Huwentoxin-iv variants and methods of use

A tiger-striped tarantula and toxin technology, which can be applied to pharmaceutical formulations, medical preparations containing active ingredients, peptide/protein components, etc., and can solve problems such as no successful reports

Inactive Publication Date: 2015-07-08
JANSSEN BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to its favorable potency and selectivity spectrum, protoxin-II has been the subject of several in vivo studies aimed at demonstrating analgesia, none of which reported success without perineurial disruption

Method used

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  • Huwentoxin-iv variants and methods of use
  • Huwentoxin-iv variants and methods of use
  • Huwentoxin-iv variants and methods of use

Examples

Experimental program
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Effect test

example 1

[0211] Design and Preparation of Huwen Tarantula Toxin-IV Variants

[0212] A single-site amino acid scanning library was prepared to replace the wild-type Ornithoctonus huwena derived from the venom of the Chinese tarantula Ornithoctonus huwena (ECLEIFKACNPSNDQCCKSSKLVCSRKTRWCKYQI; SEQ ID NO: 1). Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at each non-cysteine ​​residue. Huwentoxin-IV variants are encoded as HRV3C protease-cleavable human serum albumin (HSA) fusion proteins from N-terminus to C-terminus in the form: His 6 -HSA-(GGGGS) 4 -HRV3C cleavage site -Huwentoxin-IV variant. Each variant peptide has a residual N-terminal GP from the cleavage site after cleavage from HSA and a C-terminal GK that is the endogenous amidation recognition sequence. Single-site variants were tested in a fluorescence-based screening assay measuring their ability to inhibit veratridine-induced membrane potential, and hits were confirmed in Q...

example 2

[0223] Example 2. Characterization of Huwentoxin-IV Variants

[0224] Membrane depolarization assay

[0225] use FRET assay (fluorescence resonance energy transfer) on Tetra using DISBAC2(3) (Invitrogen, K1018) as electron acceptor and PTS18 (8-octadecyloxypyrene-1,3,6-trisodium trisulfonate ) (Sigma) as a donor, by exciting the donor at 390-420nm and measuring FRET at 515-575nm, the prepared Huwentoxin-IV variants were measured to inhibit the Nav1.7 agonist veratridine (3-veratridine Ability of redrine; Biomol, Catalog #NA125) to induce membrane depolarization.

[0226] HEK293F cells stably expressing hNav1.7 channels under G418 selection (Invitrogen) were cultured in DMEM / F12 supplemented with glutamine, 10% FBS, 1% NEAA and 400 μg / ml G-418. 50 μl of harvested cells were plated at 25,000 cells / well in poly-lysine coated 384-well black clear bottom plates. Plates were incubated at room temperature (RT) for 15 min, followed by overnight incubation at 37°C. All incuba...

example 3

[0249] Example 3. Analgesic activity of Huwentoxin-IV following intraplantar administration in rats method

[0250] Male Sprague-Dawley (CD) rats (Charles River, San Diego) weighing >300 grams were used in this study. Naive animals were trained 2 days prior to the test day (in order to reduce variability in responses). Training consisted of performing the actual trials on each animal multiple times for each rat over a duration of -1 hour. Animals first received a sharpie mark proximal to the toes in the middle of the back of the left paw to enable consistent trials of the same position of the paw. The rat was then loosely wrapped in a towel, leaving the hind paw uncovered, and the left hind paw was placed in a Randall Selitto device (Ugo-Basile Randall-Selitto Device, Analgesy- Meter) with a Sharpie marker just below the cone of the test device that contacts the paw, and pressure is increased at a steady rate through an electronic ramp with foot control until the animal ...

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Abstract

The present invention relates to Huwentoxin-IV variants, polynucleotides encoding them, methods of making and using the foregoing, and methods of alleviating pain with peptide inhibitors of Nav1.7.

Description

[0001] Cross References to Earlier Applications [0002] This application claims the benefit of U.S. Provisional Application 61 / 781,276, filed March 14, 2013, and U.S. Patent Application 13 / 833,555, filed March 15, 2013, which claim U.S. The benefit of Provisional Application 61 / 702,538 and US Provisional Application 61 / 648,871, filed May 18, 2012, the entire contents of which are hereby incorporated by reference. technical field [0003] The present invention relates to Huwentoxin-IV variants, polynucleotides encoding them, methods of making and using the foregoing, and methods of alleviating pain with peptide inhibitors of Nav1.7. Background technique [0004] Voltage-gated sodium channels (VGSCs) are present in all excitable cells, including cardiomyocytes and skeletal muscle cells as well as central and peripheral neurons. In neuronal cells, sodium channels are responsible for amplifying threshold depolarization and generating rapid rises in action potentials. As such,...

Claims

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Application Information

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IPC IPC(8): A61K38/17
CPCC07K14/43518A61P19/02A61P25/04A61P29/00A61P43/00
Inventor W.埃科特M.弗林斯帕奇M.亨特Y.刘R.内夫A.维肯登A.吉布斯
Owner JANSSEN BIOTECH INC
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