Sphingolipid-polyalkylamine-oligonucleotide compounds

一种聚烷基胺、寡核苷酸的技术，应用在基因治疗、药物组合、基因工程等方向，能够解决使用受限细胞毒性阳离子脂质等问题

Inactive Publication Date: 2016-03-30
QBI ENTERPRISES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cationic lipids have been used to deliver therapeutic oligonucleotides, however, their use is limited by cytotoxicity and the fact that cationic lipids accumulate primarily in the liver

Method used

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  • Sphingolipid-polyalkylamine-oligonucleotide compounds
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  • Sphingolipid-polyalkylamine-oligonucleotide compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0310] The selection and generation of the sense strand and the antisense strand of embodiment 1.dsRNA

[0311] 18-mer and 19-mer sequences of potential dsNAs are generated using a proprietary algorithm and the known sequence of the target gene. The antisense strand sequence generated by this method is completely or substantially complementary to the target mRNA sequence segment. In some embodiments, the antisense sequence is fully complementary to the corresponding segment of mRNA sequence. In general, double-stranded nucleic acid molecules with specific sequences selected for in vitro testing are specific for both human and a second species such as rat, mouse non-human primate or rabbit genes.

[0312] Exemplary compounds target Rac1 (Homosapiensras-related C3 botulinum toxin substrate 1 (rho family, small GTP-binding protein Rac1) (RAC1), transcript variant Rac1, mRNA) gi|156071503|ref|NM_006908.4|( SEQ ID NO: 1); PLK1 (Homosapienspolo-like kinase 1) gi|34147632|ref|NM_...

Embodiment 2

[0357] Embodiment 2: the synthesis of sphingolipid-spermine / spermidine phosphoramidite

[0358] figure 1 A scheme for the synthesis of sphingolipid-spermine / spermidine-phosphoramidites is shown. Abbreviations: TFA: trifluoroacetoxy; TBDMS: tert-butyldimethylsilyl; CC-column chromatography; MeOH-methanol; THF-tetrahydrofuran; DSC-Di(succinate)carbonate; DMAP-4-di methylaminopyridine; DCM-4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran; TBAF-tetra-n-butylammonium fluoride.

[0359] Details of the synthetic steps are provided below:

[0360] Compound 2

[0361] To a solution of D-erythro-sphingosine (1 g of compound 1) in MeOH (16 mL) was added ethyl trifluoroacetate (0.6 mL) and triethylamine (0.93 mL). The mixture was stirred for 16 hours, then the solvent was evaporated to dryness under reduced pressure to afford crude compound 2. The crude product was purified by CC (silica, DCM / MeOH as eluent).

[0362] Compound 3

[0363] Compound 2 (1 g) was disso...

Embodiment 3

[0376] Example 3: Synthesis of chimeric oligonucleotides

[0377] Incorporation of sphingolipid-polyalkylamine phosphoramidites into oligonucleotides during synthesis by conjugation to oligonucleotides, in particular into oligonucleotides that can be used to generate antisense oligonucleotide compounds or double-stranded RNA nucleic acid molecules Antisense and / or sense strand, including siRNA, siNA, anti-miR and miRNA.

[0378] For large-scale synthesis (20 μmol), sphingolipid-polyalkylamine phosphoramidites (300 mg) were dissolved in acetonitrile (1.65 ml, 0.15M). The sphingolipid-polyalkylamine coupling was performed twice (coupling time 10 min for each coupling step).

[0379] Cleavage and deprotection of siRNA-sphingolipid-polyalkylamine

[0380] For oligonucleotide strands (e.g., siRNA strands) bound to resin (344 mg), add NH in a sealed tube 4 OH (33% in water): Methylamine (33% in EtOH) (v / v; total 3.44ml) and incubated in a hot zone at 65°C for 3.5 hours. After ...

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Abstract

Provided herein are sphingolipid-polyalkylamine phosphoramidites, methods of generating sphingolipid-polyalkylamine-oligonucleotide compounds, pharmaceutical compositions comprising such compounds, and to methods of use thereof in treating cancer.

Description

[0001] sequence listing [0002] This application incorporates by reference the nucleotide sequence in the file named "250_PCT1.ST25", which is 24kb in size, created on July 27, 2014, in IBM-PC machine format, with the operating system MS-Windows Compatibility, and accompanying commits. [0003] related application [0004] This application claims the benefit of US Provisional Application No. 61 / 860,274, filed July 31, 2013, entitled "SPHINGOLIPID-POLYALKYLAMINE-OLIGONUCLEOTIDE COMPOUNDS," which is hereby incorporated by reference in its entirety for all purposes. field of invention [0005] Disclosed herein are sphingolipid-polyalkylamine-based compounds including sphingolipid-polyalkylamine phosphoramidites and methods of producing sphingolipid-polyalkylamine-oligonucleotide compounds, said sphingolipid-polyalkylamine Alkylamine-oligonucleotide compounds, pharmaceutical compositions comprising said sphingolipid-polyalkylamine-oligonucleotide compounds and methods of use th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00A61P35/00C08G73/02
CPCA61K48/0041A61K2039/55555C08G73/02C12N15/113C12N2310/14C12N2310/3515C12N2320/32A61K47/543A61P35/00C12N15/111C12N15/1135C12N15/1137C12N2310/11C12N2310/113C12N2310/141C12Y207/11021A61K48/00C12N2310/351
Inventor 莎伦·阿夫金-纳祖姆吉恩·希尔德斯海姆提塔萨·克莱门吉恩-克里斯多佛·特鲁菲特杰拉尔德·马蒂斯
Owner QBI ENTERPRISES
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