Patent Application
20170183386
This application claims benefit under 35 U.S.C. §119(a) of European Application Nos. 15177103.8 filed Jul. 16, 2015; 15177017.9 filed Jul. 16, 2015; 15177018.7 filed Jul. 16, 2015; and 15177175.5 filed Jul. 16, 2015, the contents of which are incorporated herein by reference in their entireties.
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 6, 2017, is named 048262-087760-US_SL.TXT and is 363,792 bytes in size.
In a first aspect, the invention provides a peptide, typically having 4 to 50 amino acids, and comprising an amino acid sequence selected from SEQUENCE ID NO's 1 to 1312, or a variant or thereof (hereafter “peptide of the invention”).
In one embodiment, the peptide comprises (or consists of) an amino acid sequence selected from SEQUENCE ID NO'S 1 to 151 and 707, or a variant or fragment thereof, wherein the peptide typically has anti-inflammatory activity.
In one embodiment, the peptide comprises (or consists of) an amino acid sequence selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701, or a variant or fragment thereof, wherein the peptide typically has cellular growth promoting activity.
In one embodiment, the peptide comprises (or consists of) an amino acid sequence selected from SEQUENCE ID NO'S 555 to 614 and 702 to 706, or a variant or fragment thereof, wherein the peptide typically has glucose transport promoting activity.
In one embodiment, the peptide comprises (or consists of) an amino acid sequence selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654, or a variant or fragment thereof, wherein the peptide typically has anti-bacterial activity.
In one embodiment, the peptide of the invention comprises a sequence selected from SEQUENCE ID NO's: 1 to 1312.
In one embodiment, the peptide of the invention consists of a sequence selected from SEQUENCE ID NO's: 1 to 1312.
In one embodiment, the variant of the peptide has at least 70%, 75%, 80%, 85%, 90% or 95% sequence homology with the reference peptide of the invention.
In one embodiment, the peptide of the invention is a modified peptide.
In one embodiment, the invention provides a composition comprising a peptide of the invention, or a variant or fragment thereof (hereafter “composition of the invention”).
In one embodiment, the composition comprises a peptide comprising the amino acid sequence of SEQUENCE ID NO: 41 or a variant thereof selected from SEQUENCE ID NO 706.
In one embodiment, the composition comprises a peptide comprising the amino acid sequence of SEQUENCE ID NO: 555 or a variant or fragment thereof selected from SEQUENCE ID NO'S 3, 170, 204, 213, 556, 558, 563.
In one embodiment, the composition comprises a peptide comprising the amino acid sequence of SEQUENCE ID NO: 701 or a variant or fragment thereof.
In one embodiment, the composition of the invention comprises a plurality of peptides of the invention selected from SEQUENCE ID NO'S 1 to 39, 152 to 410, 555 to 594, and 615 to 638.
In one embodiment, the composition of the invention comprises a plurality of peptides of the invention selected from SEQUENCE ID NO'S 5, 23, 22, 38, 39, 21, 258, 242, 261, 211, 222, 249, 235, 295, 283, 284, 216, 555 and 701. In one embodiment, the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the above-referenced peptides. In one embodiment, the composition comprises all of the above-referenced peptides.
In one embodiment, the composition comprises SEQUENCE ID NO's: 555 and 701, and one or more peptides (for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 peptides) selected from SEQUENCE ID NO'S 5, 23, 22, 38, 39, 21, 258, 242, 261, 211, 222, 249, 235, 295, 283, 284, 216.
In one embodiment, the composition of the invention comprises substantially all of the peptides of SEQUENCE ID NO'S 1 to 39, 152 to 410, 555 to 594, and 615 to 638.
In one embodiment, the composition of the invention comprises a plurality of peptides of the invention selected from SEQUENCE ID NO'S 40 to 151, 411 to 549, 595 to 614 and 639 to 643.
In one embodiment, the composition of the invention comprises a plurality of peptides of the invention selected from SEQUENCE ID NO'S 74, 40, 41, 502, 496, 417, 467, 448, 452, 451, 443, 447, 480, 444, 245 and 246.
In one embodiment, the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the above-referenced peptides. In one embodiment, the composition comprises all of the above-referenced peptides.
In one embodiment, the composition comprises SEQUENCE ID NO: 41, and one or more peptides (for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 peptides) selected from SEQUENCE ID NO'S 74, 40, 502, 496, 417, 467, 448, 452, 451, 443, 447, 480, 444, 245 and 246.
In one embodiment, the composition of the invention comprises substantially all of the peptides of SEQUENCE ID NO'S 40 to 151, 411 to 549, 595 to 614 and 639 to 643.
In one embodiment, the composition is a powder. In one embodiment, the composition is a liquid. In one embodiment, the liquid has a pH between 5 and 9, preferably between 6 and 8, and ideally about 7. In one embodiment, the composition is a cream. In one embodiment, the cream has a pH between 5 and 9, preferably between 6 and 8, and ideally about 7.
In one embodiment, the composition is enriched in peptides having a molecular weight of less than 10 KD. This means that the weight % of peptides in the powder having a MW of less than 10KD is greater than the weight % of peptides in the powder having a weight of greater than 10 KD. Such a composition does not exist in nature. In one embodiment, the composition is depleted in cellular debris.
In one embodiment, the invention relates to a food product comprising a composition of the invention, in which the composition is optionally in powder form.
In one embodiment, the invention relates to a personal care product comprising a composition of the invention, in which the composition is optionally in powder form.
In one embodiment, the invention relates to a pharmaceutical product comprising a composition of the invention, in which the composition is optionally in powder form.
In one embodiment, the invention relates to a nutritional or dietary supplement comprising a composition of the invention, in which the composition is optionally in powder form.
In one embodiment, the invention relates to a topical composition comprising a composition of the invention, in which the composition of the invention is optionally in powder form.
The invention also relates to a comestible product comprising a peptide of the invention. Preferably the comestible product is man-made.
Preferably, the comestible product is a food product for human or animal (mammalian) consumption.
In one embodiment the man-made comestible product is a beverage. In one embodiment the man-made comestible product is a bakery product. In one embodiment the man-made comestible product is a dairy product. In one embodiment the man-made comestible product is a snack product. In one embodiment the man-made comestible product is a baked extruded food product. In one embodiment the man-made comestible product is powdered milk. In one embodiment the man-made comestible product is an infant formula product. In one embodiment the man-made comestible product is a confectionary product. In one embodiment the man-made comestible product is a yoghurt. In one embodiment the man-made comestible product is a yoghurt drink. In one embodiment the man-made comestible product is an ice cream product. In one embodiment the man-made comestible product is a frozen food product. In one embodiment the man-made comestible product is a breakfast cereal. In one embodiment the man-made comestible product is a bread. In one embodiment the man-made comestible product is a flavoured milk drink. In one embodiment the man-made comestible product is a confectionary bar. In one embodiment the man-made comestible product is a tea or tea product. In one embodiment the man-made comestible product is a based extruded snack product. In one embodiment the man-made comestible product is a fried snack product. In one embodiment the man-made comestible product is a nutritional supplement. In one embodiment the man-made comestible product is a sports nutritional product. In one embodiment the man-made comestible product is a baby food product. In one embodiment the man-made comestible product is a speciality food product for immunocompromised individuals. In one embodiment the man-made comestible product is a food for geriatric patients.
The invention also relates to a man-made personal care product comprising a peptide of the invention.
The invention also relates to a man-made personal care product comprising a composition of peptides of the invention.
In one embodiment, the personal care product is formulated for topical delivery to the skin of a human.
In one embodiment the personal care product is a skincare product. In one embodiment the personal care product is a haircare product. In one embodiment the personal care product is a dentrifice product. In one embodiment the personal care product is a perfumery product. In one embodiment the personal care product is a deodorant product. In one embodiment the personal care product is an anti-perspirant product. In one embodiment the personal care product is a soap. In one embodiment the personal care product is a liquid soap. In one embodiment the personal care product is a cream. In one embodiment the personal care product is a lotion. In one embodiment the personal care product is a gel. In one embodiment the personal care product is a powder.
The invention also relates to a peptide or composition of the invention for use in treatment or prevention of inflammation, or an inflammatory disorder, in a mammal. In one embodiment, the peptide is selected from, or the composition comprises one or more peptides selected from, SEQUENCE ID NO'S 1 to 151 and 707.
In one embodiment the inflammation is symptomatic inflammation.
In one embodiment the inflammatory disorder is an inflammatory disorder of the joints. In one embodiment the inflammatory disorder is an inflammatory disorder of the cardiovascular system. In one embodiment the inflammatory disorder is an autoimmune disease. In one embodiment the inflammatory disorder is a lung and airway inflammatory disorder. In one embodiment the inflammatory disorder is an intestinal inflammatory disorder. In one embodiment the inflammatory disorder is dermatitis. In one embodiment the inflammatory disorder is acne vulgaris. In one embodiment the inflammatory disorder is psoriasis. In one embodiment the inflammatory disorder is rheumatoid arthritis. In one embodiment the inflammatory disorder is cardiovascular disease. In one embodiment the inflammatory disorder is atherosclerosis. In one embodiment the inflammatory disorder is Type I diabetes.
In one embodiment the inflammatory disorder is Graves disease. In one embodiment the inflammatory disorder is Guillain-Barre disease. In one embodiment the inflammatory disorder is Lupus. In one embodiment the inflammatory disorder is Psoriatic arthritis. In one embodiment the inflammatory disorder is Ulcerative colitis. In one embodiment the inflammatory disorder is asthma. In one embodiment the inflammatory disorder is cystic fibrosis. In one embodiment the inflammatory disorder is COPD. In one embodiment the inflammatory disorder is emphysema. In one embodiment the inflammatory disorder is acute respiratory distress syndrome. In one embodiment the inflammatory disorder is colitis. In one embodiment the inflammatory disorder is inflammatory bowel disease.
The invention also relates to a peptide of the invention for use in treatment or prevention of pain in a mammal. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 1 to 151 and 707.
The invention also relates to a composition of peptides of the invention for use in treatment or prevention of pain in a mammal. In one embodiment, the composition comprises one or more peptides selected from, SEQUENCE ID NO'S 1 to 151 and 707.
The invention also relates to a peptide of the invention for use in treatment or prevention of a metabolic disorder in a mammal. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 1 to 151 and 707.
The invention also relates to a composition of peptides of the invention for use in treatment or prevention of a metabolic disorder in a mammal. In one embodiment, the composition comprises one or more peptides selected from, SEQUENCE ID NO'S 1 to 151 and 707.
In one embodiment, the metabolic disorder is pre-diabetes. In one embodiment, the metabolic disorder is diabetes. In one embodiment, the metabolic disorder is Type-1 diabetes. In one embodiment, the metabolic disorder is Type-2 diabetes. In one embodiment, the metabolic disorder is metabolic syndrome. In one embodiment, the metabolic disorder is obesity. In one embodiment, the metabolic disorder is diabetic dyslipidemia. In one embodiment, the metabolic disorder is hyperlipidemia. In one embodiment, the metabolic disorder is hypertension. In one embodiment, the metabolic disorder is hypertriglyceridemia. In one embodiment, the metabolic disorder is hyperfattyacidemia. In one embodiment, the metabolic disorder is hypercholerterolemia. In one embodiment, the metabolic disorder is hyperinsulinemia. In one embodiment, the metabolic disorder is MODY.
The invention also relates to a peptide of the invention for use in maintaining or restoring gut health in a mammal. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 1 to 151 and 707.
The invention also relates to a composition of peptides of the invention for use in maintaining or restoring gut health in a mammal. In one embodiment, the composition comprises one or more peptides selected from, SEQUENCE ID NO'S 1 to 151 and 707.
