Patent Application
20230406896
The contents of the electronic sequence listing (305406_241405_SkinGenSequenceListing_ST25.txt; Size: 16.187 bytes; and Date of Creation: Sep. 16, 2022) is herein incorporated by reference in its entirety.
The present invention relates generally to the field of skin care and aesthetics, and more particularly to compositions for topical and/or transdermal use having multiple recombinant human growth factors and hyaluronic acid included therein for repairing or regenerating human cells, keratinous materials, and/or the extracellular matrix to improve any one of skin surface appearance, cutaneous signs of chronological aging and/or aging signs induced by external factors such as prolonged exposure to ultraviolet (UV) exposure, and/or impaired surface appearance of the skin.
Fine lines and wrinkles are often attributed to the loss of collagen and elastin proteins (e.g., proteins comprising the extracellular matrix (ECM)) present in the dermal layers resulting in breakdown of skin thickness and resiliency over time. The loss of collagen and ECM can be generally attributed to multiple factors including biological aging and/or exposure to various environmental factors such as UV, which may further result in photo-aging.
There are invasive and non-invasive techniques for attempting to mitigate signs of aging (fine lines, wrinkles, increased pore diameter, roughened facial texture). For example, the most common surgical interventions available for treatment of facial wrinkles include face-lifts, laser surgery, and injection therapies, including for example, dermal fillers (having crosslinked hyaluronic acid matrices) and/or BOTOX®. Although invasive surgical methods and techniques are a viable option for anti-aging, these treatments are often very painful, must be periodically repeated, and if performed improperly, have detrimental aesthetic and health complications.
In addition to the above mentioned invasive techniques, non-invasive techniques include application of topical formulations consisting of many different ingredients including, alpha/beta hydroxy, retinoic acids, and vitamins and various plant extracts. However, none of these methods are very effective in eliminating wrinkles, and often require multiple, expensive treatments. These products focus on short term skin ‘plumping’ or on having a short term effect rather than looking at helping with improving the underlying cause of the skin aging. Furthermore, some topical formulations act as skin irritants (often resulting in red, inflamed skin and/or dry skin), to elicit wound healing responses, but do not successfully replenish the thinning skin with adequate proteins for treatment and/or prevention of age-related defects. In view of the above mentioned pitfalls, U.S. Pat. No. 8,518,879 provided formulations having a conditioned medium derived from transformed eulkaryotic cells that included an exosomally secreted growth factor. However, it should be noted that formulations disclosed in U.S. Pat. No. 8,518,879 suffer from many deficiencies including potentially inducing immunogenic responses in users while also being susceptible to growth factor inactivation. Moreover, and due to constitutive transformation of the eukaryotic cells disclosed in U.S. Pat. No. 8,518,879, quality control is a major issue due to the eukaryotic cell lines potentially being mosaics, resulting in varying concentrations of the desired actives being produced as well as potentially producing spontaneous mutations/mutants resulting during cell culture of its eukaryotic cells leading to highly variable lots within its conditioned medium.
Thus, in view of the above, additional anti-aging techniques, compositions, and/or formulations are needed.
It is an object to provide topical and/or transdermal compositions/formulations having multiple recombinant human growth factors therein for repairing and/or regenerating human cells, keratinous materials, and/or the extracellular matrix to improve any one of skin surface appearance, cutaneous signs of chronological aging and/or aging signs induced by external factors such as prolonged exposure to ultraviolet (UV) exposure, and/or impaired surface appearance of the skin while concurrently avoiding and/or minimizing the problems observed with current invasive and non-invasive techniques and the inability of the body to produce the Growth Factors in endogenous amounts that adequately stimulate the cell regeneration due to the aging process. In particular, these compositions/formulations utilize multiple nano-encapsulated, recombinant human growth factors, for effective delivery, repair, and regeneration of aging skin as well as hair. These compositions may be further used in combination with other treatments and/or protocols (e.g., micro-needling protocols) to maximize aesthetic efficacy and outcome for the patient. The combination of multiple growth factors (recombinant human growth factors) as disclosed herein has a more proliferative effect on the production of cellular intermediaries needed for skin rejuvenation than, for example, formulations having a single growth factor. In particular, when combinations of multiple recombinant human growth factors are used, as disclosed herein, at therapeutic doses, they advantageously initiate skin repair and regeneration processes without inducing immunogenic responses. In certain aspects, the combination of recombinant human growth factors disclosed herein work synergistically to enhance their individual effects.
In certain aspects, disclosed is a composition for topical or transdermal use having multiple recombinant human growth factors therein for repairing or regenerating human cells, keratinous materials, and/or the extracellular matrix to improve any one of skin surface appearance, cutaneous signs of chronological aging and/or aging signs induced by external factors such as prolonged exposure to ultraviolet (UV) exposure, and/or impaired surface appearance of the skin, the composition comprising: (a) water at a concentration ranging from 55 wt % to 85 wt % of the overall composition; (b) optionally, and when present, hyaluronic acid or an acceptable salt thereof at a concentration ranging from 0.5 wt % to 6 wt % of the overall composition with a molecular weight ranging from 150 kDA to 600 kDA (and preferably uncrosslinked); and (c) at least 5 but no more than 12 recombinant human growth factors selected from sh-Polypeptide-1 (SEQ ID NO 2), sh-Polypeptide-11 (SEQ ID NO 3), sh-Polypeptide-31 (SEQ ID NO 4), sh-Oligopeptide-2 (SEQ ID NO 6), sh-Polypeptide-10 (SEQ ID NO 7), sh-Polypeptide-5 (SEQ ID NO 8), sh-Polypeptide-8 (SEQ ID NO 9), sh-Polypeptide-3 (SEQ ID NO 10), sh-Polypeptide-62 (SEQ ID NO 1), Acetyl Octapeptide-17 Amide (SEQ ID NO 5), sh-oligopeptide-1 (SEQ ID NO 11), sh-Polypeptide-4 (SEQ ID NO 12), and Acetyl sh-Oligopeptide-77 Amide (SEQ ID NO 13) in which, each growth factor, when present in the composition, is present at a concentration ranging from 0.001 wt % to 1 wt % of the overall composition. To minimize and/or avoid any unwanted immunogenic response from the user while further maximizing quality control, the recombinant human growth factors are not derived from transformed eukaryotic cells and do not include conditioned media/medium but are instead derived from transgenic bacteria and are subsequently isolated/purified therefrom. Regarding each of the compositions/formulations mentioned immediately below, each composition/formulation includes the above mentioned water, hyaluronic acid, and varying numbers of recombinant human growth factors. In certain aspects, the recombinant human growth factors of the composition are sh-Polypeptide-1 is SEQ ID NO 2, sh-Polypeptide-11 is SEQ ID NO 3, sh-Polypeptide-31 is SEQ ID NO 4, shOligopeptide-2 is SEQ ID NO 6, the sh-Polypeptide-10 is SEQ ID NO 7, sh-Polypeptide-5 is SEQ ID NO 8, sh-Polypeptide-8 is SEQ ID NO 9, sh-Polypeptide-3 is SEQ ID NO 10, sh-Polypeptide-62 is SEQ ID NO 1, Acetyl Octapeptide-17 Amide is SEQ ID NO 5, sh-oligopeptide-1 is SEQ ID NO 11, sh-Polypeptide-4 is SEQ ID NO 12, and Acetyl sh-Oligopeptide-77 Amide is SEQ ID NO 13. In other aspects, the recombinant human growth factors of the composition are when sh-Polypeptide-1 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 2, the sh-Polypeptide-11 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 3, the sh-Polypeptide-31 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 4, the shOligopeptide-2 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 6, the sh-Polypeptide-10 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 7, the sh-Polypeptide-5 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 8, the sh-Polypeptide-8 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 9, the sh-Polypeptide-3 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 10, the sh-Polypeptide-62 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 1, Acetyl Octapeptide-17 Amide comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 5, the sh-oligopeptide-1 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 11, the sh-Polypeptide-4 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 12, and the Acetyl sh-Oligopeptide-77 Amide comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 13.
