Novel peptides isolated from spider venom, and uses thereof

A venom and terminal amide technology, applied in the field of spider venom peptides, can solve the problems that neurons cannot generate action potentials, and cannot encode and transmit information.

Inactive Publication Date: 2012-07-11
ALOMONE PRECLINICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Inhibit Na V 1 channel causes neurons to be unable to generate action potentials and therefore unable to encode and transmit information

Method used

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  • Novel peptides isolated from spider venom, and uses thereof
  • Novel peptides isolated from spider venom, and uses thereof
  • Novel peptides isolated from spider venom, and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0216] Peptide purification and chemical characterization from crude venom

[0217] Peptides A, B, C, D, E and F were isolated from Pterinochilus spp. Usambara (Psp), Metal Blue spider (Hv) and RedMorph Grammostola (RMG) spider venoms using several different chromatography steps (see Figure 1- 6):

[0218] Chromatographic purification of peptide A from Psp venom

[0219] According to the procedure described in the Methods section, 100 mg of lyophilized crude venom (110 mg by Nanodrop) was loaded on a gel filtration column.

[0220] Fractions (#32-36) were collected, filtered through a 0.22 μm cellulose acetate filter, and 1 / 4 of the sample was loaded into pre-treated solvent A (in 0.1% TFA 5% ACN) equilibrated on a Phenomenex Jupiter reverse phase HPLC column (5u, C18, 300A, 250 x 4.6mm, 00G-4053-EO, S / No397274-10). Proteins were eluted by step gradient using solvent A (= 5% ACN in 0.1% TFA) and 60% ACN in 0.1% TFA as mobile phase (= buffer B), run at a constant flow rate o...

Embodiment 2

[0250] Refolding of synthetic peptides

[0251] According to Schnolzer, M. et al., (1992) In situ neutralization in BOC-chemistry solid phase peptide synthesis, Int. J. Pept. Protein Res. 40, 180-193, and Atherton, E. et al., (1989) Solid Phase Peptide Synthesis (IRL, Oxford, U.K.), produced synthetically by solid-phase synthesis procedures by chemical synthesis using BOC (tert-butoxycarbonyl) or Fmoc (9-fluorenylmethoxycarbonyl) solid-phase peptide synthesis Peptides A, B, C, D, E and F were provided as lyophilized powders with a purity of 70-95%. Each peptide was then subjected to a different refolding procedure as described below to achieve correct folding and the same biological activity as the venom-purified native peptide.

[0252] Peptide A refolding

[0253] Crude synthetic peptides were dissolved in 0.1 M Tris buffer pH 8.5 at a protein concentration of 5 mM and then reduced with 20 mM DTT for 1 hour at room temperature (RT). The reduced peptide was added to a fo...

Embodiment 3

[0279] In vitro biological activity of isolated pure peptides

[0280] For TTX-sensitive Na V 1.3 and TTX tolerant Na V 1.8 Channels were tested for blocking activity. Each of these is stably expressed in mammalian cell lines and elicits channel currents by appropriate voltage stimulation. Toxin activity is examined by measuring current before, during (usually at increasing doses) and after perfusion of the toxin. The proportion of inhibitory currents at each toxin dose was measured in at least three separate cells and the mean ± SD was plotted.

[0281] Peptide A inhibits Na V 1.3 and Na V 1.8 channels with apparent IC 50 1.75 and 2.5 μM, respectively.

[0282] Peptide B inhibits Na V 1.3 channels where the apparent IC 50 is 230 nM.

[0283] Peptide C inhibits Na V 1.3 channels where the apparent IC 50 was 1.2 μM.

[0284] Peptide D inhibits Na V 1.3 channels where the apparent IC 50 is 160 nM.

[0285] Peptide E inhibits Na V 1.3 channels where the apparent ...

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Abstract

The presently described subject matter relates to isolated spider venom peptides, which are used as potent and selective ion channel blockers, and to a composition and methods for treatment of pain.

Description

technical field [0001] The subject matter described herein relates to isolated spider venom peptides useful as potent and selective ion channel blockers, as well as compositions and methods for the treatment of neuropathic pain. Background technique [0002] According to the generation and transmission of action potentials, voltage-dependent sodium (Na V 1) Channels and in some cases low voltage activation of calcium (Ca V 3) Channels are membrane proteins that are present in all excitable cells, including neurons. Inhibit Na V 1 channel causes the neuron to be unable to generate action potentials and therefore unable to encode and transmit information. [0003] Neuropathic pain is a complex neuronal process involving peripheral nerve hyperexcitation and central nervous sensitization. Peripheral hyperexcitability may be due to ectopic spontaneous firing of damaged dorsal root ganglion (DRG) neurons, which is transmitted to the central nervous system (CNS) and perceived a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/16A61K38/00
CPCC07K14/43518A61K38/00A61P25/00A61P25/02A61P25/04A61P25/06A61P29/00A61P43/00
Inventor 阿隆·梅厄罗尼特·西姆哈·切尔基埃拉·科尔贝耶尔·兰古特尼西姆·巴扎约
Owner ALOMONE PRECLINICAL
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