Lipid membrane structure for delivery into sirna cell

A technology of structure and lipid, which is applied in the direction of liposome delivery, antibody, medical preparation of non-active ingredients, etc., and can solve the problem of reduced siRNA delivery activity and other issues

Active Publication Date: 2020-01-31
HOKKAIDO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, it was found that by miniaturizing LNP, its siRNA delivery activity was significantly reduced (Non-Patent Document 22, Non-Patent Document 23)
Overcoming this problem is extremely important in realizing an excellent siRNA delivery technology for cancer therapy, and there is currently no insight into how to overcome it

Method used

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  • Lipid membrane structure for delivery into sirna cell
  • Lipid membrane structure for delivery into sirna cell
  • Lipid membrane structure for delivery into sirna cell

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0166] The lipid compound of the present invention was synthesized according to the following scheme. When the hydrophobic scaffold is the same as YSK12 (Patent Document 6), that is, when synthesizing a lipid in which c in the general formula (A) is 0, linoleic acid (compound A) is used as a starting material. After reduction of linoleic acid with lithium aluminum hydride (compound B), activation by mesylation of the hydroxy group (compound C), bromination by exposure to magnesium bromide (compound D). Grignard reaction was carried out using δ-valerolactone as a substrate to connect two (2) linoleic acid-derived hydrophobic scaffolds (compound E). When the tertiary amino group is directly bonded to the hydrocarbon chain, the primary hydroxyl group is activated by tosylation (compound F), and the amino group is introduced by a nucleophilic substitution reaction.

[0167] On the other hand, when a tertiary amino group is bonded via an ester bond, it is connected by dehydration ...

example 2

[0404] Hydrophobic scaffolds were pH-sensitive cationic lipids derived from linoleic acid and having various hydrophilic sites. LNP is based on the method of the example (Example 2) of Patent Document 6, each pH-sensitive cationic lipid, cholesterol, methoxypolyethylene glycol 2000 dimyristoylglycerol (PEG-DMG 2000) is set as a mole Ratio 50:50:0.75-1.5, by alcohol dilution method ( figure 1 ) for modulation. The average particle diameter calculated by the phase light scattering method is 80-120nm, and the siRNA loading rate is above 90%.

[0405] The pKa of each LNP was determined using p-toluenesulfonic acid (TNS). TNS (final concentration: 0.75 μM) and LNP (final concentration: 30 μM) were mixed in a buffer adjusted to each pH, ​​and the fluorescence intensity was measured with a microplate reader. The highest value and the lowest value were set as 100% and 0% charge, respectively, and the pH at which the charge rate was 50% was set as pKa for calculation. As a result,...

example 3

[0409] Two types of hydrophilic sites, CL4 and CL15, were fixed, and the effect of changing the chemical structure of the hydrophobic scaffold was evaluated in the same manner as in Example 2. When using TNS to measure pKa, CL4 is 6.25-6.40, CL15 is 6.80-7.25, and the influence brought by the change of hydrophobic scaffold structure is not observed ( Figure 4A and 4B ). With regard to in vitro knockdown activity, derivatives other than CL15B outperformed previous CL15A with a hydrophobic scaffold ( Figure 5 ). In particular, CL15H having oleic acid as a hydrophobic scaffold exhibited about three times higher activity than CL15A. Regarding the F7 knockdown activity in vivo, the activities of the hydrophobic scaffolds C and D of CL4 and CL15 were all low, while the hydrophobic scaffold H showed the same level of activity as the hydrophobic scaffold A ( Figure 6A and Figure 6B ).

[0410] In particular, for CL4H showing high F7 knockdown activity, the formulation formul...

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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))].

Description

technical field [0001] The present invention relates to a lipid membrane structure used for intracellular delivery (delivery) of siRNA (short interfering RNA) and the like. More specifically, the present invention relates to lipid membrane structures such as liposomes that can easily deliver siRNA and the like into the nucleus of immune cells, particularly dendritic cells. [0002] This application claims priority based on Japanese Patent Application No. 2017-117708 for which it applied in Japan on June 15, 2017, and uses the content here. Background technique [0003] As a means for specifically delivering a drug to an affected area, a method of encapsulating the drug in a liposome, which is a lipid membrane structure, has been proposed. Especially in the field of treatment of malignant tumors, there are many reports on the effectiveness of liposomes encapsulating antitumor agents. In addition, as a lipid membrane structure that can be used for gene expression, a multifun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C69/30C07D295/03A61K9/127A61K31/713A61K47/18C07D211/32
CPCA61K31/713A61K47/18C07D295/03C07D211/62C07D295/15C07C219/06A61K9/1271A61K9/1272A61K9/5123C07K16/2803A61P25/28A61P9/10A61K2039/505C07K2317/21C07K2317/52C07K2317/53C07K2317/55C07K2317/565C07K2317/74C07K2317/75C07K2317/92A61K9/127C07D211/32
Inventor 原岛秀吉佐藤悠介
Owner HOKKAIDO UNIVERSITY
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