In certain aspects, the composition is an aqueous solution for topical use comprising either alone or in conjunction with microneedling (with microneedling occurring either pre-application or post-application of the composition) in which the composition includes water (as mentioned above) and at least 8 growth factors are present, hyaluronic acid at a concentration ranging from 1.5 wt % to 4.5 wt % of the overall composition; and further comprises: (i) a nanoencapsulating agent that encapsulates the recombinant human growth factors and present in the composition at an effective amount to deliver the recombinant human growth factors to cells, keratinous materials, and/or the extracellular matrix in a human, (ii) a Swertia chirata extract present at an effective amount for stimulating endogenous keratinocyte growth factor production to induce keratinocyte proliferation and epidermis regeneration to increase skin volume and/or reduce cutaneous signs of aging, (iii) an emollient, and (iv) a skin protecting agent present at an effective amount to stimulate endogenous production of collagen VII, laminin-5, and/or fibronectin. In this aspect, the Swertia chirata extract is present at a concentration ranging from 1 wt % to 8 wt % of the overall composition. In this aspect, the skin protecting agent is present at a concentration ranging from 1 wt % to 5 wt % of the overall composition. In this aspect, the skin protecting agent comprises a combination of glycerin, water, dextran, and caproyl tetrapeptide-3. In this aspect, the composition further comprises a water soluble skin conditioning agent configured to minimize enlarged pores, Lighten lax pores, improve uneven skin tone, soften fine lines and wrinkles, diminish dullness, and/or strengthen a weakened skin surface. In this aspect, the water soluble skin conditioning agent is present at a concentration ranging from 0.1 wt % to 5 wt % of the overall composition. In this aspect, the water soluble skin conditioning agent is niacinamide. In this aspect, the nanoencapsulating agent is a C1-C6 alkylene glycol (e.g., either a diol or a polyol) suitable for topical and/or transdermal use and lecithin mixture present at a ratio ranging from 4:1 to 2.3:1 of C1-C6 alkylene glycol to lecithin, and in certain aspects, the a C1-C6 alkylene glycol suitable for topical and/or transdermal use is present at a concentration of 70 wt % to 80 wt % of the overall concentration of the nanoencapsulating agent and lecithin is present at a concentration of 20 to 30 wt % of the overall concentration of the nanoencapsulating agent. In certain aspects, the C1-C6 alkylene glycol is propanediol and lecithin is a soybean (G. max) extract (or alternatively a plurality of isolates that are combined with one another) in which the lecithin comprises a mixture of phosphatidylcholine, phosphatidylinositol, phosphatidylethanoloamine, and phosphatidic acid in which phosphatidylcholine is present in the nanoencapsulating agent at a concentration of 14 wt % to 23 wt % of the overall concentration of the nanoencapsulating agent, phosphatidylinositol is present in the nanoencapsulating agent at a concentration 0.35 wt % to 0.7 wt % of the overall concentration of the nanoencapsulating agent, phosphatidylethanoloamine is present in the nanoencapsulating agent at a concentration of 1.0 wt % to 1.9 wt % of the overall concentration of the nanoencapsulating agent, and phosphatidic acid is present in the nanoencapsulating agent at a concentration of 0.15 wt % to 0.3 wt % of the overall concentration of the nanoencapsulating agent.
In additional aspects, the composition is an oil in water emulsion for topical use and transdermal absorption either alone or in conjunction with microneedling (with microneedling occurring either pre-application or post-application of the composition), the composition comprising: water (as mentioned above) and at least 8 recombinant human growth factors are present, and hyaluronic acid or an acceptable salt thereof at a concentration ranging from 0.5 wt % to 6 wt % of the overall composition with a molecular weight ranging from 150 kDA to 600 kDA (and preferably uncrosslinked), and further comprises: (i) a nanoencapsulating agent that encapsulates the recombinant human growth factors and present in an effective amount to deliver the recombinant human growth factors to cells, keratinous materials, and/or the extracellular matrix in a human; (ii) a plurality of anti-inflammatory agents and/or antioxidants at a concentration ranging from 1 wt % to 7 wt % of the overall composition; (iii) a combination of liposomally encapuslated bacterial derived photolyase(s), plant-derived roxisome(s), and bacterial derived endonuclease(s) that are present in an effective amount to prevent and/or reduce photoaging associated with ultraviolet (UV) exposure by enhancing endogenous DNA repair. In certain aspects, the growth factors and combination of photolyases, plant-derived roxisomes, and bacterial derived endonuclease improve skin after sun damage by initiating the repair and regenerative processes and help speed up the rate of improvement of the main signs of skin aging. In this aspect, the combination of liposomally encapuslated bacterial derived photolyase(s), plant-derived roxisome(s), and bacterial derived endonucleases are present at a concentration ranging from 0.3 wt % to 10 wt % of the overall composition. In this aspect, bacterial derived photolyases are present at a concentration ranging from 0.1 wt % to 3.5 wt % of the overall composition. In this aspect, the bacterial derived photolyase is a cyanobacteria photolyase. In this aspect, the cyanobacteria photolyase is an Anacystis nidulans photolyase. In this aspect, the plant derived roxisome(s) are present at a concentration ranging from 0.1 wt % to 3.5 wt % of the overall composition. In this aspect, the plant derived roxisome(s) is 8-oxo-guanine glycosylase from A. thaliana. In this aspect, the bacterial derived endonucleases are present at a concentration ranging from 0.1 wt % to 3.5 wt % of the overall composition. In this aspect, the bacterial derived endonuclease(s) are bacterial endonucleases from M. Luteus. In this aspect, the nanoencapsulating agent is a C1-C6 alkylene glycol suitable for topical and/or transdermal use and lecithin mixture present at a ratio ranging from 4:1 to 2.3:1 of C1-C6 alkylene glycol to lecithin, and in certain aspects, the a C1-C6 alkylene glycol suitable for topical and/or transdermal use is present at a concentration of 70 wt % to 80 wt % of the overall concentration of the nanoencapsulating agent and lecithin is present at a concentration of 20 to 30 wt % of the overall concentration of the nanoencapsulating agent. In certain aspects, the C1-C6 alkylene glycol is propandiol and lecithin is a soybean (G. max) extract (or alternatively a plurality of isolates that are combined with one another) in which the lecithin comprises a mixture of phosphatidylcholine, phosphatidylinositol, phosphatidylethanoloamine, and phosphatidic acid in which phosphatidylcholine is present in the nanoencapsulating agent at a concentration of 14 wt % to 23 wt % of the overall concentration of the nanoencapsulating agent, phosphatidylinositol is present in the nanoencapsulating agent at a concentration 0.35 wt % to 0.7 wt % of the overall concentration of the nanoencapsulating agent, phosphatidylethanoloamine is present in the nanoencapsulating agent at a concentration of 1.0 wt % to 1.9 wt % of the overall concentration of the nanoencapsulating agent, and phosphatidic acid is present in the nanoencapsulating agent at a concentration of 0.15 wt % to 0.3 wt % of the overall concentration of the nanoencapsulating agent. In this aspect, the composition further comprising an emollient and an emulsifier present at a concentration ranging from 6 wt % to 32 wt % of the overall composition. In this aspect, retinol is present as an antioxidant in the composition at a concentration ranging from 1 wt % to 3 wt %. In this aspect, the liposomes encapsulating bacterial derived photolyase(s), plant-derived roxisome(s), and bacterial derived endonucleases are comprised of lecithin, and the lecithin is soybean lecithin.
In yet another aspect, the composition is an aqueous solution for topical use on a human scalp either alone or in conjunction with microneedling (with microneedling occurring either pre-application or post-application of the composition), the composition comprising water and hyaluronic acid (as mentioned above) and at least 10 recombinant human growth factors, and a nanoencapsulating agent that encapsulates the recombinant human growth factors and present in an effective amount to deliver the recombinant human growth factors to the human scalp to induce hair growth and/or re-growth and/or rejuvenate scalp appearance In certain aspects, the nanoencapsulating agent includes a C1-C6 alkylene glycol suitable for topical and/or transdermal use and lecithin mixture present at a ratio ranging from 4:1 to 2.3:1 of C1-C6 alkylene glycol to lecithin, and in certain aspects, the a C1-C6 alkylene glycol suitable for topical and/or transdermal use is present at a concentration of 70 wt % to 80 wt % of the overall concentration of the nanoencapsulating agent and lecithin is present at a concentration of 20 to 30 wt % of the overall concentration of the nanoencapsulating agent. In certain aspects, the C1-C6 alkylene glycol is propanediol and lecithin is a soybean (G. max) extract (or alternatively a plurality of isolates that are combined with one another) in which the lecithin comprises a mixture of phosphatidylcholine, phosphatidylinositol, phosphatidylethanoloamine, and phosphatidic acid in which phosphatidylcholine is present in the nanoencapsulating agent at a concentration of 14 wt % to 23 wt % of the overall concentration of the nanoencapsulating agent, phosphatidylinositol is present in the nanoencapsulating agent at a concentration 0.35 wt % to 0.7 wt % of the overall concentration of the nanoencapsulating agent, phosphatidylethanoloamine is present in the nanoencapsulating agent at a concentration of 1.0 wt % to 1.9 wt % of the overall concentration of the nanoencapsulating agent, and phosphatidic acid is present in the nanoencapsulating agent at a concentration of 0.15 wt % to 0.3 wt % of the overall concentration of the nanoencapsulating agent.
