Composition comprising hydrolyzed collagen in combination with pyrroloquinoline quinone for stimulating the synthesis of collagen and regenerating skin

Abstract

A synergistic composition based on low molecular weight hydrolyzed collagen and pyrroloquinoline quinone that is suitable for healing skin lacerations or injuries and / or accelerating the cicatrization process. The composition can be formulated as a food supplement for oral administration or as a preparation for topical application for cosmetic use in the treatment of signs of skin aging or for medical use in the healing of skin injuries and / or lesions.

Description

[0001] COMPOSITION COMPRISING HYDROLYZED COLLAGEN IN COMBINATION WITH PYRROLOQUINOLINE QUINONE FOR STIMULATING THE SYNTHESIS OF COLLAGEN AND REGENERATING SKIN

[0002] The present invention relates to a composition for stimulating the synthesis of collagen and the physiological process of skin regeneration.

[0003] The present invention concerns the field of regeneration of the skin and of skin-adjacent organs, and its application is in the beauty field, in the cosmetic treatment of skin blemishes, and in the dermatological field, in the regeneration of injured skin.

[0004] In particular, the present invention relates to a composition containing biologically active ingredients in a combination that stimulates the fibroblasts to promote regeneration of the connective tissue and of the epidermis, improving the outer appearance of the skin and promoting the process of regeneration of collagen and of injured skin.

[0005] BACKGROUND ART

[0006] Collagen is one of the most-expressed proteins in the human body, where it plays a supporting role for many tissues together with elastic fibres and glycosaminoglycans, mainly hyaluronic acid. Furthermore, collagen represents about 77% of the total dry weight of the skin, not including fats, and it constitutes 90% of the fibrous components of the dermis.

[0007] Collagen fibres form a supporting structure for the skin and they also play an important role in maintaining skin elasticity. With the passing of time, an individual's collagen fibres tend to crosslink and become gradually thicker, resulting in a decrease in elasticity. As a consequence of this process, elastin fibresfibres become thicker, their crosslink increases, exacerbating the formation of skin blemishes such as wrinkles, creases and folds. For these reasons a collagen deficiency or a change in collagen turnover are generally associated with a slowing of the physiological process of regeneration of skin lesions, and with skin ageing, which is also exacerbated by extrinsic factors, principally exposure to external agents suchas the sun's radiation, smog, and smoke.

[0008] Typically in the ageing process, structural and functional changes occur in the dermis, which is a layer of connective tissue closely joined to the epidermis to form the skin, which tends to thin, lose elasticity and become drier, so resulting in the formation of wrinkles. Furthermore, the proliferation of fibroblasts in the dermis tends to decrease, and with it the capacity to synthesize new collagen also decreases.

[0009] These conditions lead to a reduction in skin firmness and tone and are a prelude to the formation of wrinkles.

[0010] It is thus evident that an insufficient turnover of collagen represents one of the main contributing factors of skin aging.

[0011] The promotion of collagen synthesis and the inhibition of excessive degradation, as found in a population of individuals aged over 50, are of interest not only on the cosmetic / aesthetic level but also on the medical level in problems linked to the cicatrization / healing process of injuries.

[0012] This process has an initial inflammation stage followed by a proliferation stage in which fibroblasts are involved in the production of collagen and of fundamental substance that will form the base structure for the overlying tissue. So in the injury healing and recovery process, collagen turnover therefore plays a very important role.

[0013] The reduction in concentrations of cutaneous collagen, or the reduced turnover, are therefore factors responsible for the structural and functional changes that contribute to cause skin aging and a slowed or inefficient injury¬ recovery process.

[0014] These are the reasons behind the recommendation to supplement the dietary regimen with suitable sources of collagen to improve its synthesis, including of cutaneous collagen, restoring its natural turnover.

[0015] This is why trends have emerged in research in cosmetics and in regenerative medicine which aim to develop preparations containing collagen to supplement deficiencies of this biomolecule, with the goal ofobtaining an improvement in the aesthetic appearance of an individual or of restoring the natural skin regeneration process.

[0016] However, the high molecular weight of the collagen contained in most of the products for internal or external use available on the market makes difficult its digestion and absorption in amounts that would effectively supplement the lost collagen and, determine a regenerative effect on the skin that would enable applications both in cosmetics and in medicine.

