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Timestamp: 2014-10-22 07:15:39
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Matched Legal Cases: ['Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60']

Patent US7566693 - Elastin protective polyphenolics and methods of using the same - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign inAdvanced Patent SearchPatentsDermal fibroblasts permanently loose their ability to synthesize elastin, the major component of elastic fibers, shortly after puberty. This progressive loss of elastic fibers cannot be replaced, resulting in the physical signs of aging. The present invention provides methods and compositions containing...http://www.google.com/patents/US7566693?utm_source=gb-gplus-sharePatent US7566693 - Elastin protective polyphenolics and methods of using the sameAdvanced Patent SearchPublication numberUS7566693 B2Publication typeGrantApplication numberUS 11/394,354Publication dateJul 28, 2009Filing dateMar 29, 2006Priority dateFeb 22, 2005Fee statusPaidAlso published asUS20060264375Publication number11394354, 394354, US 7566693 B2, US 7566693B2, US-B2-7566693, US7566693 B2, US7566693B2InventorsFelipe Jimenez, Thomas Mitts, Aleksander HinekOriginal AssigneeHuman Matrix Sciences, Llc, The Hospital For Sick ChildrenExport CitationBiBTeX, EndNote, RefManPatent Citations (32), Non-Patent Citations (100), Referenced by (2), Classifications (11), Legal Events (4) External Links: USPTO, USPTO Assignment, EspacenetElastin protective polyphenolics and methods of using the sameUS 7566693 B2Abstract Dermal fibroblasts permanently loose their ability to synthesize elastin, the major component of elastic fibers, shortly after puberty. This progressive loss of elastic fibers cannot be replaced, resulting in the physical signs of aging. The present invention provides methods and compositions containing the polyphenols ellagic acid and/or tannic acid for protection against degradation of cutaneous elastic fibers by the elastolytic enzymes. The use of ellagic acid and/or tannic acid increased the overall deposition of elastic fibers in healthy and damaged skin cells. The protection of both intra-tropoelastin and extra-cellular mature elastic fibers from proteolytic enzymes by ellagic acid and tannic acid caused an increase in the net deposition of elastic fibers. Therefore, embodiments of the present invention provide methods and composition for the treatment of skin and prevention and treatment of degradation of dermal elastic fibers.
1. A composition comprising an effective amount of a polyphenolic compound and a peptide selected from a group consisting of SEQ ID NO: 1 (Val-Gly-Ala-Met-Pro-Gly), SEQ ID NO: 2 (Val-Gly-Leu-Met-Pro-Gly), SEQ ID NO: 3 (Val-Gly-Val-Met-Pro-Gly), SEQ ID NO: 4 (Val-Gly-Ala-Ser-Pro-Gly), SEQ ID NO: 5 (Val-Gly-Ala-Ala-Pro-Gly), SEQ ID NO: 6 (Val-Gly-Leu-Ser-Pro-Gly), SEQ ID NO: 7 (Val-Gly-Leu-Ala-Pro-Gly), SEQ ID NO: 8 (Val-Gly-Val-Ser-Pro-Gly), SEQ ID NO: 9 (Ile-Gly-Ala-Met-Pro-Gly), SEQ ID NO: 10 (Ile-Gly-Leu-Met-Pro-Gly), SEQ ID NO: 11 (Ile-Gly-Val-Met-Pro-Gly), SEQ ID NO: 12 (Ile-Gly-Ala-Ser-Pro-Gly), SEQ ID NO: 13 (Ile-Gly-Ala-Ala-Pro-Gly), SEQ ID NO: 14 (Ile-Gly-Leu-Ser-Pro-Gly), SEQ ID NO: 15 (Ile-Gly-Leu-Ala-Pro-Gly), SEQ ID NO: 16 (Ile-Gly-Val-Ser-Pro-Gly), SEQ ID NO: 17 (Ile-Gly-Val-Ala-Pro-Gly) and combinations thereof.
2. The composition of claim 1, wherein said polyphenolic compound is selected from tannic acid, ellagic acid and combinations thereof.
3. The composition of claim 1, wherein said effective amount of said polyphenolic compound is from about 1 μg to about 10 μg.
4. The composition of claim 1, wherein said peptide is selected from the group consisting of SEQ ID NO: 1 (Val-Gly-Ala-Met-Pro-Gly), NO: 6 (Val-Gly-Leu-Ser-Pro-Gly), SEQ ID NO: 9 (Ile-Gly-Ala-Met-Pro-Gly), SEQ ID NO: 14 (Ile-Gly-Leu-Ser-Pro-Gly), and SEQ ID NO: 17 (Ile-Gly-Val-Ala-Pro-Gly).
5. The composition of claim 4, wherein said polyphenolic compound is selected from tannic acid, ellagic acid, and combinations thereof.
6. The composition of claim 4, wherein said effective amount of said polyphenolic compound is from about 1 μg to about 10 μg.