Such peptides can be used in personal care, supplement, food and pharmaceutical products to treat and maintain healthy levels of inflammation throughout the body. The present invention is concerned with the huge need for food-derived specific peptides and peptide compositions that reduces inflammation in a way that is able to be processed by the body without completely blocking the immune response and causing autoimmune issues and other undesirable side effects. The invention may ultimately help the 2 billion people suffering from inflammation. The invention also relates to a man-made wound treatment composition comprising a peptide of the invention. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701. The invention also relates to a man-made wound treatment composition comprising a composition of the invention. In one embodiment, the composition comprises one or more peptides selected from, SEQUENCE ID NO'S 152 to 554 and 655 to 701. Typically, the wound treatment composition is formulated for topical application to a wound. In one embodiment, the composition comprises a cream, gel, lotion, powder.
The invention also relates to a plaster, bandage or dressing suitable for application to a wound and comprising a peptide or composition of the invention. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a man-made cell culture media comprising a peptide of the invention. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701. The invention also relates to a man-made cell culture media comprising a composition of the invention. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701. In one embodiment, the cell culture media is formulated for culture of eukaryotic cells. In one embodiment, the cell culture media is formulated for culture of prokaryotic cells.
The invention also relates to a plaster, bandage or dressing suitable for application to a wound and comprising a peptide or composition of the invention.
The invention also relates to a peptide of the invention for use in promoting growth of a cell. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a peptide of the invention for use in promoting growth of a cell culture. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a peptide of the invention for use in promoting growth of a tissue. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a peptide of the invention for use in promoting growth of dermal or epithelial tissue. In one embodiment, the peptide is selected from SEQUENCE ID NO's 152 to 554 and 655 to 701.
The invention also relates to a peptide of the invention for use in promoting growth of skin. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a peptide of the invention for use in promoting growth of an organ. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a peptide of the invention for use in promoting growth of an organism. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a composition of the invention for use in promoting growth of a cell. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a composition of the invention for use in promoting growth of a cell culture. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a composition of the invention for use in promoting growth of a tissue. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a composition of the invention for use in promoting growth of epithelial tissue. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a composition of the invention for use in promoting growth of skin. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a composition of the invention for use in promoting growth of an organ. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a composition of the invention for use in promoting growth of an organism. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
In one embodiment, the cell, tissue or organism has a normal pathology (for example ageing skin). In one embodiment of the invention, the cell, tissue or skin has abnormal pathology (for example tissue damaged due to trauma, drug use, or epithelial tissue in the GI tract damaged due to an inflammatory disorder).
The growth promoting uses may be in-vivo or in-vitro uses. The growth promoting uses may involve administration to mammal externally (i.e. to the skin) or internally (i.e. to the GI tract).
The invention also relates to a peptide of the invention for use in slowing or inhibiting ageing of human skin. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a method of slowing or inhibiting ageing of human skin comprising a step of administering a peptide of the invention to the human skin. Typically, the peptide of the invention is administered topically to the skin. Administration may be by means of a plaster or patch or a formulation suitable for topical application. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a composition of the invention for use in slowing or inhibiting ageing of human skin. The invention also relates to a peptide of the invention for use in preventing or slowing ageing of the human skin. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a method of slowing or inhibiting ageing of human skin comprising a step of administering a composition of the invention to the human skin. Typically, the composition of the invention is administered topically to the skin. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a peptide of the invention for use in treatment of a wound in a mammal. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a composition of peptides of the invention for use in treatment of a wound in a mammal. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a wound treatment composition or product of the invention for use in treatment of a wound in a mammal. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a peptide of the invention for use in treatment or prevention of a disease or condition characterised by damaged epithelial cells or tissue. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
The invention also relates to a composition of peptides of the invention for use in treatment or prevention of a disease or condition characterised by damaged dermal or epithelial cells or tissue. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 152 to 554 and 655 to 701.
In one embodiment, the disease or condition characterised by damaged dermal or epithelial cells or tissue is selected from cancer, trauma
The invention also relates to a peptide of the invention for use in improving muscle status in a mammal. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 555 to 614 and 702 to 706.
The invention also relates to a composition of the invention for use in improving muscle status in a mammal. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 555 to 614 and 702 to 706.
The invention also relates to a peptide of the invention for use in promoting recovery of muscle, typically following exercise. In one embodiment, the peptide is selected from SEQUENCE ID NO'S555 to 614 and 702 to 706.
The invention also relates to a composition of the invention for use in promoting recovery of muscle, typically following exercise. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 555 to 614 and 702 to 706.
The invention also relates to a peptide of the invention for use in maintaining or restoring muscle health (for example lean tissue mass) in a mammal. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 555 to 614 and 702 to 706.
The invention also relates to a composition of peptides of the invention for use in maintaining or restoring muscle health (for example lean tissue mass) in a mammal. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 555 to 614 and 702 to 706.
The invention also relates to a peptide of the invention for use in enhancing physical performance. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 555 to 614 and 702 to 706.
The invention also relates to a composition of the invention for use in enhancing physical performance. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 555 to 614 and 702 to 706.
The invention also relates to a peptide of the invention for use in treatment or prevention of a disease or condition characterised by lethargy or low energy levels. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 555 to 614 and 702 to 706.
The invention also relates to a composition of peptides of the invention for use in treatment or prevention of a disease or condition characterised by lethargy or low energy levels. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 555 to 614 and 702 to 706.
The invention also relates to a peptide or composition of the invention for use in treating or preventing a bacterial infection in a mammal. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654.
The invention also relates to a peptide or composition of the invention for use as an antimicrobial or antibacterial agent. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654.
The invention also relates to a peptide or composition of the invention for use as a preservative. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654 . In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654.
The invention also relates to a peptide or composition of the invention for use as a preservative in a perishable product, such as a food product or a personal care composition. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654.
The invention also relates to a peptide or composition of the invention for use as an anti-bacterial agent in a personal care composition. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654.
The invention also relates to a peptide or composition of the invention for use as an anti-bacterial agent in a household cleaning product. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654.
The invention also relates to a peptide or composition of the invention for use as a plant biocidal agent. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654.
The invention also relates to a peptide of the invention for use in treatment or prevention of a disease or condition characterised by a bacterial infection. The invention also relates to a composition of peptides of the invention for use in treatment or prevention of a disease or condition characterised by a bacterial infection. In one embodiment, the bacterial infection is a MRSA infection. In one embodiment, the peptide is selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654. In one embodiment, the composition comprises one or more peptides selected from SEQUENCE ID NO'S 615 to 643 and 644 to 654.
The invention also relates to a pharmaceutical composition comprising a peptide of the invention in combination with a pharmaceutically acceptable carrier.
The invention also relates to a pharmaceutical composition comprising a composition of peptides of the invention in combination with a pharmaceutically acceptable carrier.
The invention also relates to a comestible product, for example a food product comprising a composition of the invention, for example a dairy or non-dairy product, a solid food or a beverage, a food additive or supplement. The dairy product may be a milk, a cheese, or yoghurt. In one embodiment, the food product is a snack bar. The food product may comprise any amount of the composition of the invention, for example from 0.1% to 30% (w/w).
The food product may be a Food for Specific Medicinal Purposes (FSMP) which is defined as foods that are specifically formulated, processed and intended for the dietary management of diseases, disorders or medical conditions of individuals who are being treated under medical supervision. These foods are intended for the exclusive or partial feeding of people whose nutritional requirements cannot be met by normal foods.
The invention also relates to a conjugate comprising a peptide of the invention conjugated to a binding partner. The binding partner may be selected from a drug, an agent to increase the lipophilicity of the conjugate, or an agent to prolong the plasma half-life of the peptide of the invention. In one embodiment, the peptide is modified to facilitate covalent bonding between the peptide and the binding partner.
The peptides of the invention are used in the topical cosmetic or pharmaceutical composition of this invention at cosmetically or pharmaceutically effective concentrations to achieve the desired effect; in a preferred form with regards to the total weight of the composition, between 0.00000001% (in weight) and 20% (in weight); preferably between 0.000001% (in weight) and 15% (in weight), more preferably between 0.0001% (in weight) and 10% (in weight) and even more preferably between 0.0001% (in weight) and 5% (in weight). Ideally, the peptides of the present invention are preferably used from about 0.00001% w/w to about 0.5% w/w [0.1 to 5000 ppm], and more preferably from 0.00005 w/w to about 0.05 w/w [0.5 to 500 ppm], and most preferably from about 0.0001 w/w to about 0.01 w/w of the composition [1 to 100 ppm]. Ideally, the peptides of the present invention are preferably used from about 0.0001% w/w to about 0.004% w/w of the composition.
For compositions of peptides of the invention, a typical daily dosage may be 0.2g to 100g. However, when administered as a food for special medicinal purpose, or medical food, the daily dosage may be 50-500g per day.
The dosage of compositions of the invention for use in food products and food supplements (i.e. comestible compositions) will be broadly in the 0.2-100 g/day range. In one embodiment, the daily dosage is 1-10 g/day, ideally about 3-8 g/day. In one embodiment, the daily dosage is 10-20 g/day. In one embodiment, the daily dosage is 20-30 g/day. In one embodiment, the daily dosage is 30-40 g/day. In one embodiment, the daily dosage is 10-100 g/day. In one embodiment, the daily dosage is about 5 g/day, ideally about 3-8 g/day. In one embodiment, the dosage is 2-1000 mg/day/kg body weight. In one embodiment, the dosage is 10-500 mg/day/kg body weight. In one embodiment, the dosage is 10-100 mg/day/kg body weight. In one embodiment, the dosage is 30-70 mg/day/kg body weight.
The invention also provides topical composition comprising a peptide of the invention. It will be appreciated that the topical composition may comprise a plurality of peptides, fragments and/or variants. In one embodiment, the topical composition comprises substantially all the peptides. In one embodiment, the topical composition comprises substantially all the variants.
The topical composition of the invention may be presented in a formulation selected from the group comprising creams, multiple emulsions, anhydrous compositions, aqueous dispersions, oils, milks, balsams, foams, lotions, gels, cream gels, hydro-alcoholic solutions, hydro-glycolic solutions, cosmetic, personal care product, hydrogels, liniments, sera, soaps, dusting powder, paste, semi solid formulations, liniments, serums, shampoo, conditioner, ointments, any rinse off formulation, talc, mousses, powders, sprays, aerosols, solutions, suspensions, emulsions, syrups, elixirs, polysaccharide films, patches, gel patches, bandages, an adhesive system, water-in-oil emulsions, oil-in-water emulsions, and silicone emulsions.
In an embodiment of the current invention, the emulsion contains a lipid or oil. The emulsion may be, but is not limited to, oil-in-water, water-in-oil, water-in-oil-in-water and oil-in-water-in-silcone emulsions. The emulsion may contain a humectant. The emulsion may contain an anti-foaming agent, such as silicone. The emulsion may have any suitable viscosity. Emulsions may further contain an emulsifier and/or an anti-foaming agent. Methods of preparing an emulsion are known to a person skilled in the art.
The topical composition of the invention may be incorporated into a medical device for administration. Such a device can include but is not limited to a fabric, patch, bandage, gauge, sock, tight, underwear, dressing, glove, mask, adhesive patches, non-adhesive patches, occlusive patches and microelectric patches or suitable adhesive system. In such an embodiment, the device is in direct contact with the keratinous layer such as the skin, thus releasing the peptides of the invention. It will be understood that the topical composition may be incorporated in any suitable form as detailed herein. For example, the topical composition or peptides of the invention can be incorporated into the device or be present on the surface of the device or can be in a cream, gel or wax formulation or any suitable formulation defined herein and incorporated into the device or on the surface of the device.
The device may be adapted for adhesion or attachment to the skin.
In one embodiment the device is adapted to release a constant quantity of the composition or the peptides of the invention. It will be understood that the amount of the composition contained in the sustained release system will depend, for example, on where the composition is to be administered, the kinetics and duration of the release of the composition of the invention, as well as the nature of the condition, disorder and/or disease to be treated and/or cared for. The device may be such that the composition is released by biodegradation of the device, or by friction between the device and the body, due to bodily moisture, the skin's pH or body temperature.
In an embodiment of the invention the topical composition may further comprise at least one cosmetically or pharmaceutically acceptable excipient. Excipient may be used interchangeably with functional ingredient or additive. It will be understood that although the topical compositions of the current invention can be administered alone, they will generally be administered in admixture with a cosmetic or pharmaceutical excipient. Cosmetically or pharmaceutically acceptable excipient are well known in the art and any known excipient, may be used provided that it is suitable for topical administration and is dermatologically acceptable without undue toxicity, incompatibility and/or allergic reaction.
