US 2004O175439A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0175439 A1 Cyr (43) Pub. Date: Sep. 9, 2004 (54) PLANT EXTRACTS AND COMPOSITIONS (52) U.S. Cl. ............................................. 424/725; 435/7.1 COMPRISING EXTRACELLULAR PROTEASE INHIBITORS (57) ABSTRACT (76) Inventor: Benoit Cyr, Desmaures (CA) Correspondence Address: The present invention provides a plant derived extract Nath & Associates comprising inhibitory activity against one or more extracel Sixth Floor 1030 15th Street NW lular proteases which degrade human tissue matrix. More Washington, DC 20005 (US) over, the amount of inhibitory activity in an extract can be increased by Stressing the plant prior to forming an extract. (21) Appl. No.: 10/469,402 These extracts are each prepared by a Standard process and demonstrate the ability to inhibit one or more extracellular (22) PCT Filed: Mar. 4, 2002 proteases which degrade human tissue matrix. Libraries of (86) PCT No.: PCT/CA02/00285 extracts can be prepared from Stressed and non-stressed plants, wherein each of the extracts demonstrate inhibitory (30) Foreign Application Priority Data activity against one or more extracellular protease inhibitors. Alternatively, Semi-purified and purified inhibitory com Mar. 2, 2001 (CA).......................................... 2,339,081 pounds can be isolated from the extracts following Standard Publication Classification procedures. In one aspect, these extracts with inhibitory activity can be used during protein purification to minimize (51) Int. Cl." .......................... A61K 35/78; G01N 33/53 the degradation due to extracellular proteases. Patent Application Publication Sep. 9, 2004 Sheet 1 of 3 US 2004/0175439 A1 Potential Plant Pre-Harvest Treatment Harvest Solid S1 Optional Storage Treatment Contact Solid S1 with Solvent A Extraction Process I Separate Pre-Extract A from Solid Matter S2 Potential Pre-Extract.A Contact Solid S2 with Solvent B Extraction Process II Separate Pre-Extract B a. from Solid Matter S3 Potential Pre-Extract B Contact Solid S3 with Solvent C Extraction Process III Separate Pre-Extract C w from Solid Matter S4 Potential Pre-Extract C FIGURE 1 Patent Application Publication Sep. 9, 2004 Sheet 2 of 3 US 2004/0175439 A1 Potential Pre-Extract Separation Procedure(s) Test Aliquots against panel of 2 or more Extracellular Proteases Does Extract Inconclusive demonstrate inhibitory Potential Extract is an activity against one or Extract of the more extracellular Invention proteases? Potential Extract is not an Extract of the Invention Plant remains a Potential Plant Entire Procedure may be repeated on Potential Plant under varied conditions FIGURE 2 Patent Application Publication Sep. 9, 2004 Sheet 3 of 3 US 2004/0175439 A1 One or More Plants of - the Invention Pre-Harvest Treatment Harvest Solid S1 Optional Storage Treatment Extraction Process I, II Process may be or II sequentially repeated Purification Procedure(s) Quality Control Extract Demonstrating Inhibitory Activity Against One or More Extracellular Proteases Prepare Formulation for Use FIGURE3 US 2004/0175439 A1 Sep. 9, 2004 PLANT EXTRACTS AND COMPOSITIONS normal tissues, the activity of extracellular proteases is COMPRISING EXTRACELLULAR PROTEASE tightly regulated and the breakdown/production of connec INHIBITORS tive tissue is in dynamic equilibrium, Such that there is a Slow and continual turnover due to degradation and resyn FIELD OF INVENTION thesis in the extracellular matrix of adult animals. 0001. The invention pertains to the field of protease 0007. In each of these cases, matrix components are inhibitors, Specifically inhibitors of extracellular proteases. degraded by extracellular proteolytic enzymes that are Secreted locally by cells. These proteases belong to one of four general classes: many are metalloproteinases, which BACKGROUND OF THE INVENTION depend on bound Ca" or Zn" for activity, while the others 0002 The cells of tissues are generally in contact with a are Serine, aspartic and cysteine proteases, which have a network of large extracellular macromolecules that occupies highly reactive Serine, aspartate or cysteine residue in their the Spaces in a tissue between the component cells and also respective active site (Vincenti et al., (1994) Arthritis and occupies the Space between adjacent tissues. This extracel Rheumatism, 37: 1115-1126). Together, metalloproteinases, lular matrix functions as a Scaffolding on which the cells and Serine, aspartate and cysteine proteases cooperate to degrade tissue are Supported and is involved actively in regulating matrix proteins Such as collagen, laminin, and fibronectin. interaction of the cells that contact it. The principal macro 0008. Several mechanisms operate to ensure that the