7. A method for protecting elastin fibers from degradation comprising administering a composition comprising an effective amount of a polyphenolic compound and a peptide selected from a group consisting of SEQ ID NO: 1 (Val-Gly-Ala-Met-Pro-Gly), SEQ ID NO: 2 (Val-Gly-Leu-Met-Pro-Gly), SEQ ID NO: 3 (Val-Gly-Val-Met-Pro-Gly), SEQ ID NO: 4 (Val-Gly-Ala-Ser-Pro-Gly), SEQ ID NO: 5 (Val-Gly-Ala-Ala-Pro-Gly), SEQ ID NO: 6 (Val-Gly-Leu-Ser-Pro-Gly), SEQ ID NO: 7 (Val-Gly-Leu-Ala-Pro-Gly), SEQ ID NO: 8 (Val-Gly-Val-Ser-Pro-Gly), SEQ ID NO: 9 (Ile-Gly-Ala-Met-Pro-Gly), SEQ ID NO: 10 (Ile-Gly-Leu-Met-Pro-Gly), SEQ ID NO: 11 (Ile-Gly-Val-Met-Pro-Gly), SEQ ID NO: 12 (Ile-Gly-Ala-Ser-Pro-Gly), SEQ ID NO: 13 (Ile-Gly-Ala-Ala-Pro-Gly), SEQ ID NO: 14 (Ile-Gly-Leu-Ser-Pro-Gly), SEQ ID NO: 15 (Ile-Gly-Leu-Ala-Pro-Gly), SEQ ID NO: 16 (Ile-Gly-Val-Ser-Pro-Gly), SEQ ID NO: 17 (Ile-Gly-Val-Ala-Pro-Gly) and combinations thereof to a subject in need thereof.
8. The method of claim 7, wherein said polyphenolic compound is selected from tannic acid, ellagic acid and a combination thereof.
9. The method of claim 7, wherein said effective amount of said polyphenolic compound is from about 1 μg to about 10 μg.
10. The method of claim 7, further comprising administering a stimulator of elastogenesis selected from elastin-derived peptides, plant-derived peptides, bovine-derived peptides, manganese, iron, copper and combinations thereof.
11. The method of claim 10, wherein said polyphenolic compound and said stimulator of elastogenesis are administered simultaneously.
12. The method of claim 10, wherein said polyphenolic compound and said stimulator of elastogenesis are administered sequentially.
CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 60/665,966 titled �Elastin Protective Polyphenolics� filed Mar. 29, 2005 and U.S. Provisional Patent Application No. 60/758,821 titled �Ellagic and Tannic Acids Protect Newly Synthesized Elastic Fibers from Premature Enzymatic Degradation in Dermal Fibroblast Cultures� filed Jan. 13, 2006, the disclosures of which are incorporated herein by reference in their entirety. This application also claims priority to U.S. patent application Ser. No. 11/062,377 filed Feb. 22, 2005 entitled �Compositions for Elastogenesis and Connective Tissue Treatment�, the disclosure of which is incorporated herein by reference in its entirety. This application also claims priority to U.S. Provisional Patent Application No. 60/671,557 filed Apr. 15, 2005 entitled �Plant-Derived Elastin Binding Protein Ligands and Methods of Using the Same�, U.S. Provisional Patent Application No. 60/681,600 filed May 17, 2005 entitled �Proteolytic Digest Derived from Bovine Ligamentum Nuchae Stimulates Deposition of New Elastin-Enriched Matrix in Cultures and Transplants of Human Dermal Fibroblasts� and U.S. Provisional Patent Application No. 60/737,586 filed Nov. 17, 2005 entitled �Plant-Derived Elastin Binding Protein Ligands and Methods of Using the Same�, the disclosures of which are incorporated herein by reference in their entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH PROJECTS Not Applicable
NAMES OF PARTIES SUBJECT TO A JOINT RESEARCH AGREEMENT Not Applicable
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A DISC Not Applicable
BACKGROUND Children with inherited diseases, characterized by impaired primary deposition of elastic fibers (i.e. Costello Syndrome or Cutis Laxa) develop wrinkles and deep dermal creases. Similar, but steadily developing signs of premature skin aging can also be observed in individuals with Pseudoxanthoma Elasticum and in normal persons after prolonged exposure to sun. Histological analysis of wrinkled skin demonstrates disappearance and altered organization of elastic fibers due to premature proteolytic degradation and impaired remodeling (solar elastosis) of these components of dermal extracellular matrix. This observed loss of physiologically relevant elastic fibers is also affected by the fact that fully differentiated (adult) dermal fibroblasts lose their ability to synthesize elastin and thus cannot replace damaged elastic fibers. Since elastic fibers are solely responsible for cutaneous elasticity/resilience there is an obvious need for development of methods that might protect existing elastic fibers from premature degradation by elastolytic proteinases and facilitate new elastogenesis in skin.
Topical applications of ellagic acid have been used in therapeutic preparations. Gali, et. al. demonstrated that topical applications of tannic acid practically inhibit tumor promoter-induced ornithine decarboxylase activity (ODA) in mouse epidermis in vivo suggesting that tannic acid and other polyphenols may be effective not only against skin tumor initiation and complete carcinogenesis, but also against the promotion phase of skin tumorigenesis. Moreover, tannic acid and its polyphenol derivatives have been shown to possess anti-inflammatory activities and to decrease infectivity of human cells with papiloma virus, human immunodeficiency virus, and Staphylococcus aureus. BRIEF SUMMARY OF THE INVENTION The present invention provides evidence that the effectiveness of ellagic acid or tannic acid to prevent premature proteolytic degradation of tropoelastin and fully polymerized elastin, thus facilitating more efficient elastogenesis. Thus, embodiments of the present invention provide compositions and methods for treating aging or damaged skin using ellagic acid, tannic acid, or derivatives thereof.
FIG. 3. Evaluation of the protective effect of polyphenols against elastolytic degradation of insoluble elastin. Results of in vitro assay demonstrate that samples of insoluble [3H]-labeled elastin from bovine ligamentum nuchae,pretreated with EA or TA (1 μg/mL and 10 μg/mL each) demonstrate higher resistance to proteolytic degradation by indicated enzymes belonging to three different classes of proteinases (elastases) capable of elastin degradation.