Preferably any excipient included is present in trace amounts. The amount of excipient included will depend on numerous factors, including the type of excipient used, the nature of the excipient, the component(s) of the topical composition, the amount of active or peptide in the topical composition and/or the intended use of the topical composition. The nature and amount of any excipient should not unacceptably alter the benefits of the peptides of this invention.
In an embodiment of the invention the excipient may be a suitable diluent, carrier, binder, lubricant, suspending agent, coating agent, preservative, stabilisers, dyes, vehicle, solubilising agent, base, emollient, emulsifying agent, fragrance, humectant, and/or surfactants.
Examples of suitable diluents include, but are not limited to, any diluent disclosed in disclosed in US2014120131 or US2004132667. Examples include ethanol, glycerol and water. Examples of suitable carriers include, but are not limited to, lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and any suitable carrier disclosed in US2014120131 or US2004132667.
Examples of suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol and any suitable binder disclosed in US2014120131 or US2004132667.
Examples of suitable lubricants include, but are not limited to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride and any suitable lubricant disclosed in US2014120131 or US2004132667.
The carrier may be any suitable carried known in the art or disclosed in US2014120131 or US2004132667. In some embodiments, the carrier may include, but is not limited to, a liquid, such as water, oils or surfactants, including those of petroleum, animal, plant or synthetic origin, polymer, oil, such as peanut oil, mineral oil, castor oil, soybean oil, alcohol, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glycosides, maltosides, fatty alcohols, nonoxynols, poloxamers, polyoxyethylenes, polyethylene glycols, dextrose, glycerol, or digitonin. It will be understood that the carrier will be dermatologically acceptable. Preferred carriers contain an emulsion such as oil-in-water, water-in-oil, water-in-oil-in-water and oil-in-water-in-silicone emulsions. Emulsions may further contain an emulsifier and/or an anti-foaming agent.
In an embodiment of the invention, the topical composition may further comprise one or more additional ingredients. The topical composition of the invention may be administered consecutively, simultaneously or sequentially with the one or more other additional agents. Such additional ingredients may be those of benefit to include in a topical composition, or of benefit depending on the intended use of the topical composition. The additional ingredient may be active or functional or both.
Examples of such additional ingredients include, but are not limited to, one or more of cleaning agents, conditioning agents, sunscreen, pigment, moisturiser, thickening agents, gelling agents, essential oil, astringents, pigments, anti-caking agent, anti-foaming agent, binders, additives, buffers, chelating agents, external analgesics, film formers or materials, bulking agents, polymers, opacifying agents, pH adjusters, propellants, reducing agents, sequestrants, skin bleaching and lightening agents, skin conditioning agents, aloe vera, healing agents, soothing agents, smoothing agents, pantothenic acid, treating agents, thickeners, vitamins. colourants, pharmaceuticals, antiseptic agents, antifoaming agents, buffering agents, astringents, polymers, pH adjuster, deodorant or any other dermatologically acceptable carrier or surfactant.
It is to be understood that additional ingredients listed may provide more than one benefit. The classification given herein is for clarity and convenience only and not intended to limit the additional ingredient to that particular application or category listed.
Any additional ingredients should be suitable for application to the skin without undue toxicity, incompatibility and/or allergic reaction.
In some embodiments, the additional ingredient has glucose transport activity or aids glucose transport activity. In some embodiments, the additional ingredient has anti-inflammatory activity or aids anti-inflammatory activity. In some embodiments, the additional ingredient has anti-aging activity or aids anti-aging activity. In some embodiments, the additional ingredient is for keratinous layer health and/or development, skin health and/or development, and/or muscle health, recovery and/or development. The active agent may be a pharmacological enhancer. Such active agents are known and available on the market. In such cases, the topical composition of the invention may be administered consecutively, simultaneously or sequentially with the one or more other active agents.
In some embodiments, the additional ingredient may be farnesol ([2E, 6E], -3,7,11,-trimethyl-2, 6, 10,dodecatrien-1-ol), phytantriol (3, 7, 11, 15, tetramethylhexadecane-1,2,3,-triol), desquamation actives, enzymes, enzyme inhibitors, enzyme activators, botanical extracts and marine extracts, anti-acne actives, anti-wrinkle or anti atrophy actives, anti-oxidant/radical scavengers, chelators, flavonoids, anti-inflammatory agents, anti-cellulite agents, topical anaesthetics, tanning actives, skin lightening agents, skin healing agents, bisabolol, antimicrobial or antifungal active, sunscreen actives, particulate material, conditioning agents, structuring agents, thickening agent,
The desquamation active may be any suitable agent that enhances the skin appearance or texture of the skin and is as disclosed in US2014120131 or US2004132667.
Examples of anti-acne actives are as disclosed in US2014120131 or US2004132667 and include, resorcinol, salicylic acid, erythromycin, zine, sulfur, benzoyl peroxides.
Examples of thickening agents are as disclosed in US2014120131 or US2004132667 and include carboxylic acid polymers, crosslinked polyacrylate polymers, polyacrylamide polymers, polysaccharides.
Examples of conditioning agents are as disclosed in US2014120131 or US2004132667 and include humectants, moisturiser or skin conditioner.
Examples of structuring agents are as disclosed in US2014120131 or US2004132667 and include any agent that provide rheological characteristics to the composition and contributes to the stability of the composition.
Any suitable antimicrobial or antifungal active may be used and examples are as disclosed in US2014120131 or US2004132667. Such actives are capable of destroying microbes, preventing growth or action of microbes. Examples include but are not limited to β-lactam drugs, quinolone drugs, tetracycline, erythromycin, streptomycin sulfate, salicylic acid, benzoyl peroxide.
Examples of a particulate material include metallic oxide. Examples of anti-cellulite agents include xanthine agents. Examples of tanning actives includes 1,3-dihydroxy-2-propanone and those disclosed in US2014120131 or US2004132667. Examples of topical anaesthetics include benzocaine, lidocaine and bupivacaine and those disclosed in US2014120131 or US2004132667. Examples of skin lightening agents include any agent known in the art such as kojic acid, ascorbic acid and those disclosed in US2014120131 or US2004132667.
Examples of sunscreen actives include any suitable organic or inorganic sunscreen active. Examples include metallic oxides, 2-ethylhexyl-p-methoxycinnamate and those disclosed in US2014120131 or US2004132667.
Examples of skin healing agents includes panthenoic acid as disclosed in US2014120131 or US2004132667.
Examples of anti-inflammatory agents include any agent that enhances the skin appearance, tone or colour and include but are not limited to corticosteroids, hydrocortisone, non-steroidal agents such as ibuprofen and aspirin and those disclosed in US2014120131 or US2004132667.
Examples of flavonoids includes flavanones, methoxy flavonones, unsubstituted chalcone and mixtures thereof and those disclosed in US2014120131 or US2004132667.
Examples of enzymes include lipases, proteases, catalase, super oxide-dismutase, amylase, peroxidase, glucuronidase, ceramidases, hyaluronidases. Examples of enzyme inhibitors include trypsine inhibitors, Bowmann Birk inhibitors, chymotrypsin inhibitors, botanical extracts, flavonoids, quercetin chalcone and those disclosed in US2014120131 or US2004132667 and mixtures thereof. Examples of enzyme activators include coenzyme A, Q10 (ubiquinone), glycyrrhizin, berberine, chrysin and those disclosed in US2014120131 or US2004132667 and mixtures thereof.
Examples of anti-wrinkle or anti atrophy actives include sulfur containing D and L amino acids, particular, N-acyl derivatives such as N-acetyl-L-cysteine, hydroxyl acids, phytic acid, lipoic acid, lysophosphatidic acid, skin peel agents, vitamin B3, retinoids and those disclosed in US2014120131 or US2004132667 and mixtures thereof.
The anti-oxidant/radical scavenger agent may be any agent that is useful for providing protection against UV radiation or other environmental agents which may cause skin damage such as those disclosed in US2014120131 or US2004132667. Examples of anti-oxidant/radical scavengers include ascorbic acid, its salts and derivatives (vitamin C), tocopherol its salts and derivatives (vitamin E), butylated hydroxyl benzoic acids and their salts, peroxides, gallic acids and alkyl esters, sorbic acid, lipoic acid, amines, lycine pidolate, arginine pilolate, nordihydroguaiaretic acid, bioflavonoids, curcumin, lysine, methionine, proline, superoxide dismutase, silymarin, tea extracts and mixtures thereof.
Examples of chelators include EDTA, NTA, hydoxamic acids, phytic acid, lactoferrin and those disclosed in US2014120131 or US2004132667 and mixtures thereof. A chelator means an agent capable of removing a metal ion by forming a complex so that the metal ion cannot participate in or catalyse chemical reactions. A chelator is useful for protection against UV radiation or other environmental agents that can cause skin damage.
It will be appreciated that a plurality of additional ingredients may be added. The amount of the additional ingredient may be from about 0.001% to about 50% weight of the composition, preferably, about 0.01% to about 20%, preferably about 0.1% to about 10%, about 0.5% to about 10%, about 1% to about 5%, preferably 2% weight of the composition. The amount of additional ingredient included will depend on numerous factors, including the type of additional ingredient used, the nature of the additional ingredient, the component(s) of the topical composition, the amount of active or peptide in the topical composition and/or the intended use of the topical composition. The nature and amount of any additional ingredient should not unacceptably alter the benefits of the peptides of this invention.
The topical composition may be alcohol free.
In some embodiments of the invention, the composition further comprises one or more additional active agents, in addition to the peptide of the invention (also known as the active of the composition). In addition, or alternatively, the composition may be administered with one or more other additional active agents. Typical said additional active agent is present in trace amounts only. In some embodiments, there may be no additional active agent present in the composition. The amount of additional active agent included will depend on numerous factors, including the type of additional active agent used, the nature of the additional active agent, the component(s) of the topical composition, the amount of active or peptide in the topical composition and/or the intended use of the topical composition. The nature and amount of any additional active agent should not unacceptably alter the benefits of the peptides of this invention.
It is to be understood that an ingredient that is considered to be an “active” ingredient in one product may be a “functional” or “excipient” ingredient in another and vice versa. It will also be appreciated that some ingredients play a dual role as both an active ingredient and as a functional or excipient ingredient.
Examples of the additional active agents include glucose transport promoting drugs, skin supplement, agent for treatment and/or care of the skin, anti-inflammatory agent, an anti-aging agent, a cellular growth promoting agent and pharmacological enhancers. Such agents are well known in the art and it will be appreciated that any suitable additional active agent may be used. Additional active agents for treatment and/or care of the skin may include collagen synthesis agents, retinoids, exfoliating agents, anti-cellulite agents, elastase inhibiting agents, melanin synthesis stimulating or inhibiting agents, self-tanning agents, antiaging agents, antimicrobial agents, antifungal agents, fungistatic agents, bactericidal agents, and healing agents. Active agents also include anti-inflammatory agents.
Any additional active agent should be suitable for application to the skin without undue toxicity, incompatibility and/or allergic reaction.
It will be understood that the classification given herein is for clarity and convenience only and not intended to limit the additional ingredient, excipient, or active to that particular application or category listed.
In a particularly preferred embodiment, the methods and uses of the invention involve administration of a peptide or composition of the invention in combination with one or more other active agents, for example, existing growth promoting drugs or pharmacological enhancers available on the market. In such cases, the compounds of the invention may be administered consecutively, simultaneously or sequentially with the one or more other active agents.
The effect of the current invention is accomplished by topical application or administration of the topical composition of the invention described herein to a person, animal or a patient in need of treatment or care. Topical delivery preferably means delivery to a keratinous layer such as the skin, hair and/or nails, but can also mean delivery to a body lumen lined with epithelial cells, for example the lungs or airways, the gastrointestinal tract, the buccal cavity. The effect may be confined to the surface of the skin or may be within the skin or a combination of both.
