US 20090263516A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0263516 A1 CYR (43) Pub. Date: Oct. 22, 2009 (54) PLANT EXTRACT COMPOSITION AND Publication Classification THEIR USE TO MODULATE CELLULAR (51) Int. Cl. ACTIVITY A636/8962 (2006.01) A636/00 (2006.01) (75) Inventor: Benoit CYR, St. Augustin de A6IP35/00 (2006.01) Desmaures (CA) CI2N 5/06 (2006.01) Correspondence Address: A6IR 36/3 (2006.01) SHEPPARD, MULLIN, RICHTER & HAMPTON A 6LX 36/899 (2006.01) LLP (52) U.S. Cl. ......... 424/754; 424/725; 435/375; 424/774; 990 Marsh Road 424/779; 424/755; 424/750; 424/777 Menlo Park, CA 94025 (US) (57) ABSTRACT (73) Assignee: Biopharmacopae Design Extracts from plant material, or semi-purified/purified mol International Inc., Saint-Foy (CA) ecules or compounds prepared from the extracts that demon strate the ability to modulate one or more cellular activities (21) Appl. No.: 12/263,114 are provided. The extracts are capable of slowing down, inhibiting or preventing cell migration, for example, the (22) Filed: Oct. 31, 2008 migration of endothelial cells or neoplastic cells and thus, the use of the extracts to slow down, inhibit or prevent abnormal Related U.S. Application Data cell migration in an animal is also provided. Methods of selecting and preparing the plant extracts and methods of (63) Continuation of application No. 10/526,387, filed on screening the extracts to determine their ability to modulate Oct. 6, 2005, now abandoned, filed as application No. one or more cellular activity are described. The purification or PCT/CA03/01284 on Sep. 2, 2003. semi-purification of one or more molecules from the described extracts is also contemplated as well as the use of (30) Foreign Application Priority Data these molecules, alone or in combination with an extract, to slow down, inhibit or prevent abnormal cell migration in an Aug. 30, 2002 (CA) ..................................... 24OO 936 animal. our Treatment ---...---------orasaraws were rary r Contact Solid S1 with Solvent.A Extraction Process Separate Pre-Extract. A from Solid MatterS2 Extraction Process Separate Pre-Extract B Potential Pre-ExtractB from Solid Matter S3 contact Solid S3 with : SolventC ExtractionProcess Separate Pre-Extract C Potential Pre-Extract C from Solid Matter S4 Patent Application Publication Oct. 22, 2009 Sheet 1 of 5 US 2009/0263516 A1 FIGURE 1 Potential Plant reherePre-Harvesthecarerearn seas Treatment Harvest Solid S1 Optional Storage Treatment s as a - - - - - - - - - - -27- - - - - - - - - -as a reseae as or per - our an a soapa - as a son an a-ana -- - - s Contact Solid S1 with Solvent A Extraction Process I s y Separate Pre-Extract A 0. from Solid Matter S2 Potential Pre-Extract A Contact Solid S2 with Solvent B Extraction Process II Separate Pre-Extract B PPotential Pre-Extract B from Solid Matter S3 sea n e s so was a - - - - - - - - - - - - - - - - a is an as an up a us v wear - a as a uses as us r as sar or a sur use - - Contact Solid S3 with Solvent C Extraction Process III are:-coastSeparateEs Pre-Extract C Potential(P Pre-Extractic- Solid S4 Patent Application Publication Oct. 22, 2009 Sheet 2 of 5 US 2009/0263516 A1 FIGURE 2 Potential Pre-Extract Separation Procedure(s) Potential Extract Test Aliquots against panel of 2 or more Extracellular Proteases Does Extract demonstrate inhibitory Potential Extract is an • Inconclusive activity against one or Extract of the more extracellular avention 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 Patent Application Publication Oct. 22, 2009 Sheet 3 of 5 US 2009/0263516 A1 FIGURE 3 One or More Plants of the Invention Pre-Harvest Treatinent Harvest Solid S1 Optional Storage Treatment Extraction Process I, II o Process may be sequentially repeated - Purification Procedure(s) Qualityonly aControlcore -- Extract Demonstrating Inhibitory Activity Against One or More Extracellular Proteases Prepare Formulation for Use Patent Application Publication Oct. 22, 2009 Sheet 4 of 5 US 2009/0263516 A1 Figure 4 Patent Application Publication Oct. 22, 2009 Sheet 5 of 5 US 2009/0263516 A1 Figure 5 US 2009/0263516 A1 Oct. 22, 2009 PLANT EXTRACT COMPOSITION AND 0007. The regulated turnover of extracellular matrix mac THEIR USE TO MODULATE CELLULAR romolecules is critical to a variety of important biological ACTIVITY processes. Localised degradation of matrix components is required when cells migrate through a basal lamina, as when CROSS-REFERENCE TO RELATED white blood cells migrate across the vascular basal lamina APPLICATION into tissues in response to infection or injury, or when cancer cells migrate from their site of origin to distant organs via the 0001. This application is a continuation of U.S. patent bloodstream or lymphatic vessels, during metastasis. In nor application Ser. No. 10/526,387 filed on Oct. 6, 2005, which mal tissues, the activity of extracellular proteases is tightly claims the benefit under 35 U.S.C. S.371 to PCT Application regulated and the breakdown/production of connective tissue No. PCT/CA2003/001284 filed on Sep. 2, 2003, which is in dynamic equilibrium, Such that there is a slow and claims priority to Canadian Application No. 2,400.936, filed continual turnover due to degradation and resynthesis in the on Aug. 30, 2002, all of which are incorporated herein in their extracellular matrix of adult animals. entireties. 