FIG. 5. Assessment of binding of tannic acid to collagen type I. (a) Results of triplicate (1 mg) aliquots of pure collage type I inclubated with 20 μg/ml of tannic acid for 2 hour at 37 C. Initial concentration of tannic acid was confirmed by direct spectrophotometric reading at 280 nm. This method demonstrated a dose-dependent linear increase in absorbance. At the end of incubation period the collagen type I slurries were separated by centrifugation and the concentration of TA in supernatants were spectrophotometrically determined again at 280 nm. In each experimental group means�SD were calculated and obtained values were statistically compared with beginning concentrations of both polyphenols. (b) 1 mg of collagen type I (from rat tail) sequestered 75.5�0.001% (P<0.0001) of the TA (originally 20 μg/mL) from solution, suggesting that tannic acid may also bind to collagen type I.
As used herein, the term �about� means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 50% means in the range of 45%-55%. Generally speaking, the term �tissue� refers to any aggregation of similarly specialized cells which are united in the performance of a particular function.
The term �modify� is used to convey that the present invention changes either the appearance, form, characteristics and/or the physical attributes of the tissue to which it is being provided, applied or administered. The change in form may be demonstrated by any of the following alone or in combination: enhanced appearance of the skin; increased softness of the skin; increased turgor of the skin; increased texture of the skin; increased elasticity of the skin; decreased wrinkle formation and increased endogenous elastin production in the skin, increased firmness and resiliency of the skin.
�Providing� when used in conjunction with a therapeutic means to administer a therapeutic directly into or onto a target tissue or to administer a therapeutic to a patient whereby the therapeutic positively impacts the tissue to which it is targeted. Thus, as used herein, the term �providing�, when used in conjunction with a polyphenolic compound, can include, but is not limited to, providing an polyphenolic compound into or onto the target tissue; providing a polyphenolic compound systemically to a patient by, e.g., intravenous injection whereby the therapeutic reaches the target tissue; and the like.
The term �skin� means that outer integument or covering of the body, consisting of the dermis and the epidermis and resting upon subcutaneous tissue.
As used herein, the term �therapeutic� means an agent utilized to treat, combat, ameliorate, prevent or improve an unwanted condition or disease of a patient. In part, embodiments of the present invention are directed to improve the functionality, the appearance, the elasticity, and/or the elastin content of mammalian tissue. As it applies to skin, it is measured by turgor, tone, appearance, degree of wrinkles, and youthfulness. As the term applies to blood vessels it may be measured by the degree of elasticity or proper vasomotor response (vasodilatation/vasoconstriction) of the vessel. Accordingly, therapeutic treatment of blood vessels may have implications in diseases associated with visco-elasticity, including hypertension, arteriosclerosis, angina, angiogenesis, myocardial infarction, coronary thrombosis, restenosis post angioplasty, and chronic obstructive pulmonary disease.
The terms �therapeutically effective� or �effective�, as used herein, may be used interchangeably and refer to an amount of a therapeutic composition of the present invention�e.g., a polyphenolic compound. For example, a therapeutically effective amount of a composition comprising polyphenolic compound is a predetermined amount calculated to achieve the desired effect, i.e., to effectively promote elastin production, cell proliferation, or improved appearance, or improved tissue elasticity in an individual to whom the composition is administered.
As used herein, �tissue�, unless otherwise indicated, refers to tissue which includes Elastin as part of its necessary structure and/or function. For example, connective tissue which is made up of, among other things, collagen fibrils and elastin fibrils satisfies the definition of �tissue� as used herein. Additionally, elastin appears to be involved in the proper function of blood vessels, veins, and arteries in their inherent visco-elasticity.
Skin is composed of a top layer, the epidermis, which is approximately 20 cell layers or about 0.1 mm in thickness, and a lower layer, the dermis, which is from about 1 to about 4 mm in thickness and contains small blood vessels, collagen, elastin and fibroblasts. The dermis provides structural support and nutrients to the epidermis. Aging has been shown to increase cellular heterogeneity of the epidermal layer, however, it has little effect on the thickness of the epidermal layer. The supporting dermis, on the other hand, is known to thin with age and exposure to the sun and environmental contaminants (other environmental effects on the skin are discussed in U.S. Pat. Nos. 4,938,969 and 5,140,043, the disclosure of which is herein incorporated by reference). As the dermal layer provides the support and blood supply for the epidermis, the dermal layer is important in maintaining the elasticity and appearance of the skin. Disruption of the supporting dermis leads directly to sagging and, consequently, furrowing of the epidermis, i.e., the formation of wrinkles.
A further embodiment of the present invention provides compositions comprising at least one polyphenolic compound, or derivatives thereof, preferably ellagic acid or tannic acid and optionally one stimulator of elastogenesis. A stimulator of elastogenesis may be, for example, small elastin-derived peptides including, but not limited to, ProK-60 or other small elastin-derived peptides as set forth in co-pending U.S. application Ser. No. 10/778,253 entitled �Elastin Digest Compositions and Methods Utilizing the Same� filed Feb. 13, 2004, the contents of which are herein incorporated by reference in its entirety. Such compositions may be useful to significantly increase the net deposition of insoluble elastic fibers, thereby enhancing the skin's elasticity and decreasing the appearance of fine lines and/or wrinkles. Thus, further embodiments of the present invention provide compositions and methods to compensate for the loss of elastic components in the dermis.
The result of aging on skin, whether or not it has been accelerated by environmental damage (such as radiation, pollution) is a deterioration of the dermal layer�fewer fibroblasts, less collagen, less elastin and less circulatory support. Consequently, the normal stretching and contraction of the skin leads to damage of the dermis that is not readily corrected, resulting in wrinkling. Further embodiments of the present invention provide methods and compositions for increasing the deposition of insoluble elastin fibers, therein reducing the effects of radiation, including, but not limited to, ultraviolet radiation, or other environmental damage.