The topical composition of the invention is administered in a cosmetically or pharmaceutically effective amount. In other words, in an amount that is non-toxic but sufficient amount to provide the desired effect. It will be appreciated that a person skilled in the art would be capable of determining an appropriate dose of the topical compositions of the invention to administer without undue experimentation. Alternatively, a physician will determine the actual dose that is most suitable for a patient depending on the particular condition, disease or disorder to be treated or cared for and the age, body weight and/or health of the person. It will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention. For example, the composition may be administered at a dose of from 0.01 to 50 mg/kg body weight, such as from 0.1 to 30 mg/kg, more preferably from 0.1 to 20 mg/kg body weight, more preferably from 0.1 to 10 mg/kg body weight, preferably 0.1 to 5mg/kg body weight. In an exemplary embodiment, one or more doses of 10 to 300 mg/day or more preferably, 10 to 150 mg/day, will be administered to the patient. The amount and the frequency is as best suited to the purpose. The frequency of application or administration can vary greatly, depending on the needs of each subject, with a recommendation of an application or administration range from once a month to ten times a day, preferably from once a week to four times a day, more preferably from three times a week to three times a day, even more preferably once or twice a day.
In preferred embodiments, repeated use of the topical composition is provided.
The topical composition may be applied by, but not limited to, rubbing, or massaging into the keratinous tissue, skin or area of the body to be treated or cared for. In some embodiments, the composition is left on or not removed from the area of the body. In other embodiments, the composition is removed after a period of time, such as, but not limited to, from about 2 minutes to 60 minutes, from about 5 minutes to about 30 minutes, preferably from about 10 minutes to about 20 minutes. The composition may be removed immediately after application. In some embodiments of the current invention, the composition of the invention may be applied to an area to be treated by means to achieve a greater penetration of the composition and/or peptide of the invention, such as, but not limited to, iontophoresis, sonophoresis, electroporation, microelectric patches, mechanical pressure, osmotic pressure gradient, occlusive cure, microinjections or needle-free injections by means of pressure, such as injections by oxygen pressure, or any combination thereof.
The peptides of the invention are used in the topical cosmetic or pharmaceutical composition of this invention at cosmetically or pharmaceutically effective concentrations to achieve the desired effect; in a preferred form with regards to the total weight of the composition, between 0.00000001% (in weight) and 20% (in weight); preferably between 0.000001% (in weight) and 15% (in weight), more preferably between 0.0001% (in weight) and 10% (in weight) and even more preferably between 0.0001% (in weight) and 5% (in weight).
In some embodiments of the current invention, the composition may be delivered via any one of liposomes, mixed liposomes, oleosomes, niosomes, ethosomes, millicapsules, capsules, macrocapsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, vesicles, micelles, mixed micelles of surfactants, surfactant-phospholipid mixed micelles, millispheres, spheres, lipospheres, particles, nanospheres, nanoparticles,milliparticles, solid nanopartciles as well as microemulsions including water-in-oil microemulsions with an internal structure of reverse micelle and nanoemulsions microspheres, microparticles.
A variety of methods are available for preparing liposomes. See, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787, PCT Publication No. WO 91/17424, Deamer & Bangham, Biochim. Biophys. Acta 443:629-634 (1976); Fraley, et al., PNAS 76:3348-3352 (1979); Hope et al., Biochim. Biophys. Acta 812:55-65 (1985); Mayer et al., Biochim. Biophys. Acta 858:161-168 (1986); Williams et al., PNAS 85:242-246 (1988); Liposomes (Ostro (ed.), 1983, Chapter 1); Hope et al., Chem. Phys. Lip. 40:89 (1986); Gregoriadis, Liposome Technology (1984) and Lasic, Liposomes: from Physics to Applications (1993)). Suitable methods include, for example, sonication, extrusion, high pressure/homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small liposome vehicles and ether fusion methods, all of which are well known in the art.
These delivery systems may be adapted to achieve a greater penetration of the compound and/or peptides of the invention. This may improve pharmacokinetic and pharmacodynamics properties. The delivery system may be a sustained release system wherein the compound or peptide of the invention is gradually released during a period of time and preferably with a constant release rate over a period of time. The delivery systems are prepared by methods known in the art. The amount of peptide contained in the sustained release system will depend on where the composition is to be delivered and the duration of the release as well as the type of the condition, disease and/or disorder to be treated or cared for.
The topical composition of the invention may be for human or animal usage in human and veterinary medicine.
The topical composition of the invention may be used for pharmaceutical, personal care and/or cosmetic uses.
The composition can be used to treat or care for any disease, disorder or condition of the skin, including but not limited to, psoriasis, dermatitis, allergic dermatitis, eczema, spongiosis, edema, skin cancer, ulcers, acne, scars, cellulitis, elastosis, keratosis, rosacea, varicose veins, inflammatory disorders.
The topical composition may be used to for treating or caring for visible signs of aging including but not limited to wrinkles, stretch marks and dark circles, dryness, fine lines, age spots, red blotches, sagging skin, and conditions caused by sun exposure including sunburn, stress, pollution and/diet. The topical composition may also be used for delaying, slowing or inhibiting the skins or the onset of aging. The composition may be administered by a medical device, such as a plaster or a patch as described herein.
The topical composition may be used to treat or care for a wound in a mammal. In another embodiment, the topical composition is for use in the treatment or prevention of a disease or condition characterised by damaged epithelial cells or tissue, and/or damaged dermal or epithelial cells or tissue. The disease may be but is not limited to cancer and trauma.
The topical composition may be used to treat or care for any muscle condition, to improve, muscle status in a mammal, to promote recovery of muscle, typically following exercise, to maintain or restore muscle health (for example lean tissue mass) in a mammal, to enhance physical performance, in treatment or prevention of a disease or condition characterised by lethargy or low energy levels.
The topical composition may be used to promote growth of a tissue, promote growth of epithelial tissue, promote growth of skin, promote growth of an organ, promote growth of an organism. The skin can have a normal pathology and/or an abnormal pathology.
The topical composition may also be used to treat or care for any inflammatory disorder.
A further aspect of the invention relates to a pharmaceutical composition comprising a peptide of the invention or a composition of peptides of the invention, admixed with one or more pharmaceutically acceptable diluents, excipients or carriers. Even though the peptides and compositions of the present invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine. Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the “Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and P J Weller. In particular, formulations for topical delivery are described in Topical drug delivery formulations edited by David Osborne and Antonio Aman, Taylor & Francis, the complete contents of which are incorporated herein by reference. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p hydroxybenzoic acid. Antioxidants and suspending agents may be also used. The peptide or composition of the invention may be adapted for topical, oral, rectal, parenteral, intramuscular, intraperitoneal, intra-arterial, intrabronchial, subcutaneous, intradermal, intravenous, nasal, vaginal, buccal or sublingual routes of administration. For oral administration, particular use is made of compressed tablets, pills, tablets, gellules, drops, and capsules. Preferably, these compositions contain from 1 to 250 mg and more preferably from 10-100 mg, of active ingredient per dose. Other forms of administration comprise solutions or emulsions which may be injected intravenously, intra-arterial, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. The pharmaceutical compositions of the present invention may also be in form of suppositories, vaginal rings, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders. The composition of the invention may be formulated for topical delivery. Topical delivery generally means delivery to the skin, but can also mean delivery to a body lumen lined with epithelial cells, for example the lungs or airways, the gastrointestinal tract, the buccal cavity. In particular, formulations for topical delivery are described in Topical drug delivery formulations edited by David Osborne and Antonio Aman, Taylor & Francis, the complete contents of which are incorporated herein by reference. Compositions or formulations for delivery to the airways are described in O'Riordan et al (Respir Care, 2002, Nov. 47), EP2050437, WO2005023290, US2010098660, and US20070053845. Composition and formulations for delivering active agents to the iluem, especially the proximal iluem, include microparticles and microencapsulates where the active agent is encapsulated within a protecting matrix formed of polymer or dairy protein that is acid resistant but prone to dissolution in the more alkaline environment of the ileum. Examples of such delivery systems are described in EP1072600.2 and EP13171757.1. An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. The active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
Injectable forms may contain between 10-1000 mg, preferably between 10-250 mg, of active ingredient per dose.
Compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention. Depending upon the need, the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight. In an exemplary embodiment, one or more doses of 10 to 300 mg/day or more preferably, 10 to 150 mg/day, will be administered to the patient for the treatment of an inflammatory disorder.
In a particularly preferred embodiment, the methods and uses of the invention involve administration of a peptide or composition of the invention in combination with one or more other active agents, for example, existing anti-inflammatory drugs or pharmacological enhancers available on the market. In such cases, the compounds of the invention may be administered consecutively, simultaneously or sequentially with the one or more other active agents.
In one embodiment of the invention, the peptide of the invention may be administered in the form of a conjugate comprising the peptide, and may optionally include a linker, and a partner molecule, for example a protein such as an antibody molecule intended to increase the half-life of the conjugate in-vivo. In one embodiment, the peptide may be modified to substitute one or more amino acids with amino acids employed to attach partner molecules. For example, an amino acid may be substituted with a lysine residue for the purpose of conjugating a partner molecule such as a PEG molecule.
All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.
Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:
Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term “a” or “an” used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.
As used herein, the term “comprise,” or variations thereof such as “comprises” or “comprising,” are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term “comprising” is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.
As used herein, the term “disease” is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, poisoning or nutritional deficiencies.
As used herein, the term “treatment” or “treating” refers to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s) (for example, the reduction in accumulation of pathological levels of lysosomal enzymes). In this case, the term is used synonymously with the term “therapy”.
Additionally, the terms “treatment” or “treating” refers to an intervention (e.g. the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term “prophylaxis”.
As used herein, an effective amount or a therapeutically effective amount of an agent defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition. The amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate “effective” amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure.
The term “mammal” should be understood to mean a higher mammal, especially a human. However, the term also includes non-mammalian animals such as fish.
The term “composition” should be understood to mean a composition of matter made by the hand of man and not occurring in nature. Exemplary compositions include food compositions, beverage compositions, pharmaceutical compositions, nutritional supplement compositions, personal care compositions and healthcare compositions.
The term “peptide” used herein refers to a polymer composed of 3 to 50 (or 4-50, 5-50, or 6-50) amino acid monomers typically linked via peptide bond linkage. Peptides (including fragments and variants thereof) of and for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid. For example, the peptides of and for use in the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Ill. (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984). When necessary, any of the peptides employed in the invention can be chemically modified to increase their stability. A chemically modified peptide or a peptide analog includes any functional chemical equivalent of the peptide characterized by its increased stability and/or efficacy in vivo or in vitro in respect of the practice of the invention. The term peptide analog also refers to any amino acid derivative of a peptide as described herein. A peptide analog can be produced by procedures that include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of cross-linkers and other methods that impose conformational constraint on the peptides or their analogs. Examples of side chain modifications include modification of amino groups, such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidation with methylacetimidate; acetylation with acetic anhydride; carbamylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6, trinitrobenzene sulfonic acid (TNBS); alkylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxa-5′-phosphate followed by reduction with NABH4. The guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal. The carboxyl group may be modified by carbodiimide activation via o-acylisourea formation followed by subsequent derivatization, for example, to a corresponding amide. Sulfhydryl groups may be modified by methods, such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of mixed disulphides with other thiol compounds; reaction with maleimide; maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenyl sulfonic acid, phenylmercury chloride, 2-chloromercuric-4-nitrophenol and other mercurials; carbamylation with cyanate at alkaline pH. Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides. Tryosine residues may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative. Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate. Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. Peptide structure modification includes the generation of retro-inverso peptides comprising the reversed sequence encoded by D-amino acids.
“Isolated peptide” as applied to a peptide of the invention or modified peptide of the invention typically refers to a peptide of the invention that is produced by man by means of a technical process. Thus, the peptide may be produced by means of a biotechnological process or by means of chemical synthesis.