[0017] In an attempt to overcome these limitations, currently supplements are sold for oral use or as preparations for topical use which contain collagen in hydrolyzed form.

[0018] However, taking supplements or applying topical preparations containing hydrolyzed collagen has given unsatisfactory results both in cosmetics, for example in the treatment of wrinkles, and in restorative medicine, for example, in the healing of skin injuries or lacerations.

[0019] It has in fact been observed that the oral administration of supplements containing collagen or the topical application of topical preparations brings a modest antiaging cosmetic effect and what is more, does not significantly reduce the healing times for skin lesions or lacerations.

[0020] One of the aims of the invention consists in providing a preparation based on low molecular weight hydrolyzed collagen combined with a selected biomolecule that is suitable to restore natural collagen turnover and which can be applied in cosmetics in the treatment or reduction of signs of skin ageing and in restorative medicine in the acceleration of the process of healing and recover}’ of skin injuries and lesions.

[0021] SUMMARY OF THE INVENTION

[0022] According to an aspect of the present invention, the inventors have observed that a suitable hydrolyzed collagen combined with pyrroloquinoline quinone stimulates the fibroblasts to produce substances of the extracellular matrix, in particular collagen. Furthermore, in in vitrosamples it has been observed that fibroblasts treated with hydrolyzed collagen and pyrroloquinoline quinone improve collagen turnover and reduce production of enzymes that degrade the extracellular matrix.

[0023] These effects favor the formation of a pro-anabolic and anti-catabolic environment which slows the natural process of aging and degeneration of the outermost tissues of the body, mainly the connective tissue and the cutaneous tissue.

[0024] These actions accelerate the natural collagen production process of the dermis, making the combination composition described herein suitable for applications both in cosmetics, for example in the reduction of signs of skin ageing, and in medicine, for example in accelerating the healing process of injuries.

[0025] Thus a primary aim of the present: invention is a composition comprising or consisting of low molecular weight hydrolyzed collagen in combination with pyrroloquinoline quinone in free form or in a physiologically acceptable salt or ester thereof for use in the treatment of skin lesions, lacerations or injuries and / or to cicatrize or heal injured skin.

[0026] A suitable hydrolyzed collagen contains peptides and / or polypeptides with a molecular weight in the interval described below.

[0027] Preferably the hydrolyzed collagen of the composition described herein has an average molecular weight comprised from 0.5 to 6 kDa, from 1 to 4.5 kDa, preferably from 1.5 to 2.5 kDa, more preferably 2 kDa.

[0028] The composition for the uses described herein is a combination composition wherein the active ingredients, low molecular weight hydrolyzed collagen and pyrroloquinoline quinone in free form or a physiologically acceptable salt or ester thereof, exert a synergistic action of renewal and / or regeneration of endogenous collagen, as described below.

[0029] According to some embodiments, the combination composition for any use described herein can further contain nicotinamide adenine dinucleotide, a biologically active ingredient: that further contributes to theaction of renewal and / or regeneration of collagen, and of the skin, of the two basic components of the combination composition.

[0030] According to this first aspect of the invention, the combination composition based on hydrolyzed collagen and pyrroloquinoline quinone (PQQ) or a salt or ester thereof therefore bring a synergistic activity of synthesis of new collagen which, as it builds up in the subcutaneous tissue, stretches the skin, reducing the typical signs of skin aging and of skin lesions on the human body.

[0031] The composition for the uses described herein preferably can be formulated as a food supplement: suitable for oral administration according to the appended claims 6, 7 or as nutritional water according to claim 8 or as a topical preparation or medicament for local application according to claims 9-12.

[0032] The food supplement has applications in cosmetics, preferably in medicine, in regenerative medicine or in dermatology.

[0033] The nutritional water may have application in cosmetics / aesthetic treatments, in particular in the prevention or treatment of skin blemishes and / or signs of skin ageing, and in therapy to promote and accelerate the healing of lacerations, injuries, or excessive, non-natural flaking of the skin, or of ichthyosis or xeroderma. Advantageously, the nutritional water described herein further offers an anti ageing or regenerative effect on the skin and / or on skin injuries. Preferably the anti-aging or regenerative effect is achieved by consuming nutritional water for at least 20 consecutive days, preferably at least 30-90 days, for example in an amount of from 50 to 1500ml, from 100 to 1000ml per day.