Dermatologists and cosmetologists have directed their efforts to improving the appearance of skin using agents known to stimulate the growth and proliferation of epidermal cells. Newly proliferated cells provide more structure and hold more moisture, giving the skin a younger appearance. One method of causing new skin cell proliferation is accomplished by use of an irritant or chemical peel in which the uppermost layers of the epidermis are caused to slough off, leading to proliferation and replacement with new epidermal cells. While such treatment is recognized to provide some cosmetic improvement, it does not address the major causative factor�the compromised supporting dermal layer. Thus, embodiments of the present invention also provide methods and compositions for the enhanced deposition of insoluble elastin fibers, therein providing the dermal support and elasticity necessary for smooth, supple skin.
It is another object of the present invention to provide factors, including but not limited to, a source of small elastin-derived peptides (for example, ProK-60), a manganese component (for example, Mn-PCA, manganese sulfate, manganese gluconate), an iron component (for example, ferric ammonium citrate), a copper component (for example, copper-PCA, copper sulfate), bovine-derived peptides or plant-derived peptides to promote elastogenesis for skin repair and wound healing that occurs due to photoaging processes in the skin, such as those that can occur from acute sunburn and/or chronic exposure to ultraviolet radiation. Suitable manganese and iron components are described in co-pending U.S. application Ser. No. 11/062,377 entitled �Compositions for Elastogenesis and Connective Tissue Treatment� filed Feb. 22, 2005, suitable plant-derived peptides are described in co-pending U.S. Application No. 60/671,557 entitled �Plant-Derived Elastin Binding Protein Ligands and Methods of Using the Same� filed Apr. 15, 2005, and suitable bovine-derived peptides are described in co-pending U.S. Application No. 60/681,600 entitled �Proteolytic Digest Derived from Bovine Ligamentum Nuchae Stimulates Deposition of New Elastin-Enriched Matrix in Cultures and Transplants of Human Dermal Fibroblasts� filed May 17, 2005, the contents of which are all herein incorporated by reference in their entireties.
The active ingredients described herein can be incorporated in any suitable pharmacologically acceptable carrier which is suitable for topical administration to the human skin. As such, the pharmacologically acceptable carrier must be of sufficient purity and have sufficiently low toxicity to render it suitable for administration to a human noting that, generally, the carrier can represent up to 99.99% and typically from at least approximately 80% of the total composition. Thus, the phrase �pharmaceutically acceptable� refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The pharmaceutically acceptable carriers and additives employed in the present compositions are compatible with at the tannic acid and/or ellagic acid compounds and compositions described herein containing such compounds.
Elastogenic plant-derived peptides may also be present in the composition. Such peptides are more fully described in U.S. Provisional Patent Application No. 60/671,557 filed Apr. 15, 2005 entitled �Plant-Derived Elastin Binding Protein Ligands and Methods of Using the Same�, U.S. Provisional Application No. 60/681,600 filed May 17, 2005 entitled �Proteolytic Digest Derived from Bovine Ligamentum Nuchae Stimulates Deposition of New Elastin-Enriched Matrix in Cultures and Transplants of Human Dermal Fibroblasts� and U.S. Provisional Patent Application No. 60/737,586 filed Nov. 17, 2005 entitled �Plant-Derived Elastin Binding Protein Ligands and Methods of Using the Same. Such peptides may be sextapeptide comprising the sequence X1-X2-X3-X4-X5-X6, wherein X1 is V or I, X2 is G, X3 is A, L or V, X4 is M, S, or A, X5 is P and X6 is G. Such a plant-derived peptide or synthetic plant-derived peptide may be present in the composition. In preferred embodiments, the peptide may be present in about 0.0001 to about 0.01 weight percent, more preferably from about 0.0004 to about 0.002 weight percent of the composition.
EXAMPLE 1 Materials. All chemical-grade reagents were obtained from Sigma (St. Louis, Mo.). αMEM medium, fetal bovine serum (FBS), 0.2% trypsine-0.02% EDTA and other cell culture products were obtained from GIBCO Life Technologies (Burlington, Canada). Polyclonal antibody to tropoelastin and BA4 monoclonal antibody to VGVAPG (SEQ ID NO. 1) were purchased from Elastin Products Company, Inc. (Owensville, Mo.). Monospecific polyclonal anti-AKAAAKAAAKA antibody was a gift of Dr. Barry Starcher from the University of Texas. Secondary antibody fluorescein-conjugated goat anti-rabbit (GAR-FITC) was purchased from Sigma (St. Louis, Mo.). DNeasy Tissue system for DNA assay and Rneasy Mini Kit for isolation of total RNA were purchased from Qiagen (Mississauga, Canada). Expression probe for elastin was purchased from Applied Biosystems (Foster City, Calif.). The radiolabeled reagents, [3H]-valine, and [3H]-thymidine were purchased from Amersham Canada Ltd. (Oakville, Canada).
Cell Cultures. Biological effects of ellagic acid and tannic acid were tested in cultures of dermal fibroblasts derived from punch biopsies of healthy skin from Caucasian females of different ages ranging from 4-52 years old. All fibroblasts were originally isolated by allowing them to migrate out of skin explants and then passaged by trypsinization and maintained in alpha-minimum essential medium supplemented with 20 mM Hepes, 1% antibiotics and antimycotics, 1% L-Glutamate and 2% fetal bovine serum (FBS). In all experiments, consecutive passages 3-5 were tested. Cells were densely plated (50�105 cells/dish) to reach confluency and then cultured for 7 days in the presence and absence of ellagic acid (dissolved in DMSO) and tannic acid (dissolved in water), both in concentration of 1 μg/mL. This optimal concentration was chosen after a series of pilot experiments indicated that 1 μg/ml of EA and TA induced optimal effect on net deposition of elastin and did not trigger any change in cellular proliferation rate nor affect basic metabolic performance.