The term “modified peptide” is used interchangeably with the term derivative of the peptide. The modified peptide includes a peptide which has been substituted with one or more groups as defined herein. The modification may be any modified that provides the peptides and or the composition of the invention with an increased ability to penetrate a cell. The modification may be any modification that increases the half-life of the composition or peptides of the invention. In one embodiment, the group is a protecting group. The protecting group may be an N-terminal protecting group, a C-terminal protecting group or a side-chain protecting group. The peptide may have one or more of these protecting groups. The person skilled in the art is aware of suitable techniques to react amino acids with these protecting groups. These groups can be added by preparation methods known in the art, for example the methods as outlined in paragraphs [0104] to [0107] of US2014120141. The groups may remain on the peptide or may be removed. The protecting group may be added during synthesis. In an embodiment of the invention the peptides may be substituted with a group selected from one or more straight chain or branched chain, long or short chain, saturated, or unsaturated, substituted with a hydroxyl, amino, amino acyl, sulfate or sulphide group or unsubstituted having from 1 to 29 carbon atoms. N-acyl derivatives include acyl groups derived from acetic acid, capric acid, lauric acid, myristic acid, octanoic acid, palmitic acid, stearic acid, behenic acid, linoleic acid, linolenic acid, lipoic acid, oleic acid, isosteric acid, elaidoic acid, 2-ethylhexaneic acid, coconut oil fatty acid, tallow fatty acid, hardened tallow fatty acid, palm kernel fatty acid, lanolin fatty acid or similar acids. These may be substituted or unsubstituted. When substituted they are preferably substituted with hydroxyl, or sulphur containing groups such as but not limited to SO3H, SH, or S—S. In an embodiment of the current invention, the peptide is R1—X—R2. R1 and/or R2 groups respectively bound to the amino-terminal (N-terminal) and carboxyl-terminal (C-terminal) of the peptide sequence. In one embodiment, the peptide is R1—X. Alternatively, the peptide is X—R2. Preferably, R1 is H, C1-4 alkyl, acetyl, benzoyl or trifluoroacetyl; X is the peptide of the invention; R2 is OH or NH2.
In an embodiment, R 1 is selected from the group formed by H, a non-cyclic substituted or unsubstituted aliphatic group, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, Tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and R5—CO—, wherein R5 is selected from the group formed by H, a non-cyclic substituted or unsubstituted aliphatic group, substituted or unsubstituted alicyclyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl, substituted or unsubstituted heterocyclyl and substituted or unsubstituted heteroarylalkyl; R2 is selected from the group formed by —NR3R4, —OR3 and —SR3, wherein R3 and R4 are independently selected from the group formed by H, a non-cyclic substituted or unsubstituted aliphatic group, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted aralkyl; and with the condition that R1 and R2 are not a-amino acids. In accordance with another preferred embodiment, R2 is —NR3R4, —OR 3 or —SR3 wherein R3 and R4 are independently selected from the group formed by H, substituted or unsubstituted C1-C24 alkyl, substituted or unsubstituted C2-C24 alkenyl, Tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc), substituted or unsubstituted C2-C24 alkynyl, substituted or unsubstituted C3-C 24 cycloalkyl, substituted or unsubstituted C5-C24 cycloalkenyl, substituted or unsubstituted C8-C 24 cycloalkynyl, substituted or unsubstituted C 6-C30 aryl, substituted or unsubstituted C7-C24 aralkyl, substituted or unsubstituted heterocyclyl ring of 3-10 members, and substituted or unsubstituted heteroarylalkyl of 2 to 24 carbon atoms and 1 to 3 atoms other than carbon wherein the alkyl chain is of 1 to 6 carbon atoms. Optionally, R 3 and R 4 can be bound by a saturated or unsaturated carbon-carbon bond, forming a cycle with the nitrogen atom. More preferably R 2 is —NR3R4 or —OR 3, wherein R3 and R4 are independently selected from the group formed by H, substituted or unsubstituted C1-C24 alkyl, substituted or unsubstituted C2-C24 alkenyl, substituted or unsubstituted C2-C24 alkynyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C6-C 15 aryl and substituted or unsubstituted heterocyclyl of 3-10 members, substituted or unsubstituted heteroarylalkyl with a ring of 3 to 10 members and an alkyl chain of 1 to 6 carbon atoms. More preferably R3 and R4 are selected from the group formed by H, methyl, ethyl, hexyl, dodecyl, or hexadecyl. Even more preferably R3 is H and R4 is selected from the group formed by H, methyl, ethyl, hexyl, dodecyl, or hexadecyl. In accordance with an even more preferred embodiment, R2 is selected from —OH and —NH2.
In accordance with another embodiment of this invention R i is selected from the group formed by H, acetyl, lauroyl, myristoyl or palmitoyl, and R2 is —NR3R 4 or —OR3 wherein R3 and R4 are independently selected from H, methyl, ethyl, hexyl, dodecyl and hexadecyl, preferably R2 is —OH or —NH2 More preferably, R1 is acetyl or palmitoyl and R2 is —NH2. In a preferred embodiment, the acyl group is bound to the N-terminal end of at least one amino acid of the peptide. In an embodiment of the invention, the peptide is modified to comprise a side chain protecting group. The side chain protecting group may be one or more of the group comprising benzyl or benzyl based groups, t-butyl-based groups, benzyloxy-carbonyl (Z) group, and allyloxycarbonyl (alloc) protecting group. The side chain protecting group may be derived from an achiral amino acid such as achiral glycine. The use of an achiral amino acid helps to stabilise the resultant peptide and also facilitate the facile synthesis route of the present invention. Preferably, the peptide further comprises a modified C-terminus, preferably an amidated C-terminus. The achiral residue may be alpha-aminoisobutyric acid (methylalaine). It will be appreciated that the specific side chain protecting groups used will depend on the sequence of the peptide and the type of N-terminal protecting group used.
“Conjugate”: In one embodiment of the invention the peptide is conjugated, linked or fused to a binding partner, for example one or more polyethylene glycol polymers or other compounds, such as molecular weight increasing compounds or lipophilic groups. The molecular weight increasing compound is any compound that will increase the molecular weight, typically by 10% to 90%, or 20% to 50% of the resulting conjugate and may have a molecular weight of between 200 and 20, 000, preferably between 500 and 10, 000. The molecular weight increasing compound may be PEG, any water-soluble(amphiphilic or hydrophilic) polymer moiety, homo or co-polymers of PEG, a monomethyl-subsitututed polymer of PEG (mPEG) and polyoxyethylene glycerol (POG), polyamino acids such as poly-lysine, poly-glutamic acid, poly-aspartic acid, particular those of L conformation, pharmacologically inactive proteins such as albumin, gelatin, a fatty acid, olysaccharide, a lipid amino acid and dextran. The polymer moiety may be straight chained or branched and it may have a molecular weight of 500 to 40000 Da, 5000 to 10000 Da, 10000 to 5000, Da. The compound (binding partner) may be any suitable cell penetrating compound, such as that peptide, penetratin, pep-1. The compound (binding partner) may be an antibody molecule. The compound (binding partner) may be a lipophilic moiety or a polymeric moiety. The lipophilic substituent and polymeric substituents are known in the art. The lipophilic substituent includes an acyl group, a sulphonyl group, an N atom, an O atom or an S atom which forms part of the ester, sulphonyl ester, thioester, amide or sulphonamide. The lipophilic moiety may include a hydrocarbon chain having 4 to 30 C atoms, preferably between 8 and 12 C atoms. It may be linear or branched, saturated or unsaturated. The hydrocarbon chain may be further substituted. It may be cycloalkane or heterocycloalkane. The peptide may be modified at the N-terminal, C-terminal or both. The polymer or compound (binding partner) is preferably linked to an amino, carboxyl or thio group and may be linked by N-termini or C-termini of side chains of any amino acid residue. The polymer or compound (binding partner) may be conjugated to the side chain of any suitable residue. The polymer or compound (binding partner) may be conjugated via a spacer. The spacer may be a natural or unnatural amino acid, succinic acid, lysyl, glutamyl, asparagyl, glycyl, beta-alanyl, gamma-amino butanoyl. The polymer or compound (binding partner) may be conjugated via an ester, a sulphonyl ester, a thioester, an amide, a carbamate, a urea, a sulphonamide. A person skilled in the art is aware of suitable means to prepare the described conjugate.
“Anti-inflammatory” or “anti-inflammatory activity” as applied to a peptide or fragment means a peptide or fragment that is capable of significantly reducing the secretion of TNFα by LPS-stimulated J774.2 macrophages (compared with untreated LPS-stimulated J774.2 macrophages) when the macrophages are treated with 100 μM of the peptide or fragment as described in the experimental section below.
“Glucose transport promoting” or “glucose transport promoting activity” as applied to a peptide or fragment means a peptide or fragment that is capable of increasing GLUT4 translocation into skeletal muscle compared with an untreated control when employed at a concentration of 2 μM in the in-vitro assay described below. Preferably the peptide or fragment is capable of increasing GLUT4 translocation compared with an untreated control by at least 50% (i.e a relative unit increase in GLUT4 translocation of 1% to 1.5%).
“Growth promoting” or “growth promoting activity” as applied to a peptide or fragment means a peptide or fragment that is capable of increasing elastin production or cellular proliferation of human skin treated with a 20μM solution of peptide or fragment as described in the assay below.
“Antibacterial” or “antibacterial activity” as applied to a peptide or fragment means a peptide or fragment that is capable of visibly inhibiting the growth of a bacteria in the agar-plate based growth inhibition studies described below.
A “variant” of a bioactive fragment shall be taken to mean a fragment having an amino acid sequence that is substantially identical to the reference fragment, and typically is bioactive. Thus, for example, the term should be taken to include fragments that are altered in respect of one or more amino acid residues. Preferably such alterations involve the insertion, addition, deletion and/or substitution of 5 or fewer amino acids, more preferably of 4 or fewer, even more preferably of 3 or fewer, most preferably of 1 or 2 amino acids only. Insertion, addition and substitution with natural and modified amino acids is envisaged. The variant may have conservative amino acid changes, wherein the amino acid being introduced is similar structurally, chemically, or functionally to that being substituted. Generally, the variant will have at least 70% amino acid sequence homology, preferably at least 80% sequence homology, more preferably at least 90% sequence homology, and ideally at least 95%, 96%, 97%, 98% or 99% sequence homology with the parent anti-inflammatory fragment. In this specification, the term “sequence identity” should be understand to comprise both sequence identity and similarity, i.e. a variant (or homolog) that shares 70% sequence identity with a reference sequence is one in which any 70% of aligned residues of the variant (or homolog) are identical to or conservative substitutions of the corresponding residues in the reference sequence across the entire length of the sequence. Sequence identity is the amount of characters which match exactly between two different sequences. Hereby, gaps are not counted and the measurement is relational to the shorter of the two sequences. In terms of “sequence homology”, the term should be understood to mean that a variant (or homolog) which shares a defined percent similarity or identity with a reference sequence when the percentage of aligned residues of the variant (or homolog) are either identical to, or conservative substitutions of, the corresponding residues in the reference sequence and where the variant (or homolog) shares the same function as the reference sequence. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example, one alignment program is BLAST, using default parameters. Details of these programs can be found at the following Internet address: http://www.ncbi.nlm.nih.gov/blast/Blast.cgi.
