Novel cell penetrating peptide, conjugate thereof with botulinum toxin, and use thereof

A botulinum toxin, penetrating peptide technology, applied in the direction of fusion with toxin, bacterial antigen components, medical preparations with non-active ingredients, etc., to achieve the effect of avoiding toxic effects

Active Publication Date: 2017-02-22
PROCELL THERAPEUTICS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Moreover, since the skin-penetrating and nerve terminal cell-penetrating botulinum toxin light chains or light chain derivatives are sought in the present invention, the amount of light chains or light chain derivatives should be limited to a concentration of 1-10 ppm to ensure by even toxicity-reducing methods Safety, therefore, it seems that PTD is not suitable for use as a skin penetration tool and a nerve cell penetration tool

Method used

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  • Novel cell penetrating peptide, conjugate thereof with botulinum toxin, and use thereof
  • Novel cell penetrating peptide, conjugate thereof with botulinum toxin, and use thereof
  • Novel cell penetrating peptide, conjugate thereof with botulinum toxin, and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0123] Example 1. Construction of novel cell penetrating peptides

[0124] Development of novel skin-penetrating and cell-penetrating peptides capable of transdermal delivery of the light chain of botulinum toxin. First, the structure and function of the heavy and light chains of botulinum toxin are analyzed, and sequences are selected based on the fact that the heavy chain plays an important role in the penetration of botulinum toxin type A into nerve cells. Moreover, compared with the conventional MTD sequence, a novel cell-penetrating peptide consisting of the amino acid sequence of SEQ ID NO: 1 is designed by increasing the accessibility of the cell membrane by setting amphipathic polar amino acids, improving Physical properties and solubility and by adding non-polar amino acids to provide suitable hydrophobicity for the cell membrane penetration. The cell-penetrating peptide designed according to the above description was named TD1, and its properties and structure were ...

Embodiment 2

[0125] Example 2. Demonstration of the in vitro cell penetrating potential of the cell penetrating peptide TD1 using flow cytometric analysis

[0126] Experiments were conducted using flow cytometry analysis to confirm the penetration potential of the novel cell-penetrating peptide TD1 constructed according to Example 1 against skin cells and nerve cells. In order to compare the cell penetrating properties of the cell penetrating peptide TD1, a previously developed cell penetrating peptide, kFGF4, and a representative protein translocation domain (PTD), HIV-Tat, were used as a control MTD, and each peptide Samples were fluorescently labeled with FITC and synthesized by a professional organization for peptide synthesis (GL Biochem Ltd. (Shanghai, China)).

[0127] 2.1 Quantitative analysis of cell penetration potential in keratinocytes (HaCaT cells)

[0128]To confirm the cell penetration potential in keratinocyte HaCaT cells, HaCaT cells were cultured using DMEM complete medi...

Embodiment 3

[0136] Example 3. Demonstration of the in vitro cell penetrating potential of the cell penetrating peptide TD1 using confocal microscopy

[0137] 3-1. Quantitative analysis of cell penetration potential in keratinocytes (HaCaT cells)

[0138] To demonstrate the cell penetrating potential in HaCaT cells, which is a keratinocyte line, the cells were cultured using DMEM complete medium (10% FBS, 1% penicillin / streptomycin). For microscopy, 12 mm coverslips were flame sterilized, added to each well of a 24-well plate, and the HaCaT cells were seeded into the plate and incubated for 16 to 24 hours. Each sample (reference: Vehicle, comparative: HIV-Tat&kFGF4-derived peptide, test: TD1) was added to the cells in the FBS-free medium at 3 μM and 5 μM, respectively, and the Cells were incubated for 1 hour and 3 hours, respectively. After the reaction, the medium was completely removed by aspiration, the steps of adding PBS to the plate and gently shaking the plate were repeated to was...

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Abstract

The present invention relates to: a novel cell penetrating peptide; a cell penetrating botulinum toxin recombinant protein composition in which the cell penetrating peptide and the light chain of a botulinum toxin are fused; and a use thereof and, more specifically, to a composition enabling the transdermal delivery of a cell penetrating botulinum toxin recombinant protein and capable of being locally used for various treatments of the skin and cosmetic purposes. The cell penetrating peptide-botulinum toxin recombinant protein of the present invention can be transdermally delivered, thereby having the intrinsic effect of a botulinum toxin and simultaneously having greater convenience of use, and thus can be effectively applied as a local agonist for the treatment of various diseases and aesthetic and / or cosmetic purposes.

Description

technical field [0001] The present invention relates to novel cell penetrating peptides, cell penetrating botulinum toxin recombinant proteins in which the cell penetrating peptide is conjugated to one end of the light chain of botulinum toxin, and uses thereof. Background technique [0002] Botulinum toxin is a neurotoxin produced by the Gram-positive anaerobic bacterium Clostridium botulinum, which grows in spoiled canned food or spoiled meat. Botulinum toxin is classified into 8 types of neurotoxins, seven of which (A, B, C, D, E, F, G) can induce nerve paralysis. Botulinum toxin is approximately 150 kDa in size and forms a complex of botulinum toxin and non-toxin proteins. Complexes of each of the sizes are formed to have sizes up to 900 kDa depending on the type of neurotoxin. The type and target of action and duration of activity can vary depending on the type of botulinum toxin, and botulinum toxin type A is considered a lethal biological agent. [0003] Botulinum ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K19/00C12N15/11C12N15/62C12N15/70A61K39/08A61K47/62A61P17/00
CPCA61K39/08C07K7/08C07K19/00C12N15/70A61K47/50A61K38/10A61K38/4893C12N9/52C12Y304/24069A61P17/00A61P17/04A61P21/02A61P25/04A61P25/08A61P29/00Y02A50/30A61K8/64A61Q19/00C07K14/33C07K2319/02C07K2319/21C12N15/63A61K9/0014C07K2319/55
Inventor 李秉圭李刚真金玟中朴洪圭
Owner PROCELL THERAPEUTICS
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