Also disclosed is a kit including at least one of the compositions mentioned above that is packaged within a container. The kit further includes a plurality of sterile microneedles packaged within the kit and configured to enhance transdermal delivery of the composition post-application of the composition to the human cells and/or keratinous materials by creating punctures (puncturing) in a user's stratum corneum to induce a wound healing response and to enhance delivery to improve any one of skin surface appearance, cutaneous signs of chronological aging and/or aging signs induced by external factors such as prolonged exposure to ultraviolet (UV) exposure, and/or impaired surface appearance of the skin.
Also disclosed is a method of reducing wrinkles and/or fine lines over a predetermined time period, the method comprising: (a) applying on the skin of a subject at least one of the compositions disclosed above; and (b) repeating the application of the composition to the skin of the subject at predetermined time periods thereby reducing wrinkles, fine lines, and/or the appearance thereof during the predetermined time period. In certain aspects, this method further comprises: before step (a) performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject. In certain aspects, this method further comprises after step (a) and before step (b) performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject. In certain aspects, this method further comprises after each application of the composition to the skin of the subject performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject.
Also disclose is a method for reducing pore size over a predetermined time period, the method comprising: (a) applying on the skin of a subject at least one of the compositions disclosed above; and (b) repeating the application of the composition to the skin of the subject at predetermined time periods thereby reducing pore size and/or the appearance thereof during the predetermined time period. In certain aspects, this method further comprises before step (a) performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject. In certain aspects, this method further comprises after step (a) and before step (b) performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject. In certain aspects, this method further comprises after each application of the composition to the skin of the subject performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject.
Also disclosed is a method of enhancing skin texture over a predetermined time period, the method comprising: (a) applying on the skin of a subject at least one of the compositions disclosed above; and (b) repeating the application of the composition to the skin of the subject at predetermined time periods thereby improving and/or enhancing skin texture and/or the appearance thereof during the predetermined time period. In certain aspects, this method further comprises before step (a) performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject. In certain aspects, this method further comprises after step (a) and before step (b) performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject. In certain aspects, this method further comprises after each application of the composition to the skin of the subject performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject.
Embodiments of the invention can include one or more or any combination of the above features and configurations.
Additional features, aspects and advantages of the invention will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the invention as described herein. It is to be understood that both the foregoing general description and the following detailed description present various embodiments of the invention, and are intended to provide an overview or framework for understanding the nature and character of the invention as it is claimed. The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification.
These and other features, aspects and advantages of the present invention are better understood when the following detailed description of the invention is read with reference to the accompanying drawings, in which:
The present invention will now be described more fully hereinafter with reference to the accompanying drawings in which exemplary embodiments of the invention are shown. However, the invention may be embodied in many different forms and should not be construed as limited to the representative embodiments set forth herein. The exemplary embodiments are provided so that this disclosure will be both thorough and complete, and will fully convey the scope of the invention and enable one of ordinary skill in the art to make, use and practice the invention. Like reference numbers refer to like elements throughout the various drawings.
In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.
“Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.
As used herein, the term “about” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result.
Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and thus should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within the ranges as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 1 to 5” should be interpreted to include not only the explicitly recited values of about 1 to about 5, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 3, and 4 and sub-ranges such as from 1-3, from 2-4, and from 3-5, etc. as well as 1, 2, 3, 4, and 5, individually. The same principle applies to ranges reciting only one numerical value as a minimum or a maximum. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
The compositions and methods described herein can comprise, consist of, or consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, or limitations described herein.
The term sterile, relates to an environment ensuring the safety required for preparing a composition which can be safely used by topical administration on damaged skin surfaces and/or relates to a composition which is prepared in a sterile environment or made sterile with a sterilization method which may be chosen among the ones known by the one skilled in the art. Indeed, for obvious reasons, it is essential that the compositions disclosed herein are devoid of any contaminant capable of initiating undesirable reactions and/or side effects and are initially sterile (and/or remain sterile while packaged within vials and/or containers).
The term topical refers to a composition which is intended to be applied on the skin surface (dermis) of a subject.
The term transdermal means absorbed and/or to be absorbed through a subjects epidermis and/or dermal layers.
The phrase “effective amount” as used herein relates to the amount/concentration used within the disclosed composition(s) in order to achieve the desired/specified effects.
The term/phrase “microneedling or micro-needling” relates to the puncture of the skin to various depths with very fine needles. This procedure causes a controlled injury inducing a controlled wound response in which the skin synthetizes more collagen thus having an anti-aging effect and/or improving the skin/skin appearance of the subject.
“Peptide” or “peptide sequence” may be used to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. The peptide is not limited by length, and thus “peptide” can include a peptide fragment, a polypeptide(s), and full-length proteins.
When describing variants in proteins, polypeptides, or peptides, the term “variant” refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid substitutions (i.e. a degenerate variant), substitutions within the wobble position of each codon (i.e. DNA and RNA) encoding an amino acid, amino acids added to the C-terminus of a peptide, or a peptide having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology to a reference sequence.
The terms “homology,” “identity or identical,” and “similarity” refer to the degree of sequence similarity between two peptides or between two optimally aligned nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence which can be aligned for purposes of comparison. For example, it is based upon using a standard homology software in the default position, such as BLAST, version 2.2.14. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by similar amino acid residues (e.g., similar in steric and/or electronic nature such as, for example conservative amino acid substitutions), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology/similarity or identity refers to a function of the number of similar or identical amino acids at positions shared by the compared sequences, respectfully. A sequence which is “unrelated” or “non-homologous” shares less than 40% identity, though preferably less than 25% identity with the sequences as disclosed herein.
As used herein, the term “sequence identity” means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-by-nucleotide or residue-by-residue basis) over the comparison window. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T. C, G. U. or I) or residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
It is understood that any given particular aspect of the disclosed compositions and methods can be easily compared to the specific examples and embodiments disclosed herein. By performing such a comparison, the relative efficacy of each particular embodiment can be easily determined. Particularly preferred compositions and methods are disclosed in the Examples herein, and it is understood that these compositions and methods, while not necessarily limiting, can be performed with any of the compositions and methods disclosed herein.
General Formulation/Composition
Disclosed immediately below is a general formulation and/or composition that includes chemical components and growth factors that are included within each of the specific, individual compositions/formulations disclosed in detail further below.
Each of the formulations/compositions disclosed herein generally directed to topical or transdermal use. Each composition/formulation includes multiple recombinant human growth factors (shown in Table 1 below) therein for repairing or regenerating human cells, keratinous materials, and/or the extracellular matrix to improve any one of skin surface appearance, cutaneous signs of chronological aging and/or aging signs induced by external factors such as prolonged exposure to ultraviolet (UV) exposure, and/or impaired surface appearance of the skin.
Each of these formulations are preferably water based and have water at a concentration ranging from 55 wt % to 85 wt % of the overall composition. Water concentration may be varied according to each individual composition disclosed herein, and water concentration may fall within any range of the above disclosed endpoints.
In addition, the formulations disclosed herein may include hyaluronic acid that, when included, aids in enhancing skin hydration, elasticity, and ECM repair and/or maintenance when used in an effective amount within the composition(s)/formulation(s) disclosed herein. It should be noted that hyaluronic acid (CAS No.: 9067-32-7) as disclose herein also refers to physiological acceptable salts thereof as well; these physiologically acceptable salts include sodium salt, the potassium salt, the zinc salt and the silver salt, its derivatives and mixture thereof. In certain aspects, the hyaluronic acid is preferably a low molecular weight hyaluronic acid ranging from 150 kDA to 600 kDA at a concentration ranging from 0.5 wt % to 6 wt % of the overall composition, more preferably from 1 wt % to 5 wt % the overall composition. Lower weight hyaluronic acid is preferred for the transdermal and topical applications disclosed herein. As hyaluronic acid molecular weight increases, the more difficult transdermal delivery of the hyaluronic acid becomes, which may further affect the composition efficacy and desired results (i.e, repairing or regenerating human cells, keratinous materials, and/or the extracellular matrix to improve any one of skin surface appearance, cutaneous signs of chronological aging and/or aging signs induced by external factors such as prolonged exposure to ultraviolet (UV) exposure, and/or impaired surface appearance of the skin.) disclosed herein. The hyaluronic acid disclosed herein may be uncrosslinked (non-crosslinked), crosslinked, or a combination thereof. In certain aspects, each formulation includes only uncrosslinked (non-crosslinked) hyaluronic acid, which is more easily transdermally and/or topically applied and delivered to achieve the desired effects disclose herein than crosslinked hyaluronic acid. However, in alternative aspects, the composition includes mixtures or crosslinked and uncrosslinked hyaluronic acid. The hyaluronic acid as disclosed herein may in certain aspects bind to the cell surface receptor CD 44 activating an endogenous keratinocyte proliferation cascade and up regulation of endogenous hyaluronic acid production via this keratinocyte cascade, which ultimately resulting in thickening of the epidermis and strengthening of the ECM.
