Inhibitors of extracellular proteases

a protease inhibitor and extracellular technology, applied in the field of protease inhibitors, can solve the problems that no one has used stress to improve or modify, and achieve the effect of reducing degradation

Inactive Publication Date: 2011-09-08
BIOPHARMACOPAE DESIGN INT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0053]The subject invention involves extracts from the tissues of plant species which provide inhibitory activity against extracellular proteases. In one embodiment, the present invention relates to the use of plants to produce extracts or semi-purified / purified compounds, compositions and formulations demonstrating an inhibitory activity against one or more proteases involved in the proteolytic degradation of human extracellular matrix. Such extracts, compounds, compositions and formulations derived from plant sources, optionally from water, ethanol or organic extracts prepared from said plant tissues, and fractions separable from said extracts by chromatography or centrifugal ultra-filtration or other means. In one aspect, these extracts with inhibitory activity can be used during protein purification to minimize the degradation due to extracellular proteases.
[0093]Chromatographic means include all means of carrying out chromatography known by those skilled in the art and described in G. Sofer, L. Hagel, Handbook of Process Chromatography, Academic Press, 1997. Fractionation, partial purification, and / or purification can be carried out by but not limited to regular column chromatography, flash chromatography, high performance liquid chromatography (HPLC), medium pressure liquid chromatography (MPLC), supercritical fluid chromatography (SFC), countercurrent chromatography (CCC), moving bed chromatography, simulated moving bed chromatography, expanded bed chromatography, and planar chromatography. With every chromatographic methods, sorbents that may be used include but is not limited to silica gel, alumina, fluorisil, cellulose and modified celluloses, all possible modified silica gels, all types of ion-exchange resins, all types of size exclusion gels and any other sorbents known from those skilled in the art and described in T. Hanai. HPLC: A Practical Guide. RSC Press, UK 1999. The present invention also includes the use of two or more solvent gradients to effect the fractionation, partial purification, and / or purification of said active extracts in any chromatographic methods. The solvents that may be utilized include but are not limited to hexanes, pentane, petroleum ethers, cyclohexane, heptane, diethyl ether, methanol, ethanol, isopropanol, propanol, butanol, isobutanol, tert-butanol, water, dichloromethane, dichloroethane, ethyl acetate, tetrahydrofurane, dioxane, tert-butyl methyl ether, acetone, and 2-butanone. When water or and aqueous phase is used, it may contains certain amounts of iorganic or organic salts and the pH may be adjusted to different values with an acid or a base to enhance fractionation and / or purification.

Problems solved by technology

Second, the action of proteases is confined to specific areas by various secreted protease inhibitors, such as the tissue inhibitors of metalloproteases and the serine protease inhibitors known as serpins.
Third, many cells have receptors on their surface that bind proteases, thereby confining the enzyme to where it is needed.
No one has used stress to improve or modify plants human protease inhibitor content.

Method used

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  • Inhibitors of extracellular proteases
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Examples

Experimental program
Comparison scheme
Effect test

example i

Preparation of Stressed and Non-stressed Plant Extracts

[0103]Pre-Harvest TreatmentAerian parts of a living plant are sprayed with an aqueous solution of gamma linolenic acid (6,9,12-Octadecatrienoic acid, Sigma L-2378) (stress G) or arachidonic acid (5,8,11,14-Eicosatetraenoic acid, Sigma A-3925) (stress A) (400 μM in water with 0.125% (v / v) Triton X-100) to completely cover the leaves.

Harvest Solid SI and Optional Storage Treatment

[0104]Twenty to twenty-four hours after the stress, more than 4 grams of leaves, stems, fruit, flowers, seeds or other plant parts are harvested and frozen immediately in dry ice, then transferred as soon as possible to a −20° C. freezer until use. Plant materials may be stored at −20° C. for a long period of time, more than a year, without losing inhibitory activity. Temperature is monitored to ensure a constant condition.