Variants of SEQUENCE ID NO: 555 (muscle peptide E64_SP2)
Variants of SEQUENCE ID NO: 555 (VLDLAIPVNRPGQL) including variants having 1,2 or 3 conservative amino acid substitutions, 1, 2 to 3 non-conservative amino acid substitutions, 1-2 amino acid additions, 1, 2 or 3 amino acid deletions, are provided below:
One conservative amino acid substitution:
Two conservative amino acid substitutions:
Three conservative amino acid substitutions:
One non-conservative amino acid substitution
Two non-conservative amino acid substitution
Three non-conservative amino acid substitution
One or two amino acid additions
One, two or three amino acid deletions
Variants of SEQUENCE ID NO: 41 (anti-inflammatory peptide (I_37)
Variants of SEQUENCE ID NO: 41 (RGPQQYAEWQINEK) including variants having 1,2 or 3 conservative amino acid substitutions, 1, 2 to 3 non-conservative amino acid substitutions, 1-2 amino acid additions, 1, 2 or 3 amino acid deletions, are provided below:
One conservative amino acid substitution:
Two conservative amino acid substitutions:
Three conservative amino acid substitutions:
One non-conservative amino acid substitution
Two non-conservative amino acid substitution
Three non-conservative amino acid substitution
One or two amino acid additions
One, two or three amino acid deletions
Variants of SEQUENCE ID NO: 701 (anti-ageing peptide E_1_788)
Variants of SEQUENCE ID NO: 701 (QSFLLSGNQ) including variants having 1 or 2 conservative amino acid substitutions, 1, 2 to 3 non-conservative amino acid substitutions, 1-2 amino acid additions, 1, 2 or 3 amino acid deletions, are provided below:
One conservative amino acid substitution:
Two conservative amino acid substitutions:
One non-conservative amino acid substitution
Two non-conservative amino acid substitution
One or two amino acid additions
One, two or three amino acid deletions
“Fragment” means a fragment of a peptide of the invention that typically has a bioactivity, for example anti-inflammatory activity, anti-ageing activity, glucose transport promoting activity, or anti-bacterial activity. In one embodiment, the fragment has at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids. In one embodiment, the fragment consists of at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the reference sequence. Examples of fragments of the invention are provided in SEQUENCE ID NO'S 708 to 751. Examples of fragments of SEQ ID NO: 555 include:
Examples of fragments of SEQ ID NO:701 include:
“Enriched in peptides having a molecular weight of less than 10 KD” as applied to a composition of the invention means that the dry weight % of peptides in the composition having a molecular weight of less than 10 KD is greater than the dry weight % of polypeptide/protein in the composition having a molecular weight of 10 KD or greater.
“Inflammatory disorder” means an immune-mediated inflammatory condition that affects humans and is generally characterised by dysregulated expression of one or more cytokines. Examples of inflammatory disorders include skin inflammatory disorders, inflammatory disorders of the joints, inflammatory disorders of the cardiovascular system, certain autoimmune diseases, lung and airway inflammatory disorders, intestinal inflammatory disorders. Examples of skin inflammatory disorders include dermatitis, for example atopic dermatitis and contact dermatitis, acne vulgaris, and psoriasis. Examples of inflammatory disorders of the joints include rheumatoid arthritis. Examples of inflammatory disorders of the cardiovascular system are cardiovascular disease and atherosclerosis. Examples of autoimmune diseases include Type 1 diabetes, Graves disease, Guillain-Barre disease, Lupus, Psoriatic arthritis, and Ulcerative colitis. Examples of lung and airway inflammatory disorders include asthma, cystic fibrosis, COPD, emphysema, and acute respiratory distress syndrome. Examples of intestinal inflammatory disorders include colitis and inflammatory bowel disease. Other inflammatory disorders include cancer, hay fever, periodontitis, allergies, hypersensitivity, ischemia, depression, systemic diseases, post infection inflammation and bronchitis.
The peptides and compositions of the invention may also be employed in the non-therapeutic treatment of inflammation. Examples of non-therapeutic treatment of inflammation include use to relieve normal, non-pathological, inflammation, for example inflammation in the muscles and joints following exercise.
In this specification, the term “Metabolic disorder” should be understood to include pre-diabetes, diabetes; Type-1 diabetes; Type-2 diabetes; metabolic syndrome; obesity; diabetic dyslipidemia; hyperlipidemia; hypertension; hypertriglyceridemia; hyperfattyacidemia; hypercholerterolemia; hyperinsulinemia, and MODY.
“Ani-ageing” means inhibiting or slowing the appearance of ageing of a human's skin and/or reversing the appearance of ageing. “Slowing or inhibiting ageing of the skin” means slowing or inhibiting the ageing process in the skin, and/or reversing the appearance of ageing.
“Disease or condition characterised by damaged dermal or epithelial cells or tissue” means any condition or disease that results in damaged dermal or epithelial tissue or cells or organs. One example is trauma which often results in damaged skin. Another example is an inflammatory skin condition such as psoriasis or excezma which often results in damaged skin. Another example is an inflammatory disorder of the lower intestines which can result in damaged epithelial cells/tissue lining the lower intestines. Another example is damaged epithelial cells/tissue lining the lower intestines caused by ingestion of a toxic or damaging substance, for example toxic chemicals or drugs. Another example is cancer, for example bowel cancer, which can result in damaged epithelial tissue in the bowel. Another condition is a peripheral inflammatory disorder such as atopic dermatitis which can result in damage to the skin in humans.
“Disease or condition characterised by bacterial infection” means any condition or disease characterised having a pathology caused by growth of bacteria or by bacterial infection, including for example MRSA, salmonella, listeria, bacterial pneumonia, Staphylococcal food poisoning, bacterial memingitis. Specific examples are provided on the web page of Wikipedia™ under the section “List of infectious diseases”.
“Man-made” as applied to comestible products should be understood to mean made by a human being and not existing in nature.
“Maintaining or restoring gut health” means reducing and/or regulating the pro-inflammatory response in the gut and more specifically the epithelial cells. The healthy microbiome offers some protection against pathogenic viruses and bacteria, and their presence is needed to guide the development of our immune system. It has been shown that these bacteria can react to human signals of stress, sickness, or age which can be manifested by inflammation and as a consequence switch on their virulence genes and cause or contribute to disease. Having the ability to reduce and maintain at healthy levels the inflammatory response can help maintain the healthy bacteria. Digestive problems, which comprise the number one health problem in North America, appear to be occurring with more frequency in recent years. One way to maintain digestive health is to maintain proper inflammation and intestinal flora.
“Improving muscle status” means improving the muscle health, for example promoting skeletal muscle protein synthesis, skeletal glucose absorbtion, improving lean tissue mass in therapeutic or non-therapeutic context, promoting muscle recovery generally after activity exercise, or improving muscle performance. The methods or uses may be therapeutic or non-therapeutic. The term “improving lean tissue mass status” should be understood to mean increasing lean tissue mass, or inhibiting or preventing the rate of lean tissue mass degradation.
“Promoting muscle recovery” means causing an increase in absorbtion of glucose in skeletal muscle compared with untreated skeletal muscel.
“Disease or condition characterised by lethargy or low energy levels” means any condition or disease characterised by a feeling or tiredness or low energy. Examples include allergies, asthma, anemia, cancer and its treatments, chronic pain, heart disease, infection, depression, eating disorders, grief, sleeping disorders, thyroid problems, medication side effects, alcohol use, or drug use.
“Maintaining or restoring muscle health” means helping retain or restore mammalian muscle health resulting from damage incurred during exercise. By promoting glucose transport in skeletal muscle the peptides promote recovery from exercise, and relieve muscle soreness/pain and injury connected with exercise. They can also be used to decrease and prevent muscle cramping, and to allow a faster recovery from muscle cramping. Cramping can result from physical stress, mental stress, and or Repetitive Strain Injury stress. By promoting glucose transport the peptides help reduce Myopathy of the muscle, and help prevent Sarcopenia in mammals, promote recovery from injuries during exercise, and relieve muscle soreness/pain and injury connected with exercise. The invention also relates to a peptide or composition of the invention for use in maintaining or restoring muscle health in a mammal.
In this specification, the term “substantially all” as applied to a list of peptides should be understood to mean at least 60%, 70%, 80%, 90% or 95% of the peptides.
In this specification, the term “personal care product” should be understood to mean a composition formulated for use by humans in cleaning or treating the human body, particularly the skin, teeth, nails, feet and hair. Examples include shampoo, conditioner, skin creams and lotions, powders, dentrifice, shower gel or creams, body lotion, deodorant, and anti-perspirant.
In this specification, the term “nutritional supplement” should be understood to mean a product formulated for ingestion by a mammal and intended to confer a health benefit on the recipient. The supplement can take any form, for example a solid, liquid, or powder. Examples of supplements include powders, tablets, capsules, and drinks.
A further aspect of the invention relates to a pharmaceutical composition comprising a peptide of the invention or a composition of peptides of the invention, admixed with one or more pharmaceutically acceptable diluents, excipients or carriers. Even though the peptides and compositions of the present invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine. Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the “Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and P J Weller. In particular, formulations for topical delivery are described in Topical drug delivery formulations edited by David Osborne and Antonio Aman, Taylor & Francis, the complete contents of which are incorporated herein by reference. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
The peptide or composition of the invention may be adapted for topical, oral, rectal, parenteral, intramuscular, intraperitoneal, intra-arterial, intrabronchial, subcutaneous, intradermal, intravenous, nasal, vaginal, buccal or sublingual routes of administration. For oral administration, particular use is made of compressed tablets, pills, tablets, gellules, drops, and capsules. Preferably, these compositions contain from 1 to 250 mg and more preferably from 10-100 mg, of active ingredient per dose. Other forms of administration comprise solutions or emulsions which may be injected intravenously, intra-arterial, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. The pharmaceutical compositions of the present invention may also be in form of suppositories, vaginal rings, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders. The composition of the invention may be formulated for topical delivery. Topical delivery generally means delivery to the skin, but can also mean delivery to a body lumen lined with epithelial cells, for example the lungs or airways, the gastrointestinal tract, the buccal cavity. In particular, formulations for topical delivery are described in Topical drug delivery formulations edited by David Osborne and Antonio Aman, Taylor & Francis, the complete contents of which are incorporated herein by reference. Compositions or formulations for delivery to the airways are described in O'Riordan et al (Respir Care, 2002, November 47), EP2050437, WO2005023290, US2010098660, and US20070053845. Composition and formulations for delivering active agents to the iluem, especially the proximal iluem, include microparticles and microencapsulates where the active agent is encapsulated within a protecting matrix formed of polymer or dairy protein that is acid resistant but prone to dissolution in the more alkaline environment of the ileum. Examples of such delivery systems are described in EP1072600.2 and EP13171757.1. An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. The active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required. Injectable forms may contain between 10-1000 mg, preferably between 10-250 mg, of active ingredient per dose.
Compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention. Depending upon the need, the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight. In an exemplary embodiment, one or more doses of 10 to 300 mg/day or more preferably, 10 to 150 mg/day, will be administered to the patient for the treatment of an inflammatory disorder.
In a particularly preferred embodiment, the methods and uses of the invention involve administration of a peptide or composition of the invention in combination with one or more other active agents, for example, existing anti-inflammatory drugs or pharmacological enhancers available on the market. In such cases, the compounds of the invention may be administered consecutively, simultaneously or sequentially with the one or more other active agents.
TNF-α is secreted by macrophages in response to stimulation by endotoxins such as lipopolysaccharides (LPS). TNF-α is thought to be involved in systemic inflammation and dysregulation of TNF-α production is thought to be involved in many diseases. The Biolegend assay is a sandwich ELISA kit that is designed for the accurate quantitation of human TNF-α from cell culture supernatant, serum or plasma.
THP-1 monocytes were seeded in a 96 well plate at 10,000 cells per well in RPMI containing 10% fetal calf serum (FCS), 1% Pen/strep, 1% L-glutamine, 100 nM PMA and allowed to differentiated for 72 h prior to experimentation.
Following differentiation the cells were incubated with 100 ng/ml, 10 ng/ml or 1 ng/ml synthetic peptide for 24 h respectively.
Following treatment the cells were stimulated with 10 ng/ml LPS for 5 h and the quantity of TNF-α in the supernatant determined using the Biolegend assay ELISA kit.
Results were calculated as a percentage of the untreated control. An increase in optical density reading indicates greater quantity of TNF-α release into cell culture supernatant.
The results are provided in
The effect of six synthetic peptides of the invention, SP1 to SP6 (SEQUENCE ID NO: 108, 109, 110, 111, 85 and 91) and four peptide compositions on the inflammatory response in vitro using a cell line was determined.
Peptide composition I_1_HR (Rice) contained the followings peptides (identified by SEQ ID): 116, 197, 207, 112, 211, 158, 201, 203, 114, 183, 130, 113, 182, 167, 166, 152, 220, 213, 215, 154, 219, 218, 165, 123, 185, 190, 209, 181, 198, 200, 147, 172, 184, 124, 153, 205, 115, 196, 151, 161, 160, 216, 210, 208, 146, 133, 204, 212, 206.
