
However, the mouse bone marrow cells derived from transfected with dendritic cells in the case D-MEND, 70-80% siRNA knockdown efficiency in order to achieve a higher concentration (80-120 nm) is necessary, the target of the siRNA knockdown efficiency by a factor of about 40% is also a problem that remains in the (non-patent document 9). In this way a conventional artificial in the case of using a delivery system, a typical cancer cells than in the dendritic cells tend to knock down efficiency is largely lowered, deployed in the field of pharmaceutical immune therapy siRNA are prevented.
So far, the functional nucleic acid, in particular of a given target gene expression in vivo delivery of siRNA capable of suppressing in order to achieve efficient, the number of cationic lipids have been developed. In particular, physiological pH is electrically neutral, mildly acidic endosomal pH environment such as changes in pH sensitive cationic significant development of the cationic lipid. Jayaraman et al. developed DLin-MC3-DMA, the first (F7) in mouse liver ED50 in the knock-down factors in 7 to achieve a 0.005 mg siRNA/kg (non-patent document 10). The present inventors so far unique pH sensitive cationic lipid and has developed a YSK13-C3 YSK05, F7 knockdown in achieving 0.06,0.015 mg siRNA/kg as the ED50 (non-patent document 11, Non-Patent Document 12, Non-Patent Document 13). In addition, a biodegradable Maier et al. MC3-DMA was developed to provide L319, ED50 and 0.01 mg siRNA/kg in both the high safety was reported (Non-Patent Document 14, Non-Patent Document 15, Non-Patent Document 16). However, these lipid-containing lipid nanoparticles of the endosome escape efficiency is still only about % number and not the (non-patent document 17), to further improve the bioavailability of the development of technology has been desired.
Further, Dong et al. is a lipid-like material through a high-found unique cKK-E12, F7 knockdown in ED50 was achieved in 0.002 mg siRNA/kg (non-patent document 18). On the active surface of the art literature is the most excellent, high dose toxicity in lipid biosynthesis and degradation in the safety and the like is not found.
In recent years, many cancer tissue, especially a human patient is cancerous tissue, collagen including interstitial component is very abundant, the components of the nanoparticles in cancer tissue significantly interfere with the permeability being revealed. The size of the nanoparticles in order to solve this problem very thought of as an effective strategy. In fact, Cabral et al., the diameter of the micelle polymer in the formulation of platinum about 30 nm to the sealing is controlled to be small in the cancerous tissue in the improved permeability, anti-tumor effect can be enhanced (Non-Patent Document 19) reports. SiRNA delivery in the same strategy is considered to be very effective, lipid nanoparticles (Lipid Nanoparticle: LNP) is technically difficult to be small, the report is very poor. 2 Liquid mixture can be achieved at the moment of the built-in micro flow path of the mixer which has a diameter of about 30 nm can be manufactured with good reproducibility in the LNP, in recent years have been reported (Non-Patent Document 20, Non-Patent Document 21). On the other hand, in the size of the LNP, the siRNA delivery activity has been found to significantly reduce the (non-patent document 22, Non-Patent Document 23). To overcome this problem for the treatment of cancer or siRNA delivery technique is excellent in order to achieve a very important role on the other hand, at present no knowledge about the method to overcome this is.
Scope of claims (In Japanese)請求の範囲 [請求項1]
 下記の式(I):
[化1]

〔式中、aは3~5の整数を示し;bは0又は1の整数を示し;R 1及びR 2はそれぞれ独立に下記の式(A):
[化2]

(式中、qは1~9の整数を示し;rは0又は1を示し;sは1~3の整数を示し;tは0又は1を示し;uは1~8の整数を示し;cは0又は1を示し:vは4~12の整数を示すが、bとcが同時に0となる場合には、qが3~5の整数であり、r及びtが1であり、sが1であり、かつu+vが6~10の整数である場合を除く) で表される基を示し;Xは5~7員非芳香族ヘテロ環基(ただし、当該基は炭素原子により(O-CO)b-に結合し、当該環上には1又は2個のC 1-4アルキル基又はC 2-4アルケニル基が置換していてもよい)、又は下記の式(B):
[化3]