[0034] According to an aspect, the food supplement containing the composition described herein can be introduced into the dietary regime of an individual needing to prevent or treat skin blemishes, signs of skin ageing and to accelerate healing of the skin.

[0035] In an aspect, the oral administration of the composition or of thesupplement that contains it accelerates the natural process of renewal of the dermis and of the skin, and is suitable for cosmetic or medical use.

[0036] According to another aspect, the food supplement containing the composition described herein is is useful in th medical field, in particular in regenerative medicine or in dermatology, in the repair and / or regeneration of injured cutaneous tissue, in particular in the healing of injuries.

[0037] It has in fact been observed that the oral administration of the composition described herein accelerates the natural process of healing of skin injuries or lesions.

[0038] Therefore a specific aim of the present invention is a food supplement containing the composition described herein for use in the treatment of lesions, lacerations or injuries of the skin and / or for accelerating the process of cicatrization or healing of injured skin, for example according to the appended claims 6, 7.

[0039] According to other aspects of the invention, the composition described herein may be formulated as a topical preparation, for example for the uses according to claims 9-12.

[0040] In fact it has been found that the topical preparation described herein has applications both in cosmetics, in the treatment of signs of skin ageing, and in medicine, in the treatment of injuries and / or lacerations of the skin.

[0041] In an aspect, the cosmetic, not therapeutic, use of a topical preparation comprising hydrolyzed collagen in combination with PQQ or a physiologically acceptable salt or ester thereof is disclosed.

[0042] The cosmetic and / or medical uses of the combination composition originates mainly from the stimulatory activity that promotes the formation of new' collagen which, as it builds up in the subcutaneous tissue and in the dermis, determines the stretching of the overlying cutaneous tissue, so reducing wrinkles, signs of skin ageing, and / or the depth of injuries.

[0043] A further aim of the present invention is to provide a topical preparation comprising hydrolyzed collagen in combination with PQQ or aphysiologically acceptable salt or ester thereof for use in the treatment or healing of skin injuries, for example according to claims 9-12.

[0044] These uses in cosmetics and / or medicine origin from having observed that the topical application of the topical preparation containing the composition based on hydrolyzed collagen and PQQ stimulates the renewal of the three main layers of the skin: the epidermis, the dermis and the hypodermis, particularly the dermis and hypodermis.

[0045] According to certain aspects relating to aesthetic medicine, it has been observed that the combination of the Sow molecular weight hydrolyzed collagen component as described herein with pyrroloquinoline quinone (PQQ) or a salt / ester thereof stimulates the synthesis of new collagen, promotes the repair, healing and / or regeneration of injured cutaneous tissue, and accelerates the healing process of skin lesions.

[0046] These effects have been demonstrated experimentally using a scratch test as a primary experimental model.

[0047] Advantageously, the combination composition for medical use described herein can therefore be formulated as a preparation suitable for topical application on areas of the skin affected by injuries, abscesses, skin lesions.

[0048] The combination composition for use in the treatment and healing of skin injuries also finds applications in the prevention and / or treatment of chronic injuries that tend to relapse.

[0049] The topical preparation containing the combination composition for the medical uses described herein stimulates the activity of synthesis of new type I collagen in fibroblasts, in particular in the dermis and in the hypodermis. Newly-formed collagen has a regenerative effect on the skin that makes the composition suitable for the treatment of acute and chronic injuries.

[0050] According to some aspects, the invention provides a method of treatment by therapy in the treatment: of a lesion, laceration or injury of the skin and / or amethod of cicatrizing or healing injured skin that comprises the application on the injured skin or wound of a therapeutically effective amount of a composition comprising low molecular weight hydrolyzed collagen according to one or more embodiments described herein in combination with pyrroloquinoline quinone in free form or a physiologically acceptable salt or ester thereof. According to embodiments of the method of treatment described herein, the composition further comprises nicotinamide adenine dinucleotide and / or a precursor and / or derivative thereof selected from the group consisting of nicotinamide riboside, nicotinamide mononucleotide, niacin, nicotinamide and mixtures thereof. In the method of treatment described herein said composition may be contained in a supplement, functional water or a topical preparation according to any embodiment described herein.

[0051] The present invention is described in detail below, with reference to the accompanying figures.