0008. In each of these cases, matrix components are FIELD OF INVENTION degraded by extracellular proteolytic enzymes that are secreted locally by cells. These proteases belong to one of 0002 The invention pertains to the field of modulators of four general classes: many are metalloproteinases, which cellular activity, specifically within the field of inhibitors of depend on bound Ca" or Zn" for activity, while the others extracellular proteases. are serine, aspartic and cysteine proteases, which have a highly reactive serine, aspartate or cysteine residue in their BACKGROUND OF THE INVENTION respective active site (Vincenti et al., (1994) Arthritis and 0003. The cells of tissues are generally in contact with a Rheumatism, 37: 1115-1126). Together, metalloproteinases, network of large extracellular macromolecules that occupies serine, aspartate and cysteine proteases cooperate to degrade the spaces in a tissue between the component cells and also matrix proteins such as collagen, laminin, and fibronectin. occupies the space between adjacent tissues. This extracellu 0009. Several mechanisms operate to ensure that the deg lar matrix functions as a scaffolding on which the cells and radation of matrix components is tightly controlled. First, tissue are Supported and is involved actively in regulating many proteases are secreted as inactive precursors that can be interaction of the cells that contact it. The principal macro activated locally. Second, the action of proteases is confined molecules of the extracellular matrix include the collagens to specific areas by various secreted protease inhibitors, such (the most abundant proteins in the body) and glycosami as the tissue inhibitors of metalloproteases and the serine noglycans (complex polysaccharides which are usually protease inhibitors known as serpins. These inhibitors are bonded also to protein and then termed proteoglycans). The specific for particular proteases and bind tightly to the acti macromolecules that comprise the extracellular matrix are vated enzyme to block its activity. Third, many cells have produced typically by the cells in contact therewith, for receptors on their surface that bind proteases, thereby confin example, epithelial cells in contact with a basement mem ing the enzyme to where it is needed. brane and fibroblasts embedded in connective tissue. 0010 Many pathogenic bacteria produce extracellular 0004 The glycosaminoglycan (proteoglycan) molecules metalloproteases, of which many are Zinc containing pro form a highly hydrated matrix (a gel) in which elastic or teases that can be classified into two families, the thermolysin fibrous proteins (such as collagen fibres) are embedded. The (neutral) proteases and the serralysin (alkaline) proteases. aqueous nature of the gel permits diffusion of metabolically 0011. A number of patents and publications report the required substances between the cells of a tissue and between inhibition of one or more extracellular proteases by com tissues. Additional proteins that may be found in extracellular pounds extracted from plants. For example, Sun et al., (1996) matrix include elastin, fibronectin and laminin. Phytotherapy Res., 10: 194-197, reports the inhibition invitro 0005. The term “connective tissue' refers to extracellular of stromelysin (MMP-3) and collagenase by betulinic acid matrix plus specialised cells Such as, for example, fibroblasts, extracted from Doliocarpus verruculosis. SaZuka et al. chondrocytes, osteoblasts, macrophages and mast cells found (1997) Biosci. Biotechnol. Biochem., 61: 1504-1506, reports therein. The term “interstitial tissue' is best reserved for an the inhibition of gelatinases (MMP-2 and MMP-9) and extracellular matrix that stabilises a tissue internally, filling metastasis by compounds isolated from green and black teas. the gaps between the cells thereof. There are also specialised Kumagai et al., JP 08104628 A2, Apr. 1, 1996 (CA 125: forms of extracellular matrix (connective tissue) that have 67741) reports the use of flavones and anthocyanines isolated additional functional roles-cornea, cartilage and tendon, and from Scutellaris baicanlensis roots to inhibit collagenase. when calcified, the bones and teeth. Gervasi et al., (1996) Biochem. Biophys. Res. Comm., 228: 0006. A structural form of extracellular matrix is the basal 530-538, reports the regulation of MMP-2 by some plant lamina (basement membrane). Basal laminae are thin Zones lectins and other saccharides. Dubois et al., (1998) FEBS of extracellular matrix that are found under epithelium or Lett., 427: 275-278, reports the increased secretion of delete Surrounding, for example, muscle cells or the cells that elec rious gelatinase-B (MMP-9) by some plant lectins.