(式中、dは0~3の整数を示し、R 3及びR 4はそれぞれ独立にC 1-4アルキル基又はC 2-4アルケニル基(当該C 1-4アルキル基又はC 2-4アルケニル基は1個又は2個のフェニル基で置換されていてもよい)を示すが、R 3及びR 4は互いに結合して5~7員非芳香族ヘテロ環(当該環上には1又は2個のC 1-4アルキル基又はC 2-4アルケニル基が置換していていてもよい)を形成してもよい)で表される基を示す〕
で表される脂質化合物又はその塩。

[請求項2]
 r及びtが0であり、q+s+uが8~18の整数、好ましくは10~16の整数である、請求項1に記載の脂質化合物又はその塩。

[請求項3]
 rが1であり、tが0であり、qが5~9の整数であり、s+uが5~9の整数である、請求項1又は2に記載の脂質化合物又はその塩。

[請求項4]
 vが5~12の整数である、請求項1ないし3のいずれか1項に記載の脂質化合物又はその塩。

[請求項5]
 aが4であり、bが0又は1である、請求項1ないし4のいずれか1項に記載の脂質化合物又はその塩。

[請求項6]
 bが0であり、Xが式(B)で表される基〔ただし、dは0であり、R 3及びR 4はそれぞれ独立にC 1-4アルキル基(R 3が示すC 1-4アルキル基は1個のフェニル基で置換されていてもよい)を示すか、あるいはR 3及びR 4が互いに結合する場合には、1-ピロリジニル基、1-ピペリジニル基、1-モルホリニル基、又は1-ピペラジニル基(当該1-ピロリジニル基、1-ピペリジニル基、1-モルホリニル基、又は1-ピペラジニル基は1個のC 1-4アルキル基で置換されていてよい)を形成する〕である、請求項1ないし5のいずれか1項に記載の脂質化合物又はその塩。

[請求項7]
 bが1であり、Xが式(B)で表される基〔ただし、dは0~3の整数であり、R 3及びR 4はそれぞれ独立にC 1-4アルキル基(R 3が示すC 1-4アルキル基は1個のフェニル基で置換されていてもよい)を示すか、あるいはR 3及びR 4が互いに結合する場合には、1-ピロリジニル基、1-ピペリジニル基、1-モルホリニル基、又は1-ピペラジニル基(当該1-ピロリジニル基、1-ピペリジニル基、1-モルホリニル基、又は1-ピペラジニル基は1又は2個の同一又は異なるC 1-4アルキル基で置換されていてよい)を形成する〕である、請求項1ないし5のいずれか1項に記載の脂質化合物又はその塩。

[請求項8]
 bが1であり、Xが5~7員非芳香族ヘテロ環基(当該基は炭素原子により(O-CO)b-に結合する)であり、当該5~7員非芳香族ヘテロ環基がピロリジニル基、ピペリジニル基、モルホリニル基、又はピペラジニル基(当該ピロリジニル基、ピペリジニル基、モルホリニル基、又はピペラジニル基は、1又は2個の同一又は異なるC 1-4アルキル基で置換されていてよい)である、請求項1ないし5のいずれか1項に記載の脂質化合物又はその塩。

[請求項9]
 細胞内にsiRNAを送達するための脂質膜構造体の脂質成分として用いられる、請求項1ないし8のいずれか1項に記載の脂質化合物又はその塩。

[請求項10]
 脂質成分として請求項1ないし8のいずれかに記載の脂質化合物又はその塩を含む脂質膜構造体。

[請求項11]
 リポソームである、請求項10に記載の脂質膜構造体。

[請求項12]
 siRNAが内部に封入されている請求項10又は11に記載の脂質膜構造体。

[請求項13]
 細胞内の標的遺伝子をノックダウンするために用いられる、請求項12に記載の脂質膜構造体。

[請求項14]
 前記細胞が、免疫細胞又はがん細胞である、請求項13に記載の脂質膜構造体。

[請求項15]
 患者から樹状細胞を分離・採取し、in vitroで当該樹状細胞の細胞内にsiRNAを導入した後、標的遺伝子がノックダウンされた樹状細胞をその患者に投与する免疫療法において、樹状細胞における標的遺伝子をノックダウンするために用いられる、請求項14に記載の脂質膜構造体。

  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • HOKKAIDO UNIVERSITY
  • Inventor
  • HARASHIMA Hideyoshi
  • SATO Yusuke
IPC(International Patent Classification)
Specified countries National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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