[0052] BRIEF DESCRIPTION OF THE FIGURES

[0053] Some features and advantages of the present invention will be evident from the accompanying figures wherein:

[0054] Figure 1 contains a bar chart showing the results of the in vitro comparative test of Example 2 on collagen synthesis in human dermal fibroblast (HDF) cells induced by pyrroloquinoline quinone (PQQ) alone, hydrolyzed collagen with average MW of 2kDa alone (HCOL), and a combination of hydrolyzed collagen with average MW of 2kDa and PQQ according to an embodiment of the invention. The data show that the treatment with the combination PQQ + HCOL induces an increase of 4.7 times at 48 hours and an increase of 4.3 times at 72 hours in the production of collagen compared to the cells in the untreated control sample;

[0055] Figure 2 contains a chart showing the results of the test of Example 3 on the proliferation and migration of fibroblasts, indicative of would closure. The chart show's the percentage closure of the wound at different timeintervals (12, 24, 48 hours) with PQQ alone, hydrolyzed collagen with average MW of 2kDa (HCOL) alone, the combination of PQQ+HCOL, and untreated cells (CTRL).. The chart shows that the combination of PQQ+HCOL according to an embodiment of the invention induces a faster migration of HDF cells, with consequent closure of the scratch wound by 94% in 48 hours. The percentage of cellular migration is markedly lower that for the individual components (PQQ; HCOL ) of the composition;

[0056] Figure 3 shows the ATP content in human dermal fibroblast cells in culture incubated at different doses of PQQ (1.10 μM), HCOL (0.1 and 1 mg / ml) and a combination of the two compounds PQQ+HCOL (average MW 2kDa) for 2 hours (n = 4). The synergism of action is evident from the bar chart showing the combination PQQ+HCOL;

[0057] Figure 4 contains bar charts showing the formation of new tubules and the ramification of human aortic endothelial cells ( HAEC) promoted by the combination composition according to an embodiment as reported in the experimental part described in Example 5.

[0058] Figure 5 shows the high affinity interaction between PQQ and NAMPT found according to experimental Example 5. Analyses of molecular docking in silico show a high affinity interaction between PQQ and NAMPT. The envisaged bond shape structure has shown that several amino acid residues in NAMPT stabilize hydrogen-bonded and hydrophobic interactions with the PQQ, so stabilizing the ligand in the binding pocket. DETAILED DETAILED DESCRIPTION OF THE INVENTION

[0059] According to a general aspect, the invention relates to a combination composition comprising low molecular weight hydrolyzed collagen and PQQ, optionally in the form of a salt or an ester, for example for the use as defined in the appended claim 1.

[0060] The combination composition and / or its uses defined in the appended claims brings the following synergistic effects:

[0061] - anabolic stimulation with increase in synthesis of endogenouscollagen,

[0062] - anti-catabolic protection through inhibition of degradation of the matrix,

[0063] ~ improved cellular energy.

[0064] This aspect of the invention derives from having observed that combining low molecular weight hydrolyzed collagen with PQQ results in an increased formation of new fibres of collagen and elastin and an increased formation of bonds of hydrolyzed collagen peptides with the receptors present: in the membrane of fibroblasts which stimulate the production of new collagen, elastin and hyaluronic acid, in particular in subcutaneous tissues and in the dermis. These biological effects are at the heart of an unexpected acceleration of the collagen renewal process, in particular in the dermis, which manifests as a reduction in the signs of skin aging, for example, wrinkles and folds in the skin.

[0065] Advantageously, the formation of new endogenous collagen obtained by administering or locally applying the composition described herein produces a plumping and stretching effect on the skin, which reduces the scars of injuries or lacerations of the skin and / or the signs of skin aging, giving the skin a more youthful appearance.

[0066] These effects are at the heart of the medical and / or beauty uses of the combination composition and underpin the advantages described herein.

[0067] According to some aspects, it has also been observed that the combination of low molecular weight hydrolyzed collagen with PQQ increases the formation of new fibres of collagen and elastin and favors the formation of bonds between hydrolyzed collagen peptides with the receptors present in the membrane of fibroblasts. These activities stimulate the process of skin regeneration, and accelerate the cellular metabolism in the dermis by accelerating the process of cicatrization and healing of injuries.

[0068] These effects are form the basis of the medical uses of the combination composition and of the supplement or topical preparation thatcontain it.

[0069] The composition described herein contains hydrolyzed collagen, preferably with a selected average molecular weight.

