Therapeutic peptide-based constructs

a peptide-based, construct technology, applied in the direction of peptide sources, bactericidal/permeability-increasing proteins, angiogenin, etc., can solve the problems of protamine and its limited clinical utility, and achieve the effects of inhibiting angiogenesis, and inhibiting endothelial cell proliferation

Inactive Publication Date: 2006-07-06
LITTLE ROGER +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

LPS induces the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock.
However, significant hypotensive and anaphylactoid effects of protamine have limited its clinical utility.

Method used

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  • Therapeutic peptide-based constructs

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation and Purification of Peptide-Based Constructs

[0085] This example addresses the preparation and purification of peptide-based constructs, including derivatized constructs, according to the invention.

[0086] Peptide-based constructs may be prepared according to a variety of synthetic procedures. For example, BPI-derived peptides have been prepared by solid phase peptide synthesis as described in co-assigned U.S. patent application Ser. No. 08 / 183,222 and U.S. Pat. No. 5,733,872, according to the methods of Merrifield, 1963, J. Am Chem. Soc. 85: 2149 and Merrifield et al., 1966, Anal. Chem., 38: 1905-1914 using an Applied Biosystems, Inc. Model 432 peptide synthesizer.

[0087] Alternatively, BPI-derived peptides have been synthesized on a larger scale using solid phase peptide synthesis on an Advanced Chemtech (ACT-Model 357 MPS) synthesizer utilizing a 1-Fluorenylmethyl-oxycarbonyl(Fmoc) protection strategy with a double coupling procedure employing N,N-diisopropylcarbodiim...

example 2

In Vitro HUVEC Assay

[0095] This example addresses the activity of peptide-based constructs, including derivatized constructs, in an in vitro assay of endothelial cell proliferation.

[0096] Briefly, human umbilical vein endothelial cells (HUVEC) were cultured in EGM-2 medium (Clonetics Corp., San Diego, Calif.), harvested and plated onto a 96-well culture plate. After 24 hours of quiescence, the cells were co-cultured with various peptide-based constricts in the presence of growth factors for 72 hours to test their activity. Cell proliferation was quantitated by tritiated thymidine incorporation into DNA and was expressed in counts per minute (cpm).

[0097] Details of the experimental procedures were as follows: HUVEC cells were cultured in EGM-2 media from Clonetics Corp., a subsidiary of BioWhittaker, Inc., San Diego, Calif. [EGM-2 is Endothelial Cell Basal Medium-2 (EMB-2) supplemented with growth factors (hFGF-B, VEGF, IGF-1, hEGF) ascorbic acid, heparin, GA-1000 and 2% FBS), in ...

example 3

In Vivo Matrigel® Angiogenesis Assays

[0099] This example addresses the activity of peptide-based constructs, including derivatized constructs, in in vivo assays of angiogenesis using Matrigel®.

[0100] For these experiments, peptide-based constructs according to the invention were assayed for their ability to inhibit heparin-induced angiogenesis in vivo in mice. Basement membrane matrix (Matrigel®, Becton Dickinson Labware, Mountain View, Calif.) was thawed and maintained at 4° C. and angiogenic factors were added to the gel in the liquid state generally as described in Passaniti et al., 1992, Lab. Invest. 67: 519-528. Heparin sodium (Sigma, St. Louis, Mo.) was dissolved in sterile PBS to various concentrations ranging from 1,250-10,000 U / mL, and used in these experiments at 8,000 U / mL. Human recombinant basic fibroblast growth factor (bFGF; Sigma) was reconstituted into 1. % BSA in PBS to 25 μg / mL. A volume of 2.5 μL dissolved heparin solution and 4.0 μL recombinant bFGF were added...

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Abstract

The present invention relates generally to small peptide-based constructs, including derivatized constructs, and their therapeutic uses. The sequences of these constructs are based on a reverse subsequence derived from Domain II of bactericidal / permeability-increasing protein (BPI).

Description

[0001] This is a continuation-in-part of U.S. application Ser. No. 09 / 344,219 and U.S. Application No; 09 / 344,827, each filed Jun. 25, 1999, each hereby incorporated by reference in their entirety.FIELD OF THE INVENTION [0002] The present invention relates generally to small peptide-based constructs that have 8 to 15 amino acid moieties, including derivatized constructs. The sequences of these constructs are designed and prepared based on a reverse subsequence (99-85) of amino acids identified and selected from Domain II of bactericidal / permeability-increasing protein (BPI). The invention further relates to therapeutic uses of such constructs due to their heparin-related properties of heparin binding, heparin neutralization, inhibition of endothelial cell proliferation and / or inhibition of angiogenesis, e.g., inhibition of in vivo neovascularization, including in models of chronic inflammatory disease states and metastatic tumors. BACKGROUND OF THE INVENTION [0003] Bactericidal / perm...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/10A61K38/08A61K38/00C07K14/47
CPCA61K38/08A61K38/10C07K14/4742
Inventor LITTLE, ROGERLIN, JONG-JYEGIKONYO, J.G.KINYUA
Owner LITTLE ROGER
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