Peptide composition I_2_HR (Rice) contained the followings peptides (identified by SEQ ID): 189, 177, 174, 129, 176, 202, 193, 195, 194, 192, 182, 128, 220, 127, 134, 136, 135, 180, 179, 178, 219, 218, 145, 120, 175, 190, 149, 126, 187, 191, 121, 122, 159, 132, 162, 137, 150, 186, 188, 164, 118, 125, 163, 157, 156, 117.
Peptide composition E_1_HR (Pea) contained the followings peptides (identified by SEQ 76, 106, 102, 101, 100, 92, 96, 83, 89, 90, 104, 82, 75, 79, 78, 77, 99, 103, 72, 86, 105, 94, 93, 81, 97, 80, 88, 85, 87, 71, 107, 73, 84, 98, 95.
Peptide composition E_2_HR contained homologs of the peptides of the invention.
A J774.2 mouse macrophage cell line was treated with 100 μM of each synthetic peptide (SP) and 0.5 mg/ml of each peptide composition and the effect on two pro-inflammatory markers—tumour necrosis factor α (TNFα) and interleukin-1β (IL-1β) was determined after inflammation was induced using lipopolysaccharide (LPS) as an inflammatory stimulus. A one way anova was used with the dunnett test which is a multiple comparison and compares every mean with a single control mean.
Synthetic peptides were first diluted in a suitable solvent. Dimethyl sulfoxide (DMSO) was the solvent of choice for peptides with poor predicted water solubility. Final concentration of DMSO in each well: SP1 (1_155_HR)-0.3%, SP2 (1_374_HR)-0%, SP3 (E_155_HR)-0.3%, SP4 (E_54_HR)-1%, SP5 (E_41_HR)-1%, SP6 (E_788_HR)-0.3%, positive Control-0%. Cells were first treated with 100 μM of each SP for 24 hours before an alamar blue assay was performed. No viability issues were seen with any of the peptides.
The peptide compositions were prepared by adjusting the pH to between 6-7 and sterile filtering. The effects of the peptide compositions on cell viability was determined. J774.2 macrophages were treated with 1 mg/ml and 0.5mg/ml of each peptide composition, hydrogen peroxide to induce cell death as a positive control, and a peptide known to be non-toxic as a negative control. An alamar blue assay was then performed and cell survival is shown in
The effect of the DMSO on TNFα and IL-1β secretion was determined. 1% DMSO significantly increased levels of TNFα (
THP-1 differentiated macrophages were treated with a composition of rice peptides of the invention (I_2_HR) for 24 hrs. prior to LPS stimulation were compared to untreated cells. TNF-α secretion in I_2_HR treated cells is reduced by 92% vs. untreated cells. Significant results are observed at 100 ug/ml and 500 ug/ml concentrations of I_2_HR, indicating the potency of I_2_HR.
BrDu is incorporated into newly synthesised DNA strands of actively proliferating cells. Following partial denaturation of double stranded DNA, Brdu is detected immunochemically allowing the assessment of the population of cells which are synthesizing DNA.
Human Dermal Fibroblasts (HDF—Sigma 10605a) were seeded in a 96 well plate at 10,000 cells per well in DMEM containing 10% fetal calf serum (FCS), 1% Pen/strep, 1% L-glutamine and allowed to adhere for 24 h.
Following the initial 24 h incubation the cells were incubated with 5 μg/ml, 0.5 μg/ml or 0.05 μg/ml synthetic peptide for 24 h respectively.
After 18 h incubation with synthetic peptides 20 μl BrDu reagent was added to each well. At 24 h incubation the cell were fixed and the amount of 2-DG6P was measured using the BrdU Cell Proliferation Assay, all steps were carried out according to the manufacturer's instructions. Results were calculated as a percentage of the untreated control. An increase in optical density reading indicates greater incorporation of BrDu and increase cell proliferation. The results are shown in
Hydroxyproline in tissue preparations is a direct measure of the amount of collagen present. FIRELISA Human Hydroxyproline ELISA kit assay is designed to measure hydroxyproline in tissue or peptide compositions.
Human Dermal Fibroblasts (HDF Sigma 10605a) were seeded in 24 well plates at 50,000 cells per well in DMEM containing 10% fetal calf serum (FCS), 1% Pen/strep, 1% L-glutamine and allowed to adhere for 24 h.
Following the initial 24 h incubation the cells were incubated with 5 μg/ml, 1 μg/ml or 0.1 μg/ml synthetic peptide for 96 h respectively.
After treatment the cells were lysed using 4 freeze thaw cycles in liquid nitrogen. The lysed cells were centrifuged and 50 μl/ml of each supernatant was assayed using the FIRELISA Human Hydroxyproline ELISA kit. All steps were carried out according to the manufacturer's instructions.
Results were calculated as a percentage of the untreated control. An increase in optical density reading indicates an increase collagen content. The results are shown in
Elastin is a highly elastic protein in connective tissue and allows many tissues in the body to resume their shape after stretching or contracting. FIRELISA Human Elastin ELISA kit assay is designed to measure Elastin in tissue or protein/peptide compositions.
Human Dermal Fibroblasts (HDF) were seeded in 24 well plates at 50,000 cells per well in DMEM containing 10% fetal calf serum (FCS), 1% Pen/strep, 1% L-glutamine and allowed to adhere for 24 h.
Following the initial 24 h incubation the cells were incubated with 5 μg/ml, 1 μg/ml or 0.1 μg/ml synthetic peptide for 96 h respectively.
After treatment the cells were lysed using 4 freeze thaw cycles in liquid nitrogen. The lysed cells were centrifuged and 50 μl/ml of each supernatant was assayed using the FIRELISA Human Elastin ELISA kit. All steps were carried out according to the manufacturer's instructions.
Results were calculated as a percentage of the untreated control. An increase in optical density reading indicates an increase collagen content.
The results are shown in
Incubator, Flow Laminar Chamber, Sterile Polished Plastic Rod, Pipettor, Maintenance medium, Plate 6 well, Plate 24 well.
MTT, PBS, SDS, Formaldehyde, Xylene, Ethanol absolute, Dulbecco's phosphate-buffered saline (DPBS), Metal Enhanced DAB substrate kit, ABC peroxidase staining kit, Citric acid, Sodium hydroxide 2N, Hydrogen peroxide 30%, Anti-Filaggrin, Anti-rabbit IgG-Biotin, Tween 20.
Nature: Human skin tissue 5 mm diameter
Batch number: EXP004050B009 and EXP004050B011
Provider: Laboratoire Biopredic International—8-18 rue Jean Pecker—35000 Rennes—France.
Tel: +33 (0)2.99.14.36.14—Fax: +33 (0)2.99.54.44.72.
Certificates of analysis are present in Annex 1.
Two batches are used for the assay. Batch EXP004050B005 is used for experiment day 1, and Batch EXP004050B006 is used for experiment day 5.
Maintenance Medium: Batch n°: MIL 218C
Provider: Laboratoire Biopredic International—8-18 rue Jean Pecker—35000 Rennes—France.
P9 (14-CHL-0723-09) is the Pea composition (SEQ ID Numbers:50, 85, 74, 140, 82, 136, 189, 77, 169, 149, 171, 178, 143, 127, 190, 141, 147, 133, 186, 125, 122, 119, 87, 90, 86, 89, 138, 129, 123, 120, 117, 113, 110, 121, 105, 98, 55, 161, 19, 317, 135, 130, 146, 177, 160, 170, 188, 83, 78, 36, 96, 159, 26, 330, 168, 148, 184, 151, 151, 165, 114, 284)
P10 (14-CHL-0723-010) is the Rice composition (SEQ ID Numbers: 245, 246, 263, 250, 257, 259, 276, 255, 251, 264, 256, 266, 274, 270, 269, 356, 245, 380, 262, 258, 356, 218, 252, 358, 271, 253, 344, 275, 272, 226, 224, 220, 248, 261, 265, 373, 375, 247, 249, 363, 273, 343, 273, 362)
Skin explants were prepared from abdominal plastic surgery. Some explants were delipidated with alcohol to obtain a dehydrated skin.
These explants were maintained in maintenance medium supplied by the provider Bioprédic International for 5 days. Test items are applied twice per day with 5 μL per explant. At the end of the test, viabilities controls are realized with the MTT on two explants, the third explant is fixed in the formaldehyde 4% for histology and cell staining.
For each time of analysis (D1 and D5), histologies on delipidated explants, treated explants with test items, the DMSO 0.3% control and water control, are performed.
After receipt in the laboratory, each skin explant in the maintenance medium is delipidated with 5 μL alcohol during 3 hours.
After 3 hours, all skin explants are treated two per day with test items, and they are incubated at 37° C. +/−2° C., 5% CO2 for 1 day or 5 days.
Integrity of the system is realized at day 1 and day 5 with a viability control with MTT.
Histology is realized by the laboratory Gredeco and the immunostaining to elastin and Ki67 are realized by the same laboratory. Immunostaining to filaggrin is realized by the laboratory Intertek.
The detection of elastin (rabbit monoclonal antibody, clone P15502, LSBio) is performed using an immunoperoxidase technique two layers (ABC kit, Vector Laboratories) and revealed by AEC (3-amino-9-ethylcarbazole). The immunohistochemical staining intensity in the elastic fibers is evaluated using a semi-quantitative histological score. Epithelial proliferation was analyzed by immunohistochemistry using anti-Ki67 antibody.
Immunodetection was performed using an indirect immunoperoxidase technique three layers, amplified (DAKO kit) and revealed by AEC (3-Amino-9-ethylcarbazole). Counting the number of labeled cells (keratinocytes of the basal layer of the epidermis) is performed and provides the total number of basal cells to calculate the % of labeled cells.
The specific staining of filaggrin is performed with an immunoperoxidase staining (ABC kit, Fisher). The intensity of immunohistochemical marker in the epidermis is evaluated relative to the negative control of the solvent (Water or DMSO 0.3%).
The integrity control and the viability control are present in
The elastic fibers of the dermis were revealed by staining with the catechin and morphometrically quantified by analysis by computer-assisted image. The percentage area taken up by elastic fibers in the dermis was calculated in the dermis and the average superficial dermis. Results are presents in Table 4,
Under the experimental conditions of the study, 0723-1 and 0723-3 samples show an increase by twice of elastic fibers in the superficial dermis compared to control water (Error! Reference source not found.), and an increase in the middle dermis compared to the water control at D5. The 0723-2 sample shows an increase doubled in the middle dermis at day 1 compared to control water and an increase at day 5.
The results of the immunohistochemical analysis of Ki67 are reported in Table 5 and expressed as % of labelled at the basal layer of the epidermis. The Error! Reference source not found. shows the percentage of Ki 67 cells compared to negative controls (water or DMSO). Immunohistochemical analysis of mitotic activity is shown in annex 4 with a reminder of the average for each analysed conditions.
Table 5. % of Ki67 positive cells in the basal layer of the epidermis. Orange bands correspond to samples dissolved into DMSO 0.3% instead of water.
Under the experimental conditions of the study, test item 0723-06, 0723-08, 0723-09 and 0723-010 show an increase in the number of mitotic cells compared to EGF at day 1. A decrease in the mitotic index was observed on day 5 compared to day 1 for all analysed conditions.
The decrease in this cell staining on day 5 is caused by the model. Indeed, after approximately 3 days cell turnover is exhausted on this model.
Measuring glucose uptake using 2-deoxyglucose (2-DG) is a widely accepted method used to investigate glucose uptake in skeletal muscle cells. 2-DG is taken up by glucose transporters and metabolized to 2-DG-6-phosphate (2-DG6P). The amount of accumulated non-metabolized 2-DG6P is proportional to glucose uptake by cells.
1. Human skeletal myoblasts (Sigma 150-05a) were seeded in a 96 well plate at 10,000 cells per well in Skeletal Muscle Differentiation medium and allowed to differentiated for 72 h prior to experimentation.