[0070] A suitable hydrolyzed collagen is obtained using collagen as the raw material. Collagen is a protein typically containing the amino acids glycine, proline and hydroxyproline as the primary structure in a triple helix formed by three amino acid chains.

[0071] There are many types of collagen from whicha suitable low molecular weight hydrolyzed collagen can be obtained, and among these, type I collagen and types II, III, IV, V, XI, XXIV are preferred in the scope of the invention.

[0072] A suitable native collagen, for example type I, can be extracted from several sources preferably of animal origin, in particular from bovine, pigs, sheep, chicken, ducks and fish. The principal source of extraction is bovine, for its availability and its biocompatibility.

[0073] The extraction of collagen can be done starting from various different tissues of the animal, like bones, tendons, lung tissue, connective tissue or specific organs such as eyes, for example from cattle.

[0074] The extraction of collagen can be done with conventional treatments, for example, by treating a collagen-rich tissue with an acid or an alkaline substance. Extraction of collagen by acid treatment is usually used to extract type I collagen from pig tissues or fish skin. For example, acetic acid is the most common reagent for extracting collagen. The concentration of this acid influences the final pH value, by modifying the electrostatic interaction and the structure. It also determines the solubility and the capacity’ for extraction from animal tissue. It is also possible to use a combination of acid and enzymatic treatment, which offers a more efficient extraction of collagen.

[0075] A suitable hydrolyzed collagen can also be obtained by denaturing native collagen in low molecular weight peptides.

[0076] Denaturing native collagen can be done with conventional methods.for example by means of heat treatment of the native collagen at temperatures above 40°C which yields three a chains in their random-coil conformation.

[0077] Once the chains are separated, the hydrolysis can be enzymatic, carried out using proteolytic enzymes, for example alcalase, papain, or pepsin. The product resulting from this hydrolysis is the hydrolyzed collagen made of peptides of small dimensions and with a low molecular weight. Alternatively, hydrolyzed collagen can be obtained by hydrolysis using chemical products in an acidic environment, for example with acetic acid, hydrochloric acid or phosphoric acid, or even in an alkaline environment using techniques known to the person skilled in the art.

[0078] In the present description of the invention the term “hydrolyzed collagen” preferably means a mixture of peptides with low molecular weight, preferably from 0.5 to 6 KDa, from 1 to 4.5 KDa, more preferably from 1.5 to 3KDa, for example 2KDa.

[0079] The molecular weight of hydrolyzed collagen suitable for the uses of the invention can be calculated with methods known to the person skilled in the art.

[0080] A preferred technique is SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). It is suitable for separating proteins / polypeptides with molecular weight comprised between 0.5 and 100 KDa. This technique can also be applied to determine the molecular weight of hyaluronic acid, as described herein.

[0081] According to this technique the molecules are separated based on their charge and the speed of displacement is linked to the charge of the molecule.

[0082] This method uses polyacrylamide gel (PAGE - polyacrylamide gel electrophoresis) in the presence of the anionic detergent sodium dodecyl sulfate (SDS). The polymerization in gel form of monomers of acrylamide produces linear chains. By including bisacrylamide, a three-dimensional matrix of the gel is formed. The size of the pores formed depends on theconcentration of acrylamide and on the degree of cross-linking. The first gel is the staking gel, which is a low-concentration gel (4%), while the second gel, called resolution gel, usually has a concentration of 10-12.5% and is used to separate proteins in the interval 1-100 KDa. Therefore, by varying the concentration of acrylamide and bisacrylamide in the preparation of the gel, we obtain several degrees of porosity and therefore several intervals of separation of the proteins.

[0083] An additional technique that is suitable to determine the molecular weight of hydrolyzed collagen is size exclusion chromatography together with multi-angle laser light scattering (SEC-MALS) GE Healthcare Size Exclusion Chromatography: Principles and Methods (2014). This technique can also be applied to determine the molecular weight of hyaluronic acid.

[0084] By way of example the molecular weight of a suitable collagen with low molecular weight can be measured by reproducing the techniques described in one of the following publications:

[0085] -Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680. doi: 10.1038 / 227680a0;

[0086] -Schagger H. Tricine sds-page. Nat. Protoc. 2006;1:16. doi: 10.1038 / nprot.2006.4;

[0087] -Haider S. R., Reid H. J., Sharp B. L. Tricine-sds-page. In: Kurien B. T., Scofield R. H., editors. Protein Electrophoresis: Methods and Protocols. Volume 869. Humana Press; New York, NY, USA: 2012. pp. 81–91.

