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US-12622878-B2 - Cationic lipid having cystine skeleton

US12622878B2US 12622878 B2US12622878 B2US 12622878B2US-12622878-B2

Abstract

The present invention provides, a cationic lipid represented by the formula (1) (wherein each symbol is as defined in the specification), a lipid membrane structure using the aforementioned cationic lipid, a nucleic acid-introducing agent using the aforementioned cationic lipid, and a method for introducing a nucleic acid by using the aforementioned nucleic acid-introducing agent.

Inventors

  • Shinya TAMAGAWA
  • Kota TANGE
  • Yuta Nakai
  • Masamichi Tamada
  • Hidetaka Akita
  • Hiroki Tanaka
  • Yu SAKURAI

Assignees

  • NOF CORPORATION
  • NATIONAL UNIVERSITY CORPORATION CHIBA UNIVERSITY

Dates

Publication Date
20260512
Application Date
20210319
Priority Date
20200327

Claims (16)

  1. 1 . A cationic lipid represented by the formula (1): wherein L 1a and L 1b are each independently an amide bond, a carbamate bond, or a urea bond, R 1a and R 1b are each independently an alkylene group having not more than 8 carbon atoms or an oxydialkylene group having not more than 8 carbon atoms, ka and kb are each independently 0 or 1, X a and X b are each independently a dialkylamino group having two alkyl groups each independently having 1 to 5 carbon atoms, or a 3- to 6-membered cyclic amino group, L 4a and L 4b are each independently an ester bond or an amide bond, R 2a and R 2b are each independently an alkylene group having not more than 8 carbon atoms or an oxydialkylene group having not more than 8 carbon atoms, L 2a and L 2b are each independently an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, ma and mb are each independently 0 or 1, R 3a and R 3b are each independently a divalent group derived from an aromatic compound having 3 to 16 carbon atoms and at least one aromatic ring, and optionally having a hetero atom, L 3a and L 3b are each independently an ester bond, an amide bond, a carbamate bond, an ether bond, or a urea bond, na and nb are each independently 0 or 1, R 4a and R 4b are each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms, or R 5 —CO—(CH 2 )p—, R 5 is a residue of a liposoluble vitamin having a hydroxy group or a residue of a sterol derivative having a hydroxy group, and p is 2 or 3.
  2. 2 . The cationic lipid according to claim 1 , wherein the na and nb are both 1.
  3. 3 . The cationic lipid according to claim 1 , wherein the R 3a and R 3b are each independently a group represented by the formula (2): wherein *shows a bonding position with L 3a or L 3b , ** shows a bonding position with L 2a , L 2b , L 4a , or L 4b , s is an integer of 0 to 3, t is an integer of 0 to 3, u is an integer of 0 to 4, and R 6 in the number of u are each independently a substituent.
  4. 4 . The cationic lipid according to claim 3 , wherein the s is 0.
  5. 5 . The cationic lipid according to claim 1 , wherein the ka and kb are both 0.
  6. 6 . The cationic lipid according to claim 1 , wherein the ka and kb are both 1.
  7. 7 . The cationic lipid according to claim 1 , wherein the R 5 is a residue of a liposoluble vitamin having a hydroxy group.
  8. 8 . The cationic lipid according to claim 1 , wherein the R 4a and R 4b are each independently an aliphatic hydrocarbon group having 12 to 22 carbon atoms.
  9. 9 . The cationic lipid according to claim 1 , wherein the R 4a and R 4b are each independently R 5 —CO—(CH 2 ) p —, and R 5 is a residue of a liposoluble vitamin having a hydroxy group.
  10. 10 . A lipid membrane structure comprising the cationic lipid according to claim 1 as a constituent lipid of the membrane.
  11. 11 . A nucleic acid-introducing agent comprising the cationic lipid according to claim 1 .
  12. 12 . A method for introducing a nucleic acid into a cell in vitro, comprising bringing the nucleic acid-introducing agent according to claim 11 into contact with the cell, wherein the nucleic acid is encapsulated in the nucleic acid-introducing agent.
  13. 13 . A method for introducing a nucleic acid into a target cell, comprising administering the nucleic acid-introducing agent according to claim 11 to a living organism to allow for delivery of the nucleic acid to the cell, wherein the nucleic acid is encapsulated in the nucleic acid-introducing agent.
  14. 14 . A nucleic acid-introducing agent comprising the lipid membrane structure according to claim 10 .
  15. 15 . A method for introducing a nucleic acid into a cell in vitro, comprising bringing the nucleic acid-introducing agent according to claim 14 into contact with the cell, wherein the nucleic acid is encapsulated in the nucleic acid-introducing agent.
  16. 16 . A method for introducing a nucleic acid into a target cell, comprising administering the nucleic acid-introducing agent according to claim 14 to a living organism to allow for delivery of the nucleic acid to the cell, wherein the nucleic acid is encapsulated in the nucleic acid-introducing agent.

