Peptide ligand directed drug delivery

A liposome and compound technology, applied in the field of peptide ligand-directed drug delivery, can solve the problem of weakening curative effect

Inactive Publication Date: 2010-10-06
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0001] Chemotherapy is widely used in the treatment of cancer, but its efficacy is often undercut by severe side effects due to drug toxicity to normal tissues

Method used

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  • Peptide ligand directed drug delivery
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  • Peptide ligand directed drug delivery

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Egg Yolk Phosphatidylcholine, 1,2-Distearoyl-sn-Glyceryl-3-Phosphoethanolamine (DSPE), 1,2-Distearoyl-sn-Glyceryl-3-Phosphoethanolamine-N-[ Methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[maleimide (polyethylene glycol )2000] (DSPE-PEG2000-Mal) and cholesterol were purchased from Avanti Polar Lipids (AL, USA). Lissamine TM Rhodamine B, 1,2-Dihexadecanoyl-sn-Glyceryl-3-Phosphoethanolamine (Rhodamine DHPE) and N-(Fluorescein-thiocarbamoyl)-1,2-Dihexadecanoyl- sn-glyceryl-3-phosphoethanolamine (fluorescein DHPE) was purchased from Invitrogen. N-Succinimide 3-(2-pyridinedithio)-propionate (SPDP) and tris(carboxyethyl)phosphine (TCEP) were purchased from Pierce Biotechnology, Inc. Cy5.5 Mono NHS Ester was provided by GE Healthcare. All other chemicals of analytical grade were obtained from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China).

[0079] The crystal structure of EGFR in the inactive state was downloade...

Embodiment 2

[0086] Peptide LARLLT was synthesized and purified by GL Biochem Ltd. (Shanghai), and the structure and purity were confirmed by HPLC and MS. For comparison, also synthesized named D4 * A peptide with the same amino acid composition as LARLLT, but with a scrambled sequence (RTALLL, control peptide (SEQ ID NO: 2)). To fluorescently label the two peptides, Cy5.5-NHS was mixed with LARLLT or control peptide at a molar ratio of 1:2 and incubated overnight at 25°C, protected from light. The fluorescent peptide used was not further purified.

Embodiment 3

[0088] EGF or LARLLT / control peptides were dissolved in PBS-EDTA and mixed with N-succinimide-3-(2-pyridyldithio)-propionate (SPDP) dissolved in DMSO at room temperature at 1 : 1.2 molar ratio was mixed for 1 hour and then lyophilized. At the same time, the DSPE-PEG2000-Mal lipid in chloroform was evaporated to dryness to form a thin film, and hydrated in HEPES buffer (pH 7.4) at a concentration of approximately 0.4 mM. For conjugation, thioated proteins or peptides were added to tris(2-carboxyethyl)phosphine (TCEP) solution, incubated for 1 hour at room temperature under nitrogen, and rapidly mixed with MAL-PEG2000-DSPE micellar solution to A molar ratio of 5:1 was mixed and the reaction was kept stirring overnight at 10°C under nitrogen. By HPLC analysis, almost all Mal-PEG-DSPEs were modified after this reaction. Excess protein / peptide can be removed using standard techniques, eg, through gel filtration columns, dialysis, etc.

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Abstract

Provided is a novel peptide ligand (Leu-Ala-Arg-Leu-Leu-Thr) for binding to an EGFR surface pocket based on its 3D crystal structure. When conjugated to the distal end of liposome surface PEG moieties, the peptide ligand directs liposome binding and uptake by EGFR high expressing cancer cells (H1299 and SPCA1) specifically and efficiently. The targeted delivery of liposomal anticancer drug doxorubicin results in better therapeutic efficacy towards cells in vitro. In vivo, the targeted liposomes are injected via tail vein and the time course of their distribution and accumulation in xenograft tumor tissues are studied using a live animal fluorescence imaging system. The LARLLT targeted liposomes were seen to gradually concentrate at the tumor site and be preferentially retained more than 80 hours after injection.

Description

Background of the invention [0001] In the treatment of cancer, chemotherapy is widely used, but its efficacy is often undercut by severe side effects caused by drug toxicity to normal tissues. To improve the therapeutic index of chemotherapeutic agents, many studies have focused on strategies to specifically deliver drugs to tumor tissues while avoiding delivery to normal tissues. [0002] Several liposome-based anticancer drug formulations have been developed and have been used in patients, which have better efficacy and fewer side effects than non-liposomal formulations. One of the most successful products (1) is Doxil (also known as Caelyx), which consists of PEG-coated liposomes (stealth liposomes) with prolonged serum half-life and passage through leaky vasculatures. Ability to progressively extravasate and accumulate in tumors. In addition to such passive targeting mechanisms, active targeting strategies have also been proposed, in which antibodies or targeting ligands...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/00C07K17/08A61K38/08A61K9/127A61K47/48A61P35/00C07K14/71
CPCA61K49/0041A61K49/0043A61K49/0017A61K49/0032A61K49/0056C07K7/06A61K49/0084A61K9/1271A61K47/48238B82Y5/00A61K38/00A61K47/48815A61K47/62A61K47/6911A61P35/00
Inventor 徐宇虹宋姝贤刘丹
Owner SHANGHAI JIAOTONG UNIV
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