Assessment of elastic fibers content by immunohistochemistry. Seven-day-old and 14-day-old confluent cultures of fibroblasts, which produce abundant ECM, were assessed. All cultures were fixed in cold 100% methanol at −20� C. for 30 min, then incubated for 1 hour with 2 μg/ml of polyclonal antibody to tropoelastin. Cultures were then incubated for an additional hour with appropriate fluorescein-conjugated secondary antibody (GAR-FITC). Nuclei were counterstained with propidium iodide. Morphometric analysis of five separate cultures in each experimental group, immunostained with antibodies recognizing extracellular matrix components was performed using a computerized video analysis system (Image-Pro Plus software 3.0, Media Cybernetics, Silver Spring, Md.).
Organ cultures of explants derived from surgical biopsies of human skin. In order to further test whether ellagic acid and tannic acid would penetrate into skin tissue and enhance elastogenesis, fragments of normal skin (from 30 and 34 year old females) obtained during plastic surgery procedures were tested in organ culture system. Skin fragments were cut into multiple 1 mm2 pieces and placed on top of metal grids immersed in culture medium containing 5% FBS and maintained for 10 days in the presence and absence of 1 μg/mL of ellagic acid or tannic acid alone or combined with 25 μg/ml of ProK-60. The media were changed every second day. All organ cultures were fixed in 1% buffered formalin and their transversal serial histological sections were stained with Movat's pentachrome. Morphometric analysis was performed as described above. In each analyzed group (three explants from each patient) low-power fields (1 mm2) of 20 serial sections stained with Movat's pentachrome were analyzed and all structures stained black (elastic fibers) were counted.
Assessment of tropoelastin integrity by western blots. To determine the influence of tannic acid and ellagic acid on the integrity of soluble tropoelastin, dermal fibroblasts obtained from three different donors (initially plated at 50,000 cells/dish) were cultured to confluency in medium with 5% FBS and then triplicate cultures were incubated for the next 24 hours in the presence and absence of ellagic acid or tannic acid (both in concentration 1 μg/mL). At the end of the incubation period conditioned media were collected and then the soluble proteins present in the intracellular compartments were extracted with 0.5 M acetic acid in the presence of proteinase inhibitors in the following final concentrations: 2 mM benzamidine, 2 mM EACA, 2 mM PMSF, 1 mM EDTA and 1 mg/ml Trasylol. Extraction was carried out for six hours at 4� C. and the insoluble material was pelleted by centrifugation. The supernatant was dialyzed exhaustively (4000 kDa cutoff membrane) at 4� C. against water containing proteinase inhibitors, then lyophilized. Concentrated preparations of the conditioned media and cell extracts from all analyzed cultures were analyzed for their protein content, and then samples containing equal amounts of protein (20 μg/sample) were suspended in 2�SDS sample buffer with DTT, resolved by SDS PAGE, routinely transferred to nitrocellulose and immunoblotted with specific anti-tropoelastin antibody.
Immuno-precipitation of radioactive tropoelastin-like peptides. This experiment was aimed at elucidating whether binding of ellagic acid or tannic acid to tropoelastin molecules would block two characteristic elastin domains responsible for orderly self-aggregation and cross-linking respectively. Instead of very unstable tropoelastin, triplicate samples of a [3H]-valine-labeled recombinant polypeptide containing linear amino acid sequences encoded by human tropoelastin gene exons; 20-(21-23-24)2 were used. 100 μl samples of this radioactive recombinant polypeptide modeled after human elastin dissolved in PBS (specific radioactivity 1000 CPM/sample) were incubated in the presence and absence of 1 μg/mL of ellagic acid or tannic acid, at room temperature for 6 hours. Aliquots of all control and experimental samples were then immuno-precipitated with (BA4) monoclonal antibody (recognizing VGVAPG (SEQ ID NO. 1) and other similar domains, encoded by exons 20 (SEQ ID NO. 2) and 24 (SEQ ID NO. 3), responsible for self-aggregation of tropoelastin) and with monospecific polyclonal anti-AKAAAKAAAKA antibody recognizing the exon 21- (SEQ ID NO. 4) and 23-encoded cross-linking sequences (SEQ ID NO. 5). It was anticipated that in case that binding of ellagic acid or tannic acid would block one or both of these crucial domains present in the tested radioactive recombinant peptide, it could not be immunoprecipitated with anti-VGVAPG or anti-AKAAAKAAAKA antibodies.