Description

TECHNICAL FIELD The present invention relates to a cationic lipid having a cystine skeleton, a lipid membrane structure containing the same, and use thereof. BACKGROUND ART For practicalization of nucleic acid therapy, an effective and safe nucleic acid delivery carrier is demanded. While virus vectors are nucleic acid delivery carriers with good expression efficiency, the development of non-viral nucleic acid delivery carriers that can be used more safely is ongoing. Among them, carriers using a cationic lipid are non-viral nucleic acid delivery carriers most generally used at present. Cationic lipids are largely composed of an amine moiety and a lipid moiety, wherein the amine moiety showing cationicity and a polyanion nucleic acid electrostatically interact to form a liposome or lipid membrane structure, which promotes uptake into cells and delivers the nucleic acid into cells. As known cationic lipids generally and widely used, DOTAP and DODAP can be mentioned. These known cationic lipids form a positively-charged liposome or lipid membrane structure when combined with a phospholipid, which electrostatically interacts with a nucleic acid to be able to deliver the nucleic acid to the target cells (non-Patent Literature 1). On the other hand for a lipid membrane structure using a cationic lipid to exhibit a practical effect in vivo as a nucleic acid delivery carrier, the requirements of good pharmacokinetics, specifically high stability in blood, property to highly accumulate in the target tissues such as liver, tumor, and the like need to be fulfilled. In response to this problem, the following report on examples of improving pharmacokinetics by adjusting surface pKa of lipid membrane structures. Non-Patent Literature 2 and non-Patent Literature 3 show that pharmacokinetics and distribution in each cell in the liver can be controlled by adjusting the surface pKa of lipid membrane structures. Furthermore, these literatures describe that escape of lipid membrane structures from endosomes is promoted and nucleic acids can be efficiently delivered into the cytoplasm by adjusting the pKa of lipid membrane structures to a value advantageous for endosome escape. While cationic lipids having improved pharmacokinetics have been developed as shown above, in view of the property of the nucleic acid delivery carriers that they generally introduce exogenous substances into cells, a large effect output from a small uptake amount is desired. That is, when a lipid membrane structure is used as a delivery carrier of an expression vector into cells, it is desired to increase the expression level per unit lipid membrane structure incorporated into the cells and enhance intracellular expression efficiency. To enhance the intracellular expression efficiency, it is necessary to also improve, besides pharmacokinetics, intracellular kinetics such as uptake process into cells, escape from endosome, nuclear membrane permeation, and the like (non-Patent Literature 4). There are examples in which intracellular dynamics were improved by imparting biodegradability to cationic lipids (Patent Literatures 1 to 4). These literatures describe a cationic lipid having a structure linked by a biodegradable disulfide bond. These literatures show that the cationic lipid can improve intracellular dynamics by dissociating nucleic acid from a lipid membrane structure by utilizing intracellular cleavage of a disulfide bond. In fact, it has been clarified that the cationic lipid can improve intracellular dynamics such as improvement of delivery efficiency of nucleic acid into the cytoplasm and the like, since it shows high nucleic acid delivery efficiency as compared with known cationic lipids, DOTAP and DODAP. Furthermore, the effect of reducing toxicity is expected by imparting degradability. As shown above, there are plural reports on improving the pharmacokinetics and intracellular dynamics of nucleic acid delivery carriers by adjusting the surface pKa of lipid membrane structures, introducing disulfide bonds with degradability, and the like. However, nucleic acid therapy targets a wide variety of diseases. In order to establish a treatment method suitable for each disease, it is desired to increase the lipid types available for selection and further improve the effect. CITATION LIST Patent Literature [PTL 1]U.S. Pat. No. 9,708,628[PTL 2]WO 2016/121942[PTL 3]WO 2019/188867 PTL 4 US 2018/0298379Non Patent Literature[NPL 1]Biomaterials 29(24-25): 3477-96, 2008[NPL 2]Molecular Therapy 24(4): 788-795, 2016[NPL 3]Angewante Chemie International Edition 51: 8529-8533, 2012[NPL 4]Molecular Therapy 13(4): 786-794, 2006 SUMMARY OF INVENTION Technical Problem The problem of the present invention is to provide a cationic lipid that shows good intracellular dynamics and can be used as a carrier for nucleic acid delivery, a lipid membrane structure using the aforementioned cationic lipid, a nucleic acid-introducing agent using the aforementioned cationic lip