In certain aspects and to further increase delivery of the above mentioned hyaluronic acid and when present in the compositions, the hyaluronic acid is preferably admixed with a silanol and more preferably an organosilanol for delivery and transdermal and topical absorption purposes. In particular, the silanol and/or organosilanol is methylsilanetriol (CAS No.: 4253-34-3 or CAS No. 2445-53-6), which further aids in miscibility and bioavailability of the hyaluronic acid within the compositions/formulations disclosed herein. In this aspect, the organosilanetriol (CAS No.: 4253-34-3 or methylsilanetriol CAS No. 2445-53-6) not only aids in miscibility of the hyaluronic acid within the disclosed compositions but also synergistically interacts therewith to aid in topical and/or transdermal delivery to further improve skin hydration and to further stimulate fibroblasts within the dermis to synthesize collagen, resulting in firmer skin appear. The silanol and/or organolsilanol (e.g., CAS No.: 4253-34-3 or methylsilanetriol CAS No. 2445-53-6) is preferably included with the composition at approximately 0.5 wt % to 6 wt % of the overall composition and at a ratio ranging from approximately 3:1 to 1:1 (and more preferably from 2:1 to 1:1) of hyaluronic acid to the silanol and/or organolsilanol (e.g., CAS No.: 4253-34-3 or methylsilanetriol CAS 2445-53-6). In certain aspects, the low molecular weight hyaluronic acid (150 kDA to 600 kDA) disclosed herein may be crosslinked/bonded (e.g., covalently bonded) to the silanol and/or organolsilanol (e.g., CAS No.: 4253-34-3 or methylsilanetriol CAS 2445-53-6), which may further facilitate topical and transdermal absorption thereof. In certain aspects, the admixture of hyaluronic acid with silanol and/or organolsilanol (e.g., CAS No.: 4253-34-3 or methylsilanetriol CAS 2445-53-6) is Epidermosil manufactured by Exsymol S.A.M.
In certain aspects, each formulation and/or composition disclosed herein includes at least 5 but no more than 12 recombinant human growth factors. It should be noted, and further in view of
As alluded to above and as further shown in
It should be further noted and to achieve, for example, the desired proliferative effects graphically depicted in
1Randall VA1, Jenner TJ; Stem cell factor/c-Kit signalling in normal and androgenetic alopecia hair follicles; Endocrinol. 2008 Apr; 197(1): 11-23.
29Botchkarev VA1, Botchkareva NV; Noggin is required for induction of the hair follicle growth phase in postnatal skin. FASEB J. 2001 Oct; 15(12): 2205-14.
As protein molecular weight increases, transdermal absorption and/or skin penetration is hindered and/or reduced. For example, transdermal absorption and/or skin penetration of growth factors tends to be effected when molecular weights exceed 500 Da (0.5 KDa). Furthermore, because of the delicate nature (disulfide bonding, protein folding, etc.) of recombinant human growth factors disclosed herein, these recombinant human growth factors may be susceptible to denaturation and/or decreased bioavailability and bioactivity if improperly stored. To increase transdermal absorption and/or skin penetration of the growth factors disclosed herein while further decreasing susceptibility to denaturation (and/or to maintain optimal bioavailability and bioactivity), the recombinant human growth factors disclosed herein are preferably nanoencapsulated with a nanoencapsulating agent that encapsulates the recombinant human growth factors therein. The nanoencapsulating agent is preferably present in the composition at an effective amount to deliver the recombinant human growth factors to cells, keratinous materials, and/or the extracellular matrix in a human.
In certain aspects, the nanoencapuslating agent preferably includes a mixture of a C1-C6 alkylene glycol (e.g., diols or polyols) suitable for topical and/or transdermal use in combination with lecithin. In this mixture, each component is present at a ratio ranging from 4:1 to 2.3:1 of C1-C6 alkylene glycol to lecithin (CAS No. 8002-43-5). The C1-C6 alkylene glycol suitable for topical and/or transdermal use is present at a concentration of 70 wt % to 80 wt % of the overall concentration of the nanoencapsulating agent and lecithin is present at a concentration of 20 to 30 wt % of the overall concentration of the nanoencapsulating agent. In certain aspects, the C1-C6 alkylene glycol is ethylene glycol, propanediol (propylene glycol, propane 1,2 diol) (CAS No. 504-63-2) or a combination thereof, and in preferred aspects, the C1-C6 alkylene glycol is propanediol (propylene glycol, propane 1,2 diol) present at a concentration of 70 wt % to 80 wt % of the overall concentration of the nanoencapsulating agent. It should be further noted that the lecithin in the nanoencapsulating agent is a soybean (G. max) extract (or alternatively a plurality of isolates that are combined with one another) in which the lecithin comprises a mixture of phosphatidylcholine, phosphatidylinositol, phosphatidylethanoloamine, and phosphatidic acid in which phosphatidylcholine is present in the nanoencapsulating agent at a concentration of 14 wt % to 23 wt % of the overall concentration of the nanoencapsulating agent, phosphatidylinositol is present in the nanoencapsulating agent at a concentration 0.35 wt % to 0.7 wt % of the overall concentration of the nanoencapsulating agent, phosphatidylethanoloamine is present in the nanoencapsulating agent at a concentration of 1.0 wt % to 1.9 wt % of the overall concentration of the nanoencapsulating agent, and phosphatidic acid is present in the nanoencapsulating agent at a concentration of 0.15 wt % to 0.3 wt % of the overall concentration of the nanoencapsulating agent. The nanoencapsulating agent may include additives including, for example, tocopherol at a concentration ranging from 0.15 wt % to 0.35 wt % of the overall concentration of nanoencapsulating agent and sunflower (Helianthus annuus) seed oil at a concentration ranging from 0.09 wt % to 0.25 wt % of the overall concentration of nanoencapsulating agent. In certain aspects, the nanoencapsulating agent may be Pro-Lip™Neo from Lucas Meyer Cosmetics S.A.S.
In certain aspects, all compositions/formulations disclose herein are preferably pH balanced having a pH ranging from 5.5 to 7.5 to reduce and completely avoid skin irritation attributed to application thereon. To obtain this pH balance, salt solutions including, for example, a phosphate buffered saline such as KH2PO4/K2HPO4 saline buffer may be used.
Regenerative Composition/Formulation
In certain aspects, an objective is to provide an aqueous solution for topical use (and transdermal penetration) that stimulates and improves keratinocyte proliferation, epidermis regeneration, and thickness to provide a more youthful appearance that resurfaces skin, increases skin tightness, decreases pore size, improves skin texture, and reduces vertical wrinkles, which is shown, for example, in
In this aspect, the aqueous solution for topical use includes water as a solvent at the concentrations discussed above (ranging from 55 wt % to 85 wt %) regarding the general composition/formulation, but more preferably includes from 70 wt % to 85 wt % water of the overall concentration of this composition/formulation, and most preferably includes from 75 wt % to 82.5 wt % water of the overall concentration of this composition/formulation.
In certain optional embodiments, this composition may or may not include hyaluronic acid (CAS No. 9067-32-7) and silanol and/or organosilanol (CAS No.: 4253-34-3) is methylsilanetriol (CAS No. 2445-53-6), at substantially the same concentrations as those previously discussed above. However, in certain embodiments regarding the regenerative compositions/formulations and as shown in Table 2 below, hyaluronic acid and the organosilanol is preferably excluded.