[0088] In addition, it is possible to use HPLC-MS / MS as an instrument for the identification and quantification of peptides and proteins such as collagen.

[0089] Quantification of types of collagen can also be done by means of analysis of amino acids as described, for example, in one of the following publications:

[0090] -Pataridis S., Eckhardt A., Mikulikova K., Sedlakova P., Miksik I.Identification of collagen types in tissues using HPLC-MS / MS. J. Sep. Sci.

[0091] 2008;31:3483-3488;

[0092] -Zhang G., Sun A., Li W., Liu T., Su Z. Mass spectrometric analysis of enzymatic digestion of denatured collagen for identification of collagen type. J. Chromatogr. A. 2006;1114:274-277;

[0093] -Mikulikova K., Eckhardt A., Pataridis S., Miksik I. Study of posttranslational non-enzymatic modifications of collagen using capillary electrophoresis / mass spectrometry and high-performance liquid chromatography / mass spectrometry. J. Chromatogr.

[0094] A preferred hydrolyzed collagen has a molecular weight of 2 KDa + / - 10% and the following technical characteristics:

[0095] Content of proteins / peptides 91.00– 95.00 %, Kjeldahl (N x 5.55) Protein content of the dry substance ≥ 97 %, GELITA

[0096] Dry substance (105 °C, 16 h) 90.0 - 96.0 %, GME

[0097] Ash (550 °C) < 1.5 %, USP / GME

[0098] pH (10 %, ambient temperatures) 5.5 - 6.5, Potentiometer

[0099] Viscosity (10 %, 25 °C) 1.10 – 2.00 mPa·s, GELITA method.

[0100] According to some embodiments, the collagen can be in the form of a gel, hydrogel, or dried, for example in powder form.

[0101] In certain embodiments the collagen, for example in powder form, contains hyaluronic acid which can be low molecular weight, for example from 10 to 499 kDa, medium molecular weight, for example from 500 to 2000 kDa, or high molecular weight, for example from 2000 to 10000 kDa.

[0102] In some embodiments the composition contains hydrogel based on collagen and hyaluronic acid. In some embodiments the collagen is cross¬ linked with hyaluronic acid using conventional technologies.

[0103] In hydrogels containing collagen and hyaluronic acid which are physically cross-linked, the collagen molecules aggregate owing to electrostatic and hydrophobic interactions to form fibrils, while thehyaluronic acid can be associated with the surface of the collagen fibrils or occupy the interstitial space and influence the microstructure and viscosity of the hydrogel.

[0104] The composition described herein contains hydrolyzed collagen according to any of the embodiments described herein and pyrroloquinoline quinone, a compound also known as PQQ or pyrroloquinoline-quinone 3- oxidoreductase and having the following chemical formula:

[0105]

[0106] PQQ is an enzyme belonging to the class of oxidoreductases, which catalyze the following reaction:

[0107] quinate + pyrroloquinoline-quinone ⇌ 3-dehydroquinate + reduced pyrroloquinoline-quinone.

[0108] Typically, PQQ is a cofactor with antioxidant activity.

[0109] According to the uses described herein, PQQ or a salt or esters thereof can be obtained as products available on the market and / or they can be produced using conventional methods.

[0110] Pyrroloquinoline quinone can be supplied and / or used in free form or in the form of a salt. Examples of salts of pyrroloquinoline quinone include alkali metal salts, alkaline earth metal salts, and ammonium salts thereof. An alkali metal salt is preferable.

[0111] Examples of alkali metal salts of pyrroloquinoline quinone used in the present invention are salts of sodium, potassium, lithium, cesium and rubidium. Salt of sodium or of potassium is preferred because it is easily obtainable.The alkali metal salt of pyrroloquinoline quinone can be an alkali metal salt with a substitution of from 1 to 3 alkali metals and may be any of the monoalkali metal salts, di-alkali metal salts, or tri-alkali metal salts. For example, it may be a di-alkali metal salt. In this case disodium salt or dipotassium salt are preferred. Hydrated crystals of di sodium salt are highly stable and suitable for the uses described herein.

[0112] According to some embodiments, the PQQ present in the composition of the invention has a titer higher than 99.5%, higher than 99.9%.