2. The differentiated cells were serum starved for 24 h prior to stimulation with insulin or synthetic peptides. After starvation, the serum free media was removed, cells rinsed with Phosphate Buffered Saline (PBS) and media replaced with 100 μl of Krebs-Ringer-Phosphate-HEPES (KRPH) and incubated for 1 h.
3. The cells were then stimulated with 100 nM insulin for 30 minutes or 5 μg/ml, 0.5 μg/ml or 0.05 m/ml synthetic peptide for 3 h respectively.
4. Following stimulation the cells were incubated with 10 μl/well of 2-DG solution for 40 min and glucose uptake was measured using the PrismColor Glucose Uptake Assay Kit' (Molecutools), all steps were carried out according to the manufacturer's instructions.
5. Results were calculated as a percentage of the untreated control. An increase in optical density reading indicates greater incorporation of 2-DG6P and increase in glucose uptake. All experiments were carried out in duplicate on three plates (6 wells/condition). Significance was determined using the Students t-test (*p<0.05 compared to control, **p<0.01 compared to control, *** p<0.001 compared to control)
The results are shown in
Skeletal muscle is the predominant site of glucose disposal (80%) under insulin-stimulated or post-prandial conditions. Under these conditions, transport of glucose into skeletal muscle is facilitated principally by the insulin-responsive glucose transport protein GLUT4, which translocates to the cell surface upon insulin or contractile stimulation.
We determined the effect of six synthetic peptides (SP1-6) and four peptide compositions on in vitro GLUT4 translocation using the L6 rat skeletal muscle cell line. A clone of the L6 cell line containing overexpression of GLUT4 tagged with a c-myc epitope (courtesy of Prof. Amira Klip, Hospital for Sick Children, Toronto) was used to investigate the efficacy of each synthetic peptide and peptide composition for effects on GLUT4 translocation in a dose-response design.
SP2 [SEQ ID 555] is a glucose transport promoting fragment of Pea Protein P13918, whereas peptides SP1 and SP3-SP6 are comparative peptides.
The following compositions of peptides were tested for skeletal muscle glucose transport activity in an in-vitro test:
I_2_BE (comprises peptides of SEQ ID NO: 55 and 10)
E_1_BE (comprises peptides of SEQ ID NO: 48, 49, 50, 51, 54, 58, 60, 61, 62, 63)
Cell culture
L6-GLUT4myc cells were grown in 10% FBS and 2 μg/ml blasticidin. Cells were grown for 48-72 hours before being seeded in 24-well plates at 15,000 cells per well in 2% FBS and allowed to differentiate for 6 to 8 days prior to experimentation.
L6-GLUT4myc cells were serum-starved for three hours prior to incubation with 100 nM of insulin for 30 mins, or 200, 20, 2.0 and 0.2 μM of SP, and 2, 1, 0.5 and 0.25mg/ml of peptide composition for 3 hours respectively. A 3 hour incubation period was selected based on previous findings identifying that incubation with branch chain amino acid containing di-peptides for 3 hours increases glucose uptake in L6 myotubes 1. Treatments were staggered in order to determine GLUT4myc translocation at the same time point.
The quantity of myc-tagged GLUT4 at the cell surface was measured by antibody-coupled colorimetric assay. Briefly, after incubation with either insulin for 30 mins or synthetic peptide or peptide composition for 3 hours respectively, L6-GLUT4myc cells were fixed via incubation with 3% paraformaldehyde (PFA). A 0.1 M glycine solution was then added to quench PFA and cells were blocked with 5% goat serum. The myotube monolayer was exposed to anti-myc antibody and then incubated with peroxidase conjugated donkey anti-mouse IgG. 1mL of o-phenylenediamine dihydrochloride (OPD) reagent was added to each well and this reaction was stopped by adding 250 μl/well of 3 M HCL. To determine GLUT4 translocation to cell surface, a measured aliquot of each condition was determined spectrophotometrically on a plate reader using absorbance at 492 nm.
Peptides were first diluted in a suitable solvent. Dimethyl sulfoxide (DMSO) was the solvent of choice for peptides with poor predicted water solubility. Final concentration of DMSO in each well at 200, 20, 2 and 0.2 μM for each synthetic peptide are shown in Table 6.
Peptide compositions were prepared by adjusting the pH to between 6-7 using 1 M NaOH or HCL and subsequently sterile filtered.
Synthetic peptides
In addition to an untreated control, 100 nM insulin was utilised to stimulate a maximal GLUT4 translocation response i.e. a positive control in each experiment. The average increase in cell surface GLUT4 translocation in response to 100 nM insulin was 1.72-fold when compared to untreated control (
Peptide composition E_1_BE tended to increase GLUT4 translocation at a concentration ranging from 0.25-0.5 mg/ml, however 1 and 2 mg/ml induced progressive cell death. Furthermore, there was a trend for composition I_2_BE to increase GLUT4 translocation in a dose-dependent manner (
SP2 and compositions E_1_BE and I_2_BE displayed a trend for stimulatory effect on skeletal muscle GLUT4 translocation and warrant further investigation for their potential to facilitate glucose transport in skeletal muscle.
I_2_BE or E_1_BE is administered as a solution or suspension in Purified Water. According to stability data, test item formulations at 10 mg/ml in Purified Water are stable for 10 hours at +2-+8° C. protected from light. Therefore test item formulations are kept at +2-+8° C. protected from light and used within 10 hours after preparation. Aspect of formulations and maximal duration of storage are detailed below.
Strain: BKS.Cg-Dock7m+/+Leprdb/J (db/db diabetic mice) (souche JAXTM Mice strain). Choice of species: The mouse was chosen because of its acceptance as a predictor of pharmacological effects of drugs in man and the recognition by regulatory authorities that this species is suitable for pharmacodynamic studies.
Age: 8-9 weeks on the day of randomisation.
Weight: On the day of randomisation, a maximum range of 2.5 g between each group should be achieved. The body weight of the animals on the day of randomisation will be mentioned in the report. About 10% excess animals will be ordered to allow selection of animals on the basis of body weight; if unassigned to groups, these will be available as spare animals, in case of unforeseen events.
The study involves 3 groups of 12 animals each. Groups will be as follows:
Allocation of treatment to each animal is randomly determined before the start of the study. Homogeneity of groups will be validated on the criterion of body weight and glycaemia measured on the day of randomisation.
Justification of the number of animals per group:
The number of animals per group is the minimum number enabling an accurate assessment of the pharmacokinetics profile.
Blood glucose level is measured weekly from D1 up to D29, 90 ±30 minutes after the daily treatment. A drop of blood is collected from the tail vein of non fasted db/db mice and is put on the extremity of a glucose strip (Nova Biomedical) placed into the Glucose Meter (Nova Biomedical).
Over the third week (D16-D18) and after an overnight fasting period, the OGTT is performed. After a blood glucose level measurement (predose value) and 30 minutes after the daily oral administrations of test items or vehicle, animals are dosed by the oral route with 10 mL/kg of a glucose solution at 0.2 g/mL (2 g/kg) in Purified Water. Afterwards, blood glucose level are measured following the same procedure described above, at times 15, 30, 60, 90 and 120 minutes after the glucose overload.
The effects of I_2_BE and E_1_BE on body weight and glycaemia are compared with those of the vehicle and the delta corresponding to the evolution of blood sugar in each group is calculated from D1 to D15. Evolution of blood glucose from D-5 to D1 and therefor prior to treatment shows that progression of the disease is the same in all three groups. Strong trends of activity were observed for both peptide compositions compare to control between D1 and D15 showing that both peptide compositions are able to control the evolution of blood sugar in diabetic animals.
The effects of I_2_BE and E_1_BE on body weight and glycaemia are compared with those of the vehicle using an analysis of variance for repeated measurements with a Dunnett's test in case of significance (P≦0.05). For OGTT, the results of glycemia after the glucose overload in treated animals is compared with those of the vehicle animals using an analysis of variance for repeated measurements with a Dunnett's test in case of significance (P=0.05).
Biochemical results (plasma glucose, HbA1c and insulin) are expressed as absolute values. The effects of I_2_BE and E_1_BE on biochemical parameters are compared with those of the vehicle using an analysis of variance with a Dunnett's test in case of significance (P≦0.05).
The anti-bacterial effects of peptide compositions of the invention were tested. The compositions are:
E_1_AM Contains substantially all of SEQ ID 106-251, 81, 68, 66, 106 and 107
E_2_AM Contains substantially all of SEQ ID 106-251, 81 and 68
Minimum Inhibitory Concentrations and Zone of Inhibition
MIC and MBC assays were carried out in Mueller Hinton broth previously adjusted to pH5, 7 and 9 and inoculated with 1×105 CFU/mL of each bacteria. The values shown represent the mean of three replicates performed on three independent days. Concentrations necessary to inhibit and completely halt growth are consistently lower in all strains at pH5. As the pH increases so too does the MIC and MBC suggesting that the bioactivity is improved in acidic conditions. This may be as a result of these conditions inducing a favourable isoelectric point an therefore, and enhanced electrostatic interaction between the positively charged hydrolysate and the negatively charged bacterial membrane. The zones of inhibition shown are the mean of three independent replicate experiments with the standard deviation. Values range from ˜11 mm to ˜21 mm with the best activity observed in P. aeruginosa. Each well is 8 mm in diameter alone and studies were conducted in Mueller Hinton agar at pH7.
Growth Curve in Mueller Hinton Broth pH7 at 37 degrees over 24 hours (
The peptide composition interferes with the growth of P. aeruginosa. At a concentration of 1024 μg/mL, sh_0MBH9Q extended the lag-time of P. aeruginosa by ˜10 hours. Concentrations above this resulted in complete cell death. This value corresponds to the MBC determined at pH7.
Fresh orange juice was inoculated with P. aeruginosa at 1×105 CFU/mL and plates were read at selected time points. Increased reduction in the microbial population appears to be linear with the increasing concentration of the peptide composition continues to reduce counts of P. aeruginosa over time and induces a ˜1.2 log reduction at 4096 μg/mL after 72 hours.
Total viable counts after 72 hours in P. aeruginosa (
The plate count study was conducted with peptide compositions at a concentration of 5 mg/mL. The meat used was fresh beef mince with 5% fat content.
The following peptides were tested for bacterial inhibition activity in solid and liquid media test. I_87_SF [SEQ ID 640] is an antibacterial fragment of Rice Protein P14614, whereas the remaining six peptides are comparative peptides.
E. coli 25922
Acinetobacter
baumannii
Bacterium anitratum
Compounds tested. A total of 1 compound (1 peptide) was tested. Peptide stock=5 mg/mL dissolved in DMSO.
Preparation of the peptide. The powder was reconstituted with 1.04 mL of DMSO to achieve a final concentration of 5 mg/mL. The peptide was in high purity. No precipitation problems. Antibacterial activity testing (in solid media). Bacterial inoculums were adjusted to McFarland 0.5 standard and MHA plates swabbed. Blank disks were placed in the plates and 10 μL of each compound (at 64 μg/mL—maximum concentration tested) added. Plates were incubated at 37° C. for 16-18 hours. Appropriate controls (DMSO; Mueller-Hinton media alone; and two antibiotic discs—ciprofloxacin and tetracycline) were also performed.
Results from the Testing in Agar-Plates
Determination of antibacterial activity (inhibition of growth) was performed in Mueller-Hinton plates. After incubation period results were registered and plates photographed.
E. coli ATCC 25922
A. baumannii ATCC 19606
Legend: TET—tetracycline; CIP—Ciprofloxacin; MH—Mueller-Hinton (control); NI—No Inhibition of growth
Final result: Peptide I_87_SF showed some inhibitory activity against E. coli and MRSA, but not at the levels of susceptibility. No activity was obtained against A. baumannii.
Results from the Testing in Agar-Plates (Photos)
Note: Only one set is shown since the other two sets had the same results. Control antibiotic discs (TET and CIP) were placed in the centre of each plate.
The invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.
The invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15177013.8 | Jul 2015 | EP | regional |
| 15177017.9 | Jul 2015 | EP | regional |
| 15177018.7 | Jul 2015 | EP | regional |
| 15177175.5 | Jul 2015 | EP | regional |