This composition further includes preferably at least eight of the previously mentioned recombinant human growth factors and up to twelve of the previously mentioned recombinant human growth factors with each growth factor, when present in the composition, is present at a concentration ranging from 0.001 wt % to 1 wt % of the overall composition and more preferably from 0.009 wt % to 1 wt %. The growth factors are selected to specifically achieve the above mentioned, desired effects (i.e., stimulate and improve keratinocyte proliferation, epidermis regeneration, and thickness to provide a more youthful appearance that potentially resurfaces skin, skin tightness, pore size, skin texture, and potentially reduces vertical wrinkles). In certain aspects, this composition/formulation, preferably includes each of the following recombinant human growth factors: sh-Polypeptide-1 (SEQ ID No. 2), sh-Polypeptide-11 (SEQ ID No. 3), sh-Polypeptide-31 (SEQ ID No. 4), sh-Oligopeptide-2 (SEQ ID No. 6), sh-Polypeptide-10 (SEQ ID No. 7), sh-Polypeptide-5 (SEQ ID No. 8), sh-Polypeptide-8 (SEQ ID No. 9), sh-Polypeptide-3 (SEQ ID No. 10), sh-Polypeptide-62 (SEQ ID No. 1), and Acetyl Octapeptide-17 Amide (SEQ ID No. 5), with each growth factor being present at a concentration ranging from 0.001 wt % to 1 wt % of the overall composition and more preferably from 0.009 wt % to 1 wt %. In certain aspects, this composition includes at least ten of the above mentioned recombinant human growth factors. In certain aspects, the recombinant human growth factors of the composition are when sh-Polypeptide-1 is SEQ ID NO 2, sh-Polypeptide-11 is SEQ ID NO 3, sh-Polypeptide-31 is SEQ ID NO 4, shOligopeptide-2 is SEQ ID NO 6, the sh-Polypeptide-10 is SEQ ID NO 7, sh-Polypeptide-5 is SEQ ID NO 8, sh-Polypeptide-8 is SEQ ID NO 9, sh-Polypeptide-3 is SEQ ID NO 10, sh-Polypeptide-62 is SEQ ID NO 1, Acetyl Octapeptide-17 Amide is SEQ ID NO 5, sh-oligopeptide-1 is SEQ ID NO 11, sh-Polypeptide-4 is SEQ ID NO 12, and Acetyl sh-Oligopeptide-77 Amide is SEQ ID NO 13, with each growth factor being present at a concentration ranging from 0.001 wt % to 1 wt % of the overall composition and more preferably from 0.009 wt % to 1 wt %. In other aspects, the recombinant human growth factors of the composition are when sh-Polypeptide-1 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 2, the sh-Polypeptide-11 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 3, the sh-Polypeptide-31 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 4, the shOligopeptide-2 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 6, the sh-Polypeptide-10 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 7, the sh-Polypeptide-5 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 8, the sh-Polypeptide-8 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 9, the sh-Polypeptide-3 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 10, the sh-Polypeptide-62 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 1, Acetyl Octapeptide-17 Amide comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 5, the sh-oligopeptide-1 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 11, the sh-Polypeptide-4 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 12, and the Acetyl sh-Oligopeptide-77 Amide comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 13, with each growth factor being present at a concentration ranging from 0.001 wt % to 1 wt % of the overall composition and more preferably from 0.009 wt % to 1 wt %.
The recombinant human growth factors mentioned herein for the regenerative composition/formulation are also nanoencapsulated with the same nanoencapsulating agent(s) mentioned above at substantially the same concentrations and for substantially the same reasons as those mentioned above regarding the general composition/formulation.
In certain aspects, the aqueous solution for topical use further includes a Swertia chirata extract present at an effective amount for stimulating endogenous keratinocyte growth factor production to induce keratinocyte proliferation and epidermis regeneration to increase skin volume and/or reduce cutaneous signs of aging. In certain aspects, this Swertia chirata extract may be present at a concentration ranging from 1 wt % to 8 wt % of the overall composition, and more preferably from 2.5 wt % to 5 wt % of the overall composition. In particular the Swertia chirata extract when present at the concentrations mentioned above and coupled with the recombinant human growth factors acting through a biomimetic pathway to stimulate keratinocyte growth factor (KGF) production from adipose-derived stem cells (ADSC), which advantageously improves keratinocyte proliferation and epidermis regeneration and thickness.
The aqueous solution further includes skin protecting agents that include an admixture of glycerin, water, dextran, and caproyl tetrapeptide-3 at a concentration ranging from 1 wt % to 5 wt % of the overall composition, and more preferably from 2.5 wt % to 4 wt % of the overall composition. The skin protecting agent disclosed immediately above, when included within the aqueous solution for topical use, advantageously stimulates the production of key components at the dermo-epidermal junction (collagen VII, laminin-5, and fibronectin).
The aqueous solution further includes niacinamide (vitamin B3)(CAS No. 98-92-0) at a concentration ranging from 0.1 wt % to 5 wt % of the overall composition, and more preferably 1.5 wt % to 3.5 wt % of the overall composition. When present at these concentrations, niacinamide advantageously minimizes enlarged pores, tighten lax pores, improves uneven skin tone, soften fine lines and wrinkles, diminishes skin dullness, and strengthens the skin surface.
The remainder of the aqueous solution for topical use further includes additional conditioners, emollients, and viscosity controlling agents. For example, in certain aspects, the emollients may include a mixture of polyacrylate-13, polyisobutylene, and polysorbate 20 at a concentration ranging from 1 to 5 wt % of the overall composition, and more preferably from 2 wt % to 3 wt % of the overall composition. The viscosity controlling agent is carnosine at a concentration ranging from 0.1 wt % to 1 wt % of the overall composition, and more preferably from 0.1 wt % to 0.3 wt % of the overall composition. The additional conditioners may further include a mixture of disodium EDTA, glycerin, and jojoba leaf extract at a concentration ranging from 0.02 wt % to 2 wt % of the overall composition, and more preferably from 0.05 wt % to 1 wt % of the overall composition.
This composition may be used alone or in conjunction with the other compositions/formulations disclosed herein as well as with microneedling procedures as disclosed below in the “Methods of Use” to improve the user's appearance. An exemplary regenerative composition/formulation is shown immediately below within Table 2.
Reparative Composition/Formulations
In certain aspects, the reparative compositions/formulations are oil in water emulsion for topical use and transdermal absorption disclosed herein are specifically tailored for treating skin damage, wrinkles, etc. resulting from exposure (or over exposure) to, for example, UV. To achieve this result, these compositions include the specific combination of human recombinant growth factors discussed below as well as a specific combination of enzymes (e.g., photolyases, plant-derived roxisomes, and bacterial endonucleases) as well as various anti-inflammatory agents and anti-oxidants that further activate and up-regulate various DNA repair pathways (e.g., mismatch repair pathways to remove mismatch DNA nucleotides, base-excision repair pathways to remove damaged DNA/improperly paired nucleotides (e.g., oxidized guanine resulting in improper Hoogsteen base pairing with adenine, alkylated nucleotides, and/or deaminated nucleotides) often associated with UV/DNA damage, and/or DNA damage resulting from reactive oxygen species (ROS)) to more quickly repair DNA damage (which may be associated with photoaging) and to further mitigate, reduce, and/or improve skin appearance.
In this aspect, the reparative compositions/formulations includes water as a solvent at the concentrations discussed above (ranging from 55 wt % to 85 wt %) regarding the general composition/formulation, but more preferably includes from 70 wt % to 85 wt % water of the overall concentration of this composition/formulation, and most preferably includes from 75 wt % to 82.5 wt % water of the overall concentration of this composition/formulation.
This composition includes hyaluronic acid (CAS No. 9067-32-7) and silanol and/or organosilanol (CAS No.: 4253-34-3) is methylsilanetriol (CAS No. 2445-53-6), at substantially the same concentrations as those discussed above and as further shown in Table 3 below. In certain aspects, the hyaluronic acid is present at 1 to 3 wt % of the overall composition and the organosilanol (e.g., mehylsilanetriol) is present at 1 to 3 wt % of the overall composition.