[0113] In some embodiments, the composition of the invention contains PQQ in a quantity comprised from 0.1 to 200 mg, from 1 to 100 mg, or from 5 to 20 mg.

[0114] The presence of PQQ in the composition has the twofold effect of promoting the synthesis of ATP, so stimulating mitochondrial activity, and increasing the production of cellular energy, so favoring anabolic processes like collagen synthesis.

[0115] Furthermore, the PQQ component of the composition inhibits the activity of degrading enzymes such as matrix metalloproteinases (MMPs, in particular MMP-1, MMP-9), which are responsible for the degradation of collagen in the extracellular matrix.

[0116] According to some embodiments, the composition of the invention contains PQQ in an amount comprised from 0.001 to 30% by weight, from 0.01 to 10% by weight, from 0.5 to 3% by weight, or from 0.1 to 1% by weight on the total weight of the composition.

[0117] Each of these amounts can be combined with any of the amounts of collagen described previously.

[0118] In the present invention, the term ’’synergism” or “synergistic activity" means an activity that is greater than the sum of the activities of the single active ingredient. In particular, synergism occurs when at least two substances interact in such a way that one boosts or increases one or more of their effects. As a consequence, two substances that produce similar effectssometimes produce increased effects when used simultaneously, and a quantitative evaluation is necessary to distinguish these cases from a straightforward additive action (Tallarida RJ. Drug Synergism: its detection and applications. J Pharmacol. Exp. Ther. 2001 Sept.: 298(3):865-72).

[0119] The biological effects underpinning the present invention are based on the modulation of the cellular repair and defence systems. The definition of synergism in biology is translated in terms of molecular events.

[0120] The composition for cosmetic or medical use described herein can be formulated with excipients suitable for oral administration or for topical application.

[0121] According to an aspect of the invention, a food supplement is provided for the uses according to claim 6 and / or 7.

[0122] According to certain embodiments, the food supplement contains low molecular weight hydrolyzed collagen from 0.5 to 100g, from 1 to 50g, preferably from 5 to 10 g per dose, preferably dispersed in a physiologically acceptable vehicle.

[0123] In some embodiments, the supplement may comprise one or more further biologically active substances or ingredients.

[0124] According to a preferred embodiment, the composition or the food supplement further contains nicotinamide adenine dinucleotide NAD or NADH. It has been observed that the presence of NAD or NADH in the composition and / or supplement results in an increase in the synergism of action found by combining low molecular weight hydrolyzed collagen and PQQ according to any of the embodiments described herein.

[0125] According to embodiments, the composition, the food supplement, the nutritional water or the topical preparation described herein can further contain a precursor and / or derivative of NAD or NADH chosen from the group consisting of nicotinamide riboside, nicotinamide mononucleotide, niacin, nicotinamide and mixtures thereof.

[0126] In some embodiments, the composition, the food supplement, thenutritional water or the topical preparation further comprises one or more vitamins, for example, vitamins in group B, folic acid, niacin, vitamin A, vitamin C, vitamin PP, the vitamins in group B being preferred According to some embodiments, the composition of the invention further comprises one or more trace elements and / or minerals such as salts of Mg, K, Na, Zn, Fe, Cr, Se, Mn and others, typically in the form of salts with inorganic salts, organic salts, and in amino-chelated forms.

[0127] The term "vehicle" as used in the present description indicates a medium, excipient, diluent with which the association of therapeutic or active ingredients is administered.

[0128] Any vehicle and / or excipient suitable for the desired form of preparation for administration to humans is envisaged for use with the compounds described in the present invention.

[0129] For the purposes of the present application, the term "physiologically acceptable" is used to mean edible substances that are approved by the health authorities for use and / or in the formulation of nutritional compositions or food compositions or nutraceuticals.

[0130] In the present application, the term "combination" means that one or more active ingredients are added or mixed with each other or with one or other ingredients.

[0131] The term "combination" must not be understood to mean that the active ingredients are associated with each other with the formation of bonds of a chemical nature, for example, covalent bonds or other types of bonds.

[0132] The compositions or supplements of the invention are suitable for foodstuff, nutritional, dietetic or pharmaceutical use in mammals, in particular in humans.

[0133] The composition, the food supplement and the topical preparation described herein may assume a wide variety of forms of preparation, according to the desired administration route.

[0134] The composition and the food supplement or topical preparation thatthey contain preferably are in solid form.