Proteolytic degradation protection assay of insoluble elastin. To determine whether ellagic acid or tannic acid may directly protect fully cross-linked �insoluble elastin� against elastolytic activity of several elastases, an in vitro assay measuring degradation of an insoluble [3H]-elastin substrate was used. Briefly, insoluble elastin was purified from bovine ligamentum nuchae using a modification of the hot alkali technique and was shown by amino acid analysis to be free of microfibrillar protein and other contaminants. Sequencing of insoluble, digested elastin was performed using an Applied Biosystems model 473A protein sequencer equipped with a model 610A data analysis program. The stock of this pure insoluble elastin preparation was washed twice with water and twice with acetonitrile and then labeled with sodium [3H]-borohydride and stored at −20� C. Before each experiment, the [3H]-elastin substrate suspended in PBS was boiled for 5 min and extensively washed to remove all unbound radioactivity. Then, its 100 μg aliquots (specific activity 300 CPM/1 μg) were suspended in serum free culture medium and pre-incubated for 1 hour in the presence and absence of 1 μg/mL or 10 μg/mL of ellagic acid or tannic acid. All samples of radioactive elastin were then submitted to three 5 min washes in serum free culture medium prior to their 18 hour incubation at 37� C. with aliquots (50 ng) of human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), MMP-2, or Papaine dissolved in the assay buffer (50 mM Tris-HCL, pH 7.5 containing 150 mM NaCl, 10 mM CaCl2, 0.02% Brij and 0.02% sodium azide). Each treatment was tested in quadruplicate samples. At the end of the incubation, all samples were microcentrifuged (8000�g for 5 min) and 100 μl aliquots of supernatant containing the solubilized degradation products were mixed with 4 ml scintillation fluid and counted in triplicates in liquid scintillation counter. The radioactivity (cpm/sample) released into the supernatant reflecting the degradation of [3H]-elastin substrate was assessed and the mean and standard deviations were calculated from sixtiplicate assessments from 3 different experiments
Assessement of TA and EA binding to insoluble elastin. In order to directly show that both polyphenols bind to elastin, triplicate (1 mg) aliquots of our above mentioned preparation of pure insoluble elastin were incubated with 20 μg/ml of ellagic acid or tannic acid for 2 hr at 37� C. The initial concentration of both polyphenols were confirmed by a direct spectrophotometric reading at 280 nm. This method demonstrated a dose-dependent linear increase in absorbancy. At the end of incubation period the insoluble elastin slurries were separated by centrifugation and the concentrations of polyphenols in supernatants were determined again. The detected differences between the initial and final concentration of polyphenols in supernatants from particular samples directly indicated that both ellagic acid and tannic acid bound to elastin slurries during the incubation period. In each experimental group means�SD were calculated and obtained values were statistically compared with beginning concentrations of both polyphenols.
Statistical analysis. In all above mentioned quantitative assays, means and standard deviations (expressed as Mean�SD) were calculated and statistical analyses were carried out by ANOVA to establish whether detected differences were statistically significant.
Ellagic acid and tannic acid enhance deposition of elastin by dermal fibroblasts. Results of immunohistochemical analysis (FIGS. 1 a and b) and quantitative assessment of metabolically labeled insoluble elastin (FIG. 1 c) indicated that 7 day old monolayer cultures of dermal fibroblasts maintained with ellagic acid or tannic acid contain thicker elastic fibers and a higher net content of NaOH-insoluble elastin than untreated control cultures. Moreover, results of morphometric analysis demonstrated that both ellagic acid and tannic acid caused a significant (p<0.005) increase (67�6% and 96�12% respectively) in net elastogenesis observed in organ cultures of human skin explants maintained for 10 days with 5% FBS. Explants maintained for 10 days in culture media containing tannic acid contain thicker and longer elastic fibers than those present in explants maintained only in control medium or medium with ProK-60 (data not shown). Interestingly, the presence of tannic acid seems to particularly enhance elastogenesis in cells protruding from the stratum basale, toward the papillary dermis, and in cells surrounding small capillaries. Results of semi-quantitative PCR and Northern blotting indicated, however, that treatment of cultured dermal fibroblasts with ellagic acid or tannic acid did not induce any increase in the transcription of their elastin gene (data not shown) nor change their proliferation rate, as assessed by incorporation of radioactive thymidine and total DNA content (data not shown). Despite this finding, results of western blotting, with anti-tropoelastin antibody, showed that both cell extracts and conditioned media, of dermal fibroblasts incubated with ellagic acid or tannic acid, contained more intact 70 kDa tropoelastin and less immuno-detectable degradation products of lower molecular weight than untreated counterparts (data not shown). This finding gave evidence that both polyphenols protected newly produced tropoelastin from premature intracellular and pericellular degradation by endogenous proteinases.
Ellagic acid and tannic acid bind to elastin and tropoelastin. Results of a spectrophotometric assay (displaying a linear concentration curve for both ellagic acid and tannic acid at an absorbance of 280 nm), comparing concentrations of both polyphenols before and after incubation with insoluble elastin, demonstrated that 1 mg of pure insoluble elastin, isolated from ligamentum nuchae, absorbed 87�3% of the tannic acid and 81�2% of the ellagic acid in solutions having had initial concentrations of 20 μg/mL of each polyphenol. This finding implied that both polyphenols bind to insoluble elastin. Additional results showed that preincubation of a [3H]-valine-labeled recombinant peptide, containing the most characteristic hydrophobic and cross-link generating domains of tropoelastin, with ellagic acid and tannic acid did not preclude its effective (practically identical) immunoprecipitation with respective anti-VGVAPG and anti-AKAAAKAAAKA antibodies (data not shown). This indicated that these polyphenols associate with and protect tropoelastin in a way which does not obscure hydrophobic domains (eg. VGVAPG (SEQ ID NO. 1)), necessary for self-aggregation, nor KAAAK (SEQ ID NO. 6)sequences participating in crosslinking.