This composition further includes preferably at least eight of the previously mentioned recombinant human growth factors and up to twelve of the previously mentioned recombinant human growth factors with each growth factor, when present in the composition, is present at a concentration ranging from 0.001 wt % to 1 wt % of the overall composition and more preferably from 0.009 wt % to 1 wt %. The growth factors are selected to specifically achieve the above mentioned, desired effects (i.e., stimulate and improve keratinocyte proliferation, epidermis regeneration, and thickness to provide a more youthful appearance that potentially resurfaces skin, skin tightness, pore size, skin texture, and potentially reduces vertical wrinkles). In certain aspects, this composition/formulation, preferably includes each of the following recombinant human growth factors: sh-Polypeptide-1 (SEQ ID No. 2), sh-Polypeptide-11 (SEQ ID No. 3), sh-Polypeptide-31 (SEQ ID No. 4), sh-Oligopeptide-2 (SEQ ID No. 6), sh-Polypeptide-10 (SEQ ID No. 7), sh-Polypeptide-5 (SEQ ID No. 8), sh-Polypeptide-8 (SEQ ID No. 9), sh-Polypeptide-3 (SEQ ID No. 10), sh-Polypeptide-62 (SEQ ID No. 1), and Acetyl Octapeptide-17 Amide (SEQ ID No. 5), with each growth factor being present at a concentration ranging from 0.001 wt % to 1 wt % of the overall composition and more preferably from 0.009 wt % to 1 wt %. In certain aspects, this composition includes at least ten of the above mentioned recombinant human growth factors. In certain aspects, the recombinant human growth factors of the composition are when sh-Polypeptide-1 is SEQ ID NO 2, sh-Polypeptide-11 is SEQ ID NO 3, sh-Polypeptide-31 is SEQ ID NO 4, shOligopeptide-2 is SEQ ID NO 6, the sh-Polypeptide-10 is SEQ ID NO 7, sh-Polypeptide-5 is SEQ ID NO 8, sh-Polypeptide-8 is SEQ ID NO 9, sh-Polypeptide-3 is SEQ ID NO 10, sh-Polypeptide-62 is SEQ ID NO 1, Acetyl Octapeptide-17 Amide is SEQ ID NO 5, sh-oligopeptide-1 is SEQ ID NO 11, sh-Polypeptide-4 is SEQ ID NO 12, and Acetyl sh-Oligopeptide-77 Amide is SEQ ID NO 13, with each growth factor being present at a concentration ranging from 0.001 wt % to 1 wt % of the overall composition and more preferably from 0.009 wt % to 1 wt %. In other aspects, the recombinant human growth factors of the composition are when sh-Polypeptide-1 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 2, the sh-Polypeptide-11 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 3, the sh-Polypeptide-31 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 4, the shOligopeptide-2 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 6, the sh-Polypeptide-10 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 7, the sh-Polypeptide-5 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 8, the sh-Polypeptide-8 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 9, the sh-Polypeptide-3 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 10, the sh-Polypeptide-62 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 1, Acetyl Octapeptide-17 Amide comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 5, the sh-oligopeptide-1 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 11, the sh-Polypeptide-4 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 12, and the Acetyl sh-Oligopeptide-77 Amide comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 13, with each growth factor being present at a concentration ranging from 0.001 wt % to 1 wt % of the overall composition and more preferably from 0.009 wt % to 1 wt %.
The recombinant human growth factors mentioned herein for the regenerative composition/formulation are also nanoencapsulated with the same nanoencapsulating agent(s) mentioned above at the same concentrations and for substantially the same reasons as those mentioned above regarding the general composition/formulation.
In addition and to further promote DNA repair (e.g., via base excision repair and/or mismatch repair pathways) and reduction and repair of UV associated photoaging, this composition further include a combination of liposomally encapuslated bacterial derived photolyase(s), plant-derived roxisome(s), and bacterial derived endonuclease(s) that are present in an effective amount prevent and/or reduce photoaging associated with ultraviolet (UV) exposure by enhancing endogenous DNA repair. The combination of liposomally encapuslated bacterial derived photolyase(s), plant-derived roxisome(s), and bacterial derived endonucleases are present at a concentration ranging from 0.3 wt % to 10 wt % of the overall composition. In certain aspects, the bacterial derived photolyases (also referred to as “photosomes”) are present at a concentration ranging from 0.1 wt % to 3.5 wt % of the overall composition, and preferably are bacterial derived photolyase is a cyanobacteria photolyase in which the cyanobacteria photolyase is an Anacystis nidulans photolyase. The photolyases are generally referred to and may be obtained as “photosomes” from Barnet Products Corporation and includes a mixture of plankton extract (CAS No. 37290-70-3) and lecithin (8002-43-5) that encapsulates the plankton extract therein. Within this composition, plant derived roxisome(s) are present at a concentration ranging from 0.1 wt % to 3.5 wt % of the overall composition in order to combat DNA damage resulting from endogenous reactive oxygen species (ROS) that oxidize guanine into 8-oxo-guanine. These plant derived roxisome(s) are preferably 8-oxo-guanine glycosylase from A. thaliana and may be further obtained from Barnet Products Corporation generally as “Roxisomes” (including a mixture of A. thaliana extract (CAS No. 89958-13-4), lecithin (CAS No. 8002-43-5), and water (CAS No. 7732-18-5)). Bacterial derived endonucleases (also referred to as “Ultrasomes”) are present at a concentration ranging from 0.1 wt % to 3.5 wt % of the overall composition in which the bacterial derived endonuclease(s) are bacterial endonucleases from M. Luteus and may further reduce expression of tumor necrosis factor (TNF) and interleukin-1, 6, and 8 often associated with UV damage. These ultrasomes may also be obtained from Barnet Products. In certain aspects, the liposomal encapsulating agent that encapsulates each of the derived photolyase(s), plant-derived roxisome(s), and bacterial derived endonuclease(s) may be lecithin and may be substantially identical to the nanoencapsulating agent(s) disclosed above. Furthermore, in certain aspects, the photolyase(s), plant-derived roxisome(s), and bacterial derived endonuclease(s) may be encapsulated within the same micelles the recombinant human growth factors within these compositions.
To further enhance this compositions reparative characteristics a plurality of anti-inflammatory agents and/or antioxidants at a concentration ranging from 1 wt % to 10 wt % of the overall composition is also included, which aid in reducing the deleterious effects of ROS and/or mitigate and/or reduce endogenous inflammatory pathways. For example, within this plurality of anti-inflammatory agents and/or antioxidants an Evodia rutaecarpa fruit extract (e.g., Evodiox®) is present at a concentration ranging from 0.1 wt % to 3 wt % of the overall composition (and more preferably 0.5 wt % to 3 w %); this extract exhibits strong anti-inflammatory properties. In addition, thiotaine (INCI Name: Ergothioneine; CAS No. 497-30-3) is preferably included at a concentration ranging from 0.1 to 3 wt % of the overall composition (and more preferably 0.5 wt % to 3 wt %), which further mitigates and/or aids in repair of DNA damage associated with ROS, especially ROS that oxidize guanine into 8-oxo-guanine leading to Hoogsteen base pairing during, for example, DNA replication. Retinol is also included within the plurality of anti-inflammatory agents and/or antioxidants at a concentration ranging from 1 wt % to 3 wt % of the overall concentrations.
Additional additives, emollients, preservatives, and conditioners may also be included and comprise the remainder of the reparative composition/formulation. For example, in certain aspects, this composition includes emollient and an emulsifier present at a concentration ranging from 6 wt % to 32 wt % of the overall composition
This composition may be used alone or in conjunction with the other compositions/formulations disclosed herein as well as with microneedling procedures as disclosed below in the “Methods of Use”. An exemplary reparative composition/formulation is shown immediately below within Table 3.
Hair/Scalp Composition/Formulation
Also disclosed is an aqueous solution for topical use on a human scalp comprising at least 10 recombinant human growth factors (and up to 12 recombinant growth factors), and a nanoencapsulating agent that encapsulates the recombinant human growth factors and present in an effective amount to deliver the recombinant human growth factors to the human scalp to rejuvenate scalp appearance as well as to improve and/or restore hair growth.
In this aspect, the hair/scalp compositions/formulations include water as a solvent at the concentrations discussed above (ranging from 90 wt % to 98 wt %) regarding the general composition/formulation, but more preferably includes from 95 wt % to 97.5 wt % water of the overall concentration of this composition/formulation.