[0105]Stressed and non-stressed plant specimens are collected as wet samples and stored at −20° C. for various periods of time, and ar...

example ii

In vitro Enzyme Inhibition Assays

[0113]The inhibitory activity of sample compositions towards human MMP-1, human MMP-2, human MMP-3, human MMP-9, human cathepsin-B, human cathepsin-D, human cathepsin-G, human cathepsin-L, human cathepsin-K, human leukocyte elastase (HLE), bacteria clostripain and bacteria subtilisin can be determined using either fluorogenic substrates or the FASC assay.

Measurement of Human MMP-1, -2, -3 and -9 Activity with Fluorogenic Peptidic Substrates

[0114]MMP-1, -2, -9 are purified from natural sources (human immortalized cell lines: 8505C (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH) for MMP-1, HT-1080 (ATCC, Manassas, Va.) for MMP-2 and THP-1 (ATCC, Manassas, Va.) for MMP-9) as described in literature and based on protocols found in I. M. Clark: Matrix metalloproteinases protocol>>, Humana Press (2001). Recombinant human MMP-3 is overexpressed in E. Coli and purified according to Windsor L J, Steele D L (2001), Methods Mol Biol 151:191-205. P...

example iii

Examplary Purification of Inhibitory Activityfound in an Extract

[0124]Extracts were separated by HPLC on an Agilent 1100 system (San Fernando, Calif.). Briefly, 100 μl of a crude extract prepared as described in Example I was applied on a C18 reverse-phase column (Purospher RP-18 5 μm, 4.0×125 mm (HP), Agilent, San Fernando, Calif.). Elution of compounds was achieved with a linear gradient of 10-85% acetonitrile. Fractions were collected, evaporated, resuspended in aqueous buffer and then reanalysed for their inhibition activity on specific enzymes as already described. Fractions of interest (demonstrating a biological activity) where then reisolated at a larger scale for further analysis and characterization.

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Abstract

Provided is a plant derived extract including inhibitory activity against one or more extracellular proteases which degrade human tissue matrix. Moreover, the amount of inhibitory activity in an extract can be increased by stressing the plant prior to forming an extract. These extracts are each prepared by a process and demonstrate the ability to inhibit one or more extracellular proteases which degrade human tissue matrix. Libraries of extracts can be prepared from stressed and non-stressed plants, where each of the extracts demonstrate inhibitory activity against on or more extracellular protease inhibitors. Alternatively, semi-purified and purified inhibitory compounds can be isolated from the extracts. In one aspect, these extracts with inhibitory activity can be used during protein purification to minimize degradation due to extracellular proteases.

Description

CROSS-REFERENCE TO EARLIER FILED APPLICATIONS[0001]This is a Continuation Application of U.S. patent application Ser. No. 11 / 878,978, filed Jul. 30, 2007, which was a Continuation Application of U.S. patent application Ser. No. 10 / 469,402, filed Aug. 29, 2003 and claims benefit of International Application No. PCT / CA02 / 00285 filed Mar. 4, 2002, the contents of each of which are hereby incorporated by reference in their entirety.FIELD OF INVENTION[0002]The invention pertains to the field of protease inhibitors, specifically inhibitors of extracellular proteases.BACKGROUND OF THE INVENTION[0003]The cells of tissues are generally in contact with a network of large extracellular macromolecules that occupies the spaces in a tissue between the component cells and also occupies the space between adjacent tissues. This extracellular matrix functions as a scaffolding on which the cells and tissue are supported and is involved actively in regulating interaction of the cells that contact it. T...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/99A61K36/00A61P43/00G01N33/53
CPCA61K8/97A61K36/00A61K2800/782A61K36/185A61Q19/08C12N9/99A61K2300/00A61K8/9761A61K8/9767A61K8/9789A61K8/9794A61P43/00A61K36/704
Inventor CYR, BENOIT
Owner BIOPHARMACOPAE DESIGN INT
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