molecules of the extracellular matrix include the collagens degradation of matrix components is tightly controlled. (the most abundant proteins in the body) and glycosami First, many proteases are Secreted as inactive precursors that noglycans (complex polysaccharides which are usually can be activated locally. Second, the action of proteases is bonded also to protein and then termed proteoglycans). The confined to specific areas by various Secreted protease macromolecules that comprise the extracellular matrix are inhibitors, Such as the tissue inhibitors of metaloproteases produced typically by the cells in contact there with, for and the Serine protease inhibitors known as Serpins. These example, epithelial cells in contact with a basement mem inhibitors are specific for particular proteases and bind brane and fibroblasts embedded in connective tissue. tightly to the activated enzyme to block its activity. Third, 0003) The glycosaminoglycan (proteoglycan) molecules many cells have receptors on their Surface that bind pro form a highly hydrated matrix (a gel) in which elastic or teases, thereby confining the enzyme to where it is needed. fibrous proteins (such as collagen fibers) are embedded. The 0009. Many pathogenic bacteria produce extracellular aqueous nature of the gel permits diffusion of metabolically metalloproteases, of which many are Zinc containing pro required Substances between the cells of a tissue and teases that can be classified into two families, the thermol between tissues. Additional proteins that may be found in ysin (neutral) proteases and the Serralysin (alkaline) pro extracellular matrix include elastin, fibronectin and laminin. teaSeS. 0004. The term “connective tissue' refers to extracellular 0010) A number of patents and publications report the matrix plus specialised cells Such as, for example, fibro inhibition of one or more extraceliular proteases by com blasts, chondrocytes, osteoblasts, macrophages and mast pounds extracted from plants. For example, Sun et al., cells found therein. The term “interstitial tissue’ is best (1996) Phytotherapy Res., 10: 194-197, reports the inhibi reserved for an extracellular matrix that Stabilizes a tissue tion in vitro of stromelysin (MMP-3) and collagenase by internally, filling the gaps between the cells thereof. There betulinic acid extracted from Doliocarpus venuculosis. are also specialized forms of extracellular matrix (connec Sazuka et al., (1997) BioSci. Biotechnol. Biochem., 6.1: tive tissue) that have additional functional roles-cornea, 1504-1506, reports the inhibition of gelatinases (MMP-2 cartilage and tendon, and when calcified, the bones and and MMP-9) and metastasis by compounds isolated from teeth. green and black teas. Kumagai et at JP 08104628 A2, Apr. 0005. A structural form of extracellular matrix is the 1, 1996 (CA 125: 67741) reports the use of flavones and basal lamina (basement membrane). Basal laminae are thin anthocyanines isolated from Scutellaris baican lensis roots Zones of extracellular matrix that are found under epithelium to inhibit collagenase. Gervasi et al., (1996) Biochem. Bio or Surrounding, for example, muscle cells or the cells that phys. Res. Comm., 228: 530-538, reports the regulation of electrically insulate nerve fibres. Generally Speaking, basal MMP-2 by some plant lectins and other saccharides. Dubois laminae Separate cell layers from underlying Zones of con et al., (1998) FEBS Lett., 427: 275-278, reports the increased nective tissue or Serve as a boundary between two cell layers secretion of deleterious gelatinase-B (MMP-9) by some wherein a basal lamina can Serve as a pathway for invading plant lectins. Nagase et al.(1998) Planta Med., 64: 216-219, cells associated with pathologic processes, or for Structural reports the weak inhibition of collagenase (MMPs) by organisation associated with tissue repair (i.e. as a blueprint delphinidin, a flavonoid isolated from Solanum melongena. from which to regenerate original tissue architecture and 0011. Other reports discuss the use of extracts to inhibit morphology). extracellular proteases. For example, Asano et al., (1998) 0006 The regulated turnover of extracellular matrix mac Immunopharmacology, 39: 117-126, reports the inhibition romolecules is critical to a variety of important biological of TNF-C. production using Tripterygium wilfordii Hook F. processes. Localised degradation of matrix components is extracts. Maheu et al., (1998) Arthritis Rheumatol., 41: required when cells migrate through a basal lamina, as when 81-91, reports the use of avocado/Soybean non-Saponifiable white blood cells migrate across the vascular basal lamina extracts in the treatment of arthritis. Makirnura etal., (1993) into tissues in response to infection or