In this composition, at least 10 recombinant human growth factors are present and are selected from the following: sh-Polypeptide-1 (SEQ ID No. 2), sh-Polypeptide-11 (SEQ ID No. 3), sh-Polypeptide-31 (SEQ ID No. 4), sh-Oligopeptide-2 (SEQ ID No. 6), sh-Polypeptide-10 (SEQ ID No. 7), sh-Polypeptide-5 (SEQ ID No. 8), sh-Polypeptide-8 (SEQ ID No. 9), sh-Polypeptide-3 (SEQ ID No. 10), sh-Polypeptide-62 (SEQ ID No. 1), Acetyl Octapeptide-17 Amide (SEQ ID No. 5), sh-oligopeptide-1 (SEQ ID No. 11), sh-Polypeptide-4 (SEQ ID No. 12), and Acetyl sh-Oligopeptide-77 Amide (SEQ ID No. 13). When present in the composition, each growth is included at a concentration ranging from 0.001 wt % to 1 wt % of the overall composition and more preferably from 0.009 wt % to 1 wt %.. In certain aspects, the recombinant human growth factors of the composition are when sh-Polypeptide-1 is SEQ ID NO 2, sh-Polypeptide-11 is SEQ ID NO 3, sh-Polypeptide-31 is SEQ ID NO 4, shOligopeptide-2 is SEQ ID NO 6, the sh-Polypeptide-10 is SEQ ID NO 7, sh-Polypeptide-5 is SEQ ID NO 8, sh-Polypeptide-8 is SEQ ID NO 9, sh-Polypeptide-3 is SEQ ID NO 10, sh-Polypeptide-62 is SEQ ID NO 1, Acetyl Octapeptide-17 Amide is SEQ ID NO 5, sh-oligopeptide-1 is SEQ ID NO 11, sh-Polypeptide-4 is SEQ ID NO 12, and Acetyl sh-Oligopeptide-77 Amide is SEQ ID NO 13, with each growth factor being present at a concentration ranging from 0.001 wt % to 1 wt % of the overall composition and more preferably from 0.009 wt % to 1 wt %. In other aspects, the recombinant human growth factors of the composition are when sh-Polypeptide-1 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 2, the sh-Polypeptide-11 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 3, the sh-Polypeptide-31 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 4, the shOligopeptide-2 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 6, the sh-Polypeptide-10 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 7, the sh-Polypeptide-5 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 8, the sh-Polypeptide-8 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 9, the sh-Polypeptide-3 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 10, the sh-Polypeptide-62 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 1, Acetyl Octapeptide-17 Amide comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 5, the sh-oligopeptide-1 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 11, the sh-Polypeptide-4 comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 12, and the Acetyl sh-Oligopeptide-77 Amide comprises a sequence at least 90%, 95%, or 98% identical to SEQ ID NO 13, with each growth factor being present at a concentration ranging from 0.001 wt % to 1 wt % of the overall composition and more preferably from 0.009 wt % to 1 wt %.
This composition further includes hyaluronic acid (CAS No. 9067-32-7) and silanol and/or organosilanol (CAS No.: 4253-34-3) is methylsilanetriol (CAS No. 2445-53-6), at substantially the same concentrations as those previously discussed above and for reasons similar to those disclosed above as well as for the scalp rejuvenation and restore hair growth purposes disclosed herein for this specific composition.
This composition may be used alone or in conjunction with the other compositions/formulations disclosed herein as well as with microneedling procedures as disclosed below in the “Methods of Use”. An exemplary reparative composition/formulation is shown immediately below within Table 4.
%
-
.000
Alcohol (and) D
Acid (and) Water
1-6
520-46-6
77
2-18-
Acid (and)
(and) Water (and) Butylene Glycol
0
-7,
253-
-
7732-18-
07-
-0
ing
yle
Glycol (and) 1,2-hexanediol (and) Sodium Phosphate
322-
-
-2 (and) sh-Polypeptide-
(and) sh-
(and) sh-Polypeptide-
(and) sh-Polypeptide-10 (and) sh-
(and) Ace
sh-O
Amide (and) A
O
peptide-17
indicates data missing or illegible when filed
Kits
In certain aspects, each of the compositions/formulations disclosed herein are preferably sterile. These compositions/formulations may be further packaged within single use vials and/or containers (single use disposable vials and/or disposable containers), or alternatively may be packaged within multi-use vials and/or containers. In certain aspects, any one of the above mentioned compositions/formulations may be packaged alone within a kit. Alternatively, any one of the above compositions/formulations may be packaged within a kit that further includes microneedles. The number of microneedles provided in the kit sufficiently corresponds with the composition amount packaged within the kit.
In certain aspects, the kit may include at least two compositions selected from the regenerative composition/formulation, the reparative composition/formulation, and the hair/scalp composition/formulation as disclosed above. In this aspect, the kit may further include microneedles that sufficiently correspond with the composition amount included within the kit in which the microneedles may be further used with the compositions as disclosed herein (e.g., as disclosed within the “Methods of Use” below).
In certain additional aspects, the kit may include all three of the regenerative composition/formulation, reparative composition/formulation, and the hair/scalp composition/formulation as disclosed above, and the kit may further include microneedles that sufficiently correspond with the composition amount included within the kit and that may be further used with the compositions as disclosed herein (e.g., as disclosed within the “Methods of Use” below).
Methods of Use
Also disclosed is a method of reducing wrinkles and/or fine lines over a predetermined time period, the method comprises (a) applying on the skin of a subject at least one of the compositions disclosed above; and (b) repeating the application of the composition to the skin of the subject at predetermined time periods (e.g., once a day or twice a day) thereby reducing wrinkles, fine lines, and/or the appearance thereof during the predetermined time period. In certain aspects, the compositions are applied twice a day (e.g., morning and at night). In certain aspects, this method further comprises: before step (a) (e.g., either immediately before or within 5 to 10 minutes before) performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject. In certain aspects, this method further comprises after step (a) and before step (b) (e.g., either immediately before or within 5 to 10 minutes before) performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject. In certain aspects, the microneedling procedure is performed at least once per week but no more than twice per week depending on whether the subject can tolerate this procedure twice per week.
Also disclosed is a method for reducing pore size over a predetermined time period, the method comprises (a) applying on the skin of a subject at least one of the compositions disclosed above; and (b) repeating the application of the composition to the skin of the subject at predetermined time periods (e.g., once a day or twice a day) thereby reducing pore size and/or the appearance thereof during the predetermined time period. In certain aspects, the compositions are applied twice a day (e.g., morning and at night). In certain aspects, this method further comprises before step (a) (e.g., either immediately before or within 5 to 10 minutes before) performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject. In certain aspects, this method further comprises after step (a) and before step (b) (e.g., either immediately before or within 5 to 10 minutes before) performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject. In certain aspects, the microneedling procedure is performed at least once per week but no more than twice per week depending on whether the subject can tolerate this procedure twice per week.
Also disclosed is a method of enhancing skin texture over a predetermined time period, the method comprises (a) applying on the skin of a subject at least one of the compositions disclosed above; and (b) repeating the application of the composition to the skin of the subject at predetermined time periods thereby improving and/or enhancing skin texture and/or the appearance thereof during the predetermined time period. In certain aspects, the compositions are applied twice a day (e.g., morning and at night). In certain aspects, this method further comprises before step (a) (e.g., either immediately before or within 5 to 10 minutes before) performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject. In certain aspects, this method further comprises after step (a) and before step (b) (e.g., either immediately before or within 5 to 10 minutes before) performing a microneedling procedure on the subject to increase delivery efficacy of the composition to the subject. In certain aspects, the microneedling procedure is performed at least once per week but no more than twice per week depending on whether the subject can tolerate this procedure twice per week. In certain aspects, a user's scalp appearance may be rejuvenated and/or a user's hair may be re-grown by applying the hair composition/formulation disclosed above to a user's scalp and using substantially the same steps and procedures as those disclosed immediately above within the “Methods of Use” section (for reducing wrinkles, reducing pore size, and improving skin texture respectively).
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, and methods described and claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for.
Cell Culture Data Comparing Proliferative Effects Of One Recombinant Human Growth Factor Versus A Combination Of Ten Recombinant Human Growth Factors (Proof of Concept)
Formulating Solution(s)
A. After formulation, aliquot into 50 mL conical tube and store at 4° C.
Test Procedure:
1. Culture Balb/c-3T3 cell with 10% FBS in DMEM high glucose culture media onto 75U-flask. Proceed subculture when flask reaches 80-90% confluency.
2. Cell seeding (Day #1)
Formulate a cell suspension liquid with 2.0×104 cells/0.1 ml per well. Move onto a 96well plate and fill the empty wells with 200 pl of PBS. Incubate for 24 hours. (37° C., 5% CO2 incubator)
Clinical Tests and Results
A study was conducted to evaluate the efficacy and safety of an exemplary topical/transdermal composition having recombinant human growth factors as disclosed herein and in conjunction with a micro needling procedure to evaluate efficacy for anti-aging and anti-wrinkle benefits in healthy female subjects.
Primary Objectives:
To evaluate the reduction in fine lines and wrinkles defined time points in comparison to the baseline (i.e., pre-treatment).
To evaluate the improvement in evenness of skin tone, skin clarity and age spots.
To evaluate improved skin texture and improved skin elasticity/firmness.
Secondary Objectives:
To evaluate skin hydration.
To evaluate the product safety by skin tolerance evaluation performed by dermatologist.
Assessment Parameters:
The Dermatologists' assessment for fine lines and wrinkles and the overall improvement in skin texture, age spots and pigmentation and hydration level are shown in
Subject self-assessment (not shown) for perceived improvement in fine lines and wrinkles, overall improvement in age spots and pigmentation, skin texture and moisturization.