Ellagic acid and tannic acid enhance elastogenic effect of selected stimulators of elastogenesis. Results of immunostaining and metabolic labeling established that addition of ellagic acid or tannic acid to fibroblast cultures simultaneously treated with known stimulators of elastin gene expression, ProK-60 or Ferric Ammonium Chloride (FAC), significantly enhanced their net deposition of insoluble elastin as estimated in 7 day old monolayer cultures (FIG. 4). Morphometric analysis additionally demonstrated that both polyphenols significantly (p<0.02) enhanced elastogenic effect of ProK-60 observed in 10 day-old organ cultures of human skin explants (Ellagic Acid=22�4% and Tannic Acid=35�6%). Representative micrographs depicting synergistic effect of ProK-60 and tannic acid are presented in FIG. 4 d. Discussion. Results demonstrate that the two polyphenols, ellagic acid and tannic acid, used in concentration of 1 μg/ml, did not modulate cellular proliferation of normal human dermal fibroblasts, despite the fact that anti-proliferative properties of both of these compounds were reported in cultures of various normal and malignant cell lines when used in higher doses. Dermal fibroblasts treated with both acids did not demonstrate any increase in levels of elastin mRNA yet facilitated a significant increase in net elastic fiber content detected by immunochemistry and metabolic labeling of insoluble elastin. It was speculated that both ellagic acid and tannic acid might bind to intracellular tropoelastin and to newly assembled crosslinked elastin and protect them from proteolytic degradation by fibroblast-secreted proteolytic enzymes engaged in early remodeling of extacellular matrix. The hypothesis regarding potential preferential binding of both polyphenols to intracellular tropoelastin and extracellular elastin polymer was based on a previously described observation that addition of 0.25% tannic acid to glutaraldehyde fixative dramatically enhanced contrast of intracellular secretory vesicles containing tropoelastin and contrast of extracellular elastic fibers in tissues observed under the electron microscope. In fact, in the pre-immunostaining era, treatment with tannic acid became a widely accepted method for ultrastructural identification of elastic fibers that were previously described as �electron lucent and amorphous� under electron microscopy. The hypothesis was further supported by results of our pulse-and-chase experiment where ellagic acid and tannic acid pre-treated tropoelastin and insoluble elastin, deposited by dermal fibroblasts, remained resistant against non-specific endogenous degradation in the absence of ellagic acid or tannic acid in culture medium. Results also show that pre-incubation of [3H]-labeled pure insoluble elastin, with either ellagic acid or tannic acid, significantly reduced its rate of degradation (in the absence of either polyphenol in the digest buffer) by several exogenous elastolytic enzymes including porcine pancreatic elastase, human leukocyte elastase, papaine and the UVB-inducible MMP-2. Results of this protection assay are consistent with recent observations that addition of tannic acid to the glutaraldehyde fixation process increased the stability of porcine aortic explants exposed to pancreatic elastase digestion. Moreover, the spectrophotometric study demonstrated that pure insoluble elastin, isolated from ligamentum nuchae, binds both ellagic acid and tannic acid, sequestering them from their solvent solutions. Results of this study are in further agreement the observation of similar binding of tannic acid by pure aortic elastin over time.
It has been previously shown that plant derived polyphenols, tannins and their synthetic derivatives, can inhibit human leukocyte elastase and MMP−2/−9 activity in several tumor cells. Other studies directly demonstrated that tannic acid specifically inhibits the chymotrypsin-like activity of purified 20S proteasomes and the activity of tissue-type plasminogen activator, urokinase-type plasminogen activator and plasmin activity. It was speculated that a certain fraction of the described protective effect may be due to a direct inhibition of proteolytic enzymes by both polyphenols absorbed by or released from the elastin substrate:
EXAMPLE 2 In order to directly prove that tannic acid also binds to collagen type 1, triplicate (1 mg) aliquots of pure collagen type I were incubated with 20 μg/ml of TA for 2 h at 37� C. The initial concentration of tannic acid was confirmed by direct spectrophotometric reading at 280 nm. This method adopted from Gori et al. demonstrated a dose-dependent linear increase in absorbance. At the end of incubation period the collagen type I slurries were separated by centrifugation and the concentration of tannic acid in supernatants were spectrophotometrically determined again at 280 nm. In each experimental group means�SD were calculated and obtained values were statistically compared with beginning concentrations of both polyphenols.
Results. As shown in FIG. 5, the binding studies demonstrate that 1 mg of collagen type I (from rat tail) sequestered 75.5�0.001% (P<0.0001) of the tannic acid (originally 20 μg/mL) from solution, suggesting that tannic acid may also bind to collagen type I.