Instrumental measurements and assessments using: Antera (3D imaging) are shown in
Image based comparative assessments using VISIA images (CR imaging) are shown in
Selection Criteria:
A total of twelve female subjects (as shown in the Table immediately below) were required to complete the study. Female subject falling within the age group of 35-60 and Fitzpatrick skin types III-V were required for the study along with having aging concerns and/or signs of aging. Nineteen subjects were initially screened and enrolled after satisfying inclusion and exclusion criteria. Seventeen subjects were used in the study, which are as follows:
Twelve subjects completed the study. Two subjects dropped out of the study due to adverse events (dry and/or inflamed skin) while three additional subjects were dropped from the study due to failure to follow-up and/or maintain the below described protocol.
Products Used:
1 mm micro needles (Hydra 20 Micro Needle as shown in
Study Outline:
There were a total of 8 visits:
The study was conducted for a period of approximately 3 months for each subject and included a total of 8 visits i.e., visit 1 (day 0, screening, and enrollment assessment visit) and on visits 2, 3, 4, 5, 6 and 7 all assessments were performed followed by micro-needling procedure. Visit 8 was the final assessment visit; there was no procedure on this assessment visit.
A wash out period of 7 days and preparatory period of 2 days was maintained between visit 1 and visit 2. A sufficient number of healthy female subjects of 35-60 (inclusive both ages) years of age was screened, so that total 10 subjects complete Part I (exploratory pilot study) of the study. The subjects who satisfied inclusion and exclusion criteria were enrolled after obtaining informed consent.
Prior to the baseline assessment visit, there was a wash out period of 1 week. During their wash out phase the subjects received a skin care kit containing cleanser, day cream (with sunscreen) and a night cream (with moisturizer). The last two days of this period was regarded as the preparatory phase of the study during which the subjects stopped the day cream and night cream.
First anti-aging hydra needle procedure was performed at the site by a trained designee on visit 2, after baseline assessment. On visits 3, 4, 5, 6 and 7 subjects underwent dermatological assessment; subject self-assessment for perceived improvement, safety assessment, and instrumental assessment followed by anti-aging hydra needle procedure.
On the days when the subjects were not visiting the study center, they underwent Anti-aging hydra needle procedure by trained personnel appointed by the study center at their home every alternate day except on Sundays.
At Site (by Investigator)
Subjects visited the site every two weeks. The micro needling procedure was performed (using 1 mm needle) after topical anesthetic cream application. The anesthetic cream was applied in thin film without occlusion. Following the micro needling procedure, the exemplary topical/transdermal composition was applied to the subject on and around the site of micro needling.
At Home (self)—Twice a week
Procedure was performed by the subject (self) using 0.5/0.6 mm needle.
Subject visited the site at day 3 for training.
First self-procedure was done at the site under the supervision of the investigator.
The composition was applied immediately after the procedure and not 12 hrs prior to the procedure.
Procedure was performed in the late evening.
Data Processing and Statistical Methodology
As discussed further below, data was compiled using multiple different assessments (i.e., dermatological assessment, subject-self assessment, and multiple different image assessments) to obtain statistical data regarding whether the exemplary compositions exhibited advantageous effects during the study while concurrently minimizing subjective bias via the use of these multiple different assessment techniques.
Data was captured in electronic case report form using a mobile app built by MSCR. Different types of edit check were applied to avoid discrepancy while entering the data. After completion of the study, data was exported into Excel® and then the data was checked again as a process of data validation. After completion of data validation, the data was locked for analysis.
For Efficacy—The data of all subjects who successfully completed the study was considered for the statistical analysis. Data of 12 subjects was considered for the statistical analysis.
Product Safety—The data of all the subjects available on each assessment visit was considered which included lost to follow up, drop outs till their last participation visit.
Descriptive statistical analysis was carried out in the present study. In all analysis, a 2-sided significance level of 5% (p-value<0.05) was used to determine significance levels.
Results on continuous measurements were presented on Mean±SD and results on categorical measurements were presented in Number (%). Significance was assessed at 5% level of significance. Paired t-test was performed to find out efficacy in comparison to baseline on continuous scales such as instrumental assessments.
Statistical figures:
Statistical software: The Statistical software, R-ver.3.1.2 was used for the analysis of the data and Microsoft word and Excel have been used to generate graphs, tables etc.
Dermatological Assessment:
Fine line(s) and wrinkles (
Subject Self-Assessment (Data Not Shown):
There was a significant improvement perceived for all the six parameters (fine lines and wrinkles, evenness of skin tone, skin clarity, age spots, skin firmness and hydration of skin) when compared to baseline (data not shown).
Instrumental measurement using Antera (3D Imaging)
(
Wrinkle Assessment: Mild to moderate wrinkle on the forehead was noted to reduce significantly by week 12 (final assessment visit;
There was a significant reduction in wrinkles (fine, mild and deep) on Crow's feet observed at all the given time points when compared to baseline (
There was a significant reduction in wrinkles (mild and deep) on nasolabial fold which was observed at all the given time points when compared to baseline (
Texture Assessment: There was a significant improvement in skin texture (mild and deep) on forehead which observed at all the given time points when compared to baseline (
Significant improvement in texture (fine, mild and deep) on Crow's feet was observed at all the given time points when compared to baseline (
Significant improvement in skin texture (mild and deep) on nasolabial fold was observed at all the given time points when compared to baseline (
Instrumental measurement using Visia (CR Imaging):
Wrinkle Assessment: There was a significant decrease in the percentage of wrinkles that was observed at a given test site on week 8 when compared to week 1 (
Texture Assessment: There was a significant improvement noted in the percentage of texture of the skin on three given time point (week 4 to week 12) when compared to baseline (
Pore Assessment: There was a significant decrease noted in the percentage of pore spread at a given test site on all the three visit (week 4 to week 12) when compared to baseline (
Image Based Comparative Assessment:
There was a significant reduction of wrinkles observed at Crow's feet, Nasolabial folds and Frown lines (
There was no significant change observed in the age spots, evenness of skin and skin texture when compared to baseline.
The study was conducted to evaluate the efficacy and safety of skin care regimen in adjunction with the micro needling procedure for providing anti-ageing and anti-wrinkle benefits in healthy female subjects.
As per the dermatological evaluations, there was significant improvement in Fine line & wrinkles, evenness of skin tone, age spots, skin smoothness, skin firmness and hydration of skin when compared to baseline. There was suggestively significant improvement noted for skin clarity on final assessment visit (week 12).
As per the subject self-assessment, there was a significant improvement noted for all the six parameters (fine line & wrinkles, evenness of skin tone, skin clarity, age spots, skin firmness and hydration of skin) at time points (week 2 to week 12) when compared to baseline.
As per instrumental assessment (Antera (3D Imaging)).
Wrinkle: There was a significant reduction in mild and fine wrinkles at week 12 (final assessment visit) observed on all the tested sites namely, forehead, crow's feet and nasolabial fold when compared to baseline. There was significant reduction in deep wrinkle for test sites (crow's feet and nasolabial fold) at week 12 (final assessment visit) when compared to baseline and suggestive reduction for test site (Forehead) at week 12 when compared to baseline.
Texture: There was a significant improvement observed in skin texture (mild and deep) on forehead, crow's feet and nasolabial fold at all the given time points when compared to baseline. The improvement in the fine skin texture on crow's feet and nasolabial fold was found to be significant at all-time points when compared to baseline however the improvement in the fine skin texture on forehead was found to be suggestively significant at week 4 when compared to baseline.
As per instrumental assessment Visia (CR Imaging), a significant reduction in the percentage of wrinkles was observed at week 8 when compared to week 1. Also, there was a significant improvement noted in the percentage of texture & pore spread on three given time points (week 4, week 8 and week 12) when compared to baseline.
As per image based comparative assessment, a significant reduction of wrinkle was observed at Crow's feet, Nasolabial folds and Frown lines, evenness of skin and skin texture at week 12 when compared to baseline. However, there was no change noted in the age spots when compared to baseline.
Product Safety: Two subjects (S02 and S06) could not tolerate the Micro needling procedure and developed dry and irritable skin post first procedure. As per the dermatologists opinion this may be a result of severe skin dryness in combination with topical anesthetic product. The protocol was modified accordingly and was thereafter well tolerated by rest of the subjects who participated in the study. Subjects 02 and 06 were followed-up till complete resolution of skin reaction which resolved with.
The foregoing description provides embodiments of the invention by way of example only. It is envisioned that other embodiments may perform similar functions and/or achieve similar results. Any and all such equivalent embodiments and examples are within the scope of the present invention and are intended to be covered by the appended claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2021/000149 | 3/19/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62991854 | Mar 2020 | US |