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS4254105Jul 10, 1978Mar 3, 1981The Lion Dentifrice Co., Ltd.Suntan lotionUS4938969Nov 14, 1988Jul 3, 1990Milor Scientific, Ltd.Method for the treatment of aging or photo-damaged skinUS5006331Oct 26, 1988Apr 9, 1991Gaskin Frances CCompositions and method of strengthening hairUS5079010Sep 22, 1989Jan 7, 1992Siegfreid NattererAqueous solution of trace elements or salts at acid ph; suntan lotionUS5087442Oct 4, 1989Feb 11, 1992Otsuka Pharmaceutical Co., Ltd.Containing iron citrate compound, sodium carbonate and/or bicarbonate, acid neutralizing agentUS5140043Apr 17, 1989Aug 18, 1992Duke UniversityUsing 1:1 ratio of acid to water and pH of no more than 3.5; sunblock compositionsUS5296500Jul 30, 1993Mar 22, 1994The Procter & Gamble CompanyUse of N-acetyl-cysteine and derivatives for regulating skin wrinkles and/or skin atrophyUS6475501Oct 19, 2000Nov 5, 2002The Procter & Gamble CompanyAntiviral compositions for tissue paperUS6964954 *Oct 25, 2002Nov 15, 2005L'orealUse of DHEA derivatives on keratinous substancesUS7252834Mar 23, 2006Aug 7, 2007Clemson University Research Foundation (Curf)Applying a formulation including a phenolic compound directly to connective tissue, the composition having a pH between about 4 and about 9, wherein the connective tissue comprises elastin and wherein the phenolic compound comprises a hydrophobic core that includes more than one phenolic groupUS20020028254May 18, 2001Mar 7, 2002Isabelle NonotteTopically administering manganese and/or at least one organic and/or inorganic salt thereof.US20020127256 *Feb 25, 2002Sep 12, 2002Howard MuradCompositions and methods for treating dermatological disordersUS20030054021 *Jun 14, 2002Mar 20, 2003Maria Dalko7-oxo-DHEA compounds for treating keratinous conditions/afflictionsUS20030069171Nov 5, 2002Apr 10, 2003Petito George D.Mixture of glucosamine salt, chondroitin sulfate, collagen and sodium hyaluronate for treating bone disordersUS20040120918 *Dec 19, 2003Jun 24, 2004Sederma S.A.S.Cosmetic or dermopharmaceutical compositions of ceramides and polypeptidesUS20040153145 *Nov 24, 2003Aug 5, 2004Clemson UniversityFixation method for bioprosthesesUS20040162232Feb 13, 2004Aug 19, 2004Thomas MittsTo stimulate the endogenous production of Elastin or appear to enhance the elasticity of the skinUS20050208150Feb 22, 2005Sep 22, 2005Thomas MittsCompositions for elastrogenesis and connective tissue treatmentUS20080050346May 17, 2006Feb 28, 2008Felipe JimenezElastin producing fibroblast formulations and methods of using the sameDE10043466A1Sep 4, 2000Mar 28, 2002Johannes WohlrabNon-irritating topical preparation for treating or preventing skin disorders, e.g. atopic dermatitis, psoriasis or aging symptoms, containing manganese salt to stimulate urea synthesis by keratinocytesEP1145709A1Apr 14, 2000Oct 17, 2001Laboratoires SerobiologiquesUse of natural products to prepare cosmetic compositionsEP1284133B1Aug 18, 2001Oct 11, 2006Cognis IP Management GmbHMixtures of active agentsFR7302M Title not availableGB2376886A Title not availableJP2001072571A Title not availableJP2002205913A Title not availableJP2003128511A Title not availableJP2006160685A Title not availableJPH07138142A Title not availableJPS63313707A Title not availableWO1996019182A1Dec 21, 1995Jun 27, 1996Cosmederm TechnologiesFormulations and methods for reducing skin irritationWO2004047620A2Nov 25, 2003Jun 10, 2004Univ ClemsonFixation method for bioprostheses* Cited by examinerNon-Patent CitationsReference1Aessopos et al., Elastic tissue abnormalities resembling pseudoxanthoma elasticum in beta thalassemia and the sickling syndromes, Blood, vol. 99, No. 1, pp. 30-35, Jan. 1, 2002.2Akiyama et al., Antibacterial action of several tannins against Staphylococcus aureus, 2001, J. 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Pharmacol. 66:907-915.100Zhang et al., Inhibition of liver microsomal cytochrome P450 activity and metabolsim of the tobacco-specific nitrosamine NNK by capsaicin and ellagic acid, 1993, Anticancer Res. 13(6A):2341-2346.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS8114829Jul 15, 2009Feb 14, 2012Human Matrix Sciences, LlcElastin protective polyphenolics and methods of using the sameUS8642578Oct 25, 2007Feb 4, 2014Human Matrix Sciences, LlcElastin protective polyphenolics and methods of using the sameClassifications U.S. Classification514/1.1, 530/300International ClassificationC07K14/00Cooperative ClassificationA61K31/366, A61K31/353, A61K31/7048, A61K38/39European ClassificationA61K38/39, A61K31/7048, A61K31/353, A61K31/366Legal EventsDateCodeEventDescriptionJan 28, 2013FPAYFee paymentYear of fee payment: 4Sep 18, 2012CCCertificate of correctionOct 4, 2007ASAssignmentOwner name: THE HOSPITAL FOR SICK CHILDREN, CANADAFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HINEK, ALEKSANDER;THE HOSPITAL FOR SICK CHILDREN;REEL/FRAME:019922/0325;SIGNING DATES FROM 20070928 TO 20071002Owner name: THE HOSPITAL FOR SICK CHILDREN,CANADAFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HINEK, ALEKSANDER;THE HOSPITAL FOR SICK CHILDREN;SIGNED BETWEEN 20070928 AND 20071002;US-ASSIGNMENT DATABASE UPDATED:20100223;REEL/FRAME:19922/325Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HINEK, ALEKSANDER;THE HOSPITAL FOR SICK CHILDREN;SIGNED BETWEEN 20070928 AND 20071002;US-ASSIGNMENT DATABASE UPDATED:20100525;REEL/FRAME:19922/325Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HINEK, ALEKSANDER;THE HOSPITAL FOR SICK CHILDREN;SIGNED BETWEEN 20070928 AND 20071002;REEL/FRAME:19922/325Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:HINEK, ALEKSANDER;THE HOSPITAL FOR SICK CHILDREN;SIGNINGDATES FROM 20070928 TO 20071002;REEL/FRAME:019922/0325Apr 24, 2007ASAssignmentOwner name: HUMAN MATRIX SCIENCES, LLC, CALIFORNIAFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:MITTS, THOMAS;JIMENEZ, FELIPE;REEL/FRAME:019199/0874;SIGNING DATES FROM 20070403 TO 20070410Owner name: THE UNIVERSITY OF TORONTO, THE HOSPITAL FOR SICK CFree format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:HINEK, ALEKSANDER;REEL/FRAME:019199/0831Effective date: 20070402RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google