[0135] When the food supplement is presented in solid form it can be in the form of a tablet, a capsule, a powder, or granules.

[0136] Preparations in solid form can comprise one or more vehicles / carriers such as for example amides, sugars, micro-crystalline cellulose, and optionally diluents, granulation agents, lubricants, binding agents, di sintegrati on agents.

[0137] According to some embodiments the tablet, pill, capsule, granulate, or powder can also contain a binding agent such as tragacanth, acacia, com starch or gelatin; optionally excipients such as dicalcium phosphate; a disintegration agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharine. If desired, the tablets can be coated using conventional techniques.

[0138] When the unitary form is a capsule, it can contain, in addition to the materials of the type mentioned above, a liquid vehicle such as a fatty oil.

[0139] According to certain embodiments, the composition or food supplement of the invention contains a cellulose-based excipient that comprises i) organic esters of cellulose for example selected from cellulose acetate, cellulose propionate, cellulose triacetate, cellulose acetate propionate, cellulose acetate butyrate, ii) inorganic esters of cellulose for example chosen from nitrocellulose, cellulose sulfate, iii) esters of cellulose chosen from a) alkyl esters of cellulose for example selected from methylcellulose, ethyl cellulose, ethyl methyl cellulose; b) hydroxy alkyl esters of cellulose for example selected from hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, hydroxyethylethylcellulose; c) carboxy alkyl cellulose for example carboxymethylcellulose, salts and mixtures thereof. In certain embodiments the cellulose-based excipients are cross-linked with physiologically acceptable cross-linking agents.In the composition, food supplement or topical preparation there may also be flavoring agents, preservatives, coloring agents and the like.

[0140] In certain embodiments the composition or food supplement of the invention is in solid form, for example in granulate or powder form containing excipients preferably like those described. According to preferred embodiments, the composition of the inven tion is in granulate form.

[0141] According to certain embodiments, the composition or food supplement is in a form for modulated or controlled release of the active ingredients. By way of example the composition can be a retard release, rapid release or slow-fast release formulation. Preferably the modulated or controlled release formulations contain one or more cellulose-based excipients, in particular of the type described previously.

[0142] In certain embodiments the composition or food supplement of the invention comprises a hydrogenated fatty acid as an excipient, preferably having a chain with 3-20 carbon atoms, 14-18 carbon atoms. A typical example of a suitable hydrogenated fatty acid is hydrogenated palm oil.

[0143] When the composition or food supplement is presented in liquid form it can be in the form of a suspension, an emulsion, or a solution. In these cases the vehicle is liquid and can be chosen for example from water, glycols, oils, alcohols and mixtures thereof.

[0144] The food supplement in liquid form is preferably water-based.

[0145] In some embodiments, the active ingredients contained in the composition or food supplement or topical preparation of the present invention can be combined or mixed as active ingredients in an intimate mixture with a suitable edible vehicle and / or an excipient according to conventional pharmaceutical techniques and conventional food / nutrition industry.

[0146] The compositions can be suitably presented in a single pharmaceutical form and prepared using any of the methods that are well known in pharmaceutical or food technology.The amount of active compound in the compositions or food supplement of the invention is such as to obtain a dose that is preventive and effective in the reduction of signs of skin aging and in the regeneration of injured skin or dermis.

[0147] In some embodiments, the composition or food supplement of the invention further comprises one or more additional components such as additives, fillers, stabilizers, emulsifiers, texturizing agents, film-forming agents, plasticizers, wetting agents and thickeners.

[0148] Various other materials can be present as coatings or to modify the physical shape of the pharmaceutical unit. For example, tablets can be coated with shellac, sugar or both. To prevent disintegration during transit through the upper part of the gastrointestinal tract, the composition can be a formulation with an enteric or gastro-resistant coating.

[0149] In certain embodiments wherein the formulation is in liquid form, for example, in the case of an aqueous solution, a syrup or elixir, it can contain, in addition to the association of active ingredients, sucrose as a sweetening agent, methyl and propyl parabens as preservatives, a coloring agent, and a flavoring agent such as cherry or orange flavor.

[0150] In some embodiments, in the compositions and food supplements of the present invention, the active ingredients are normally formulated in dosage units. The dosage unit can contain low molecular weight hydrolyzed collagen from 0.5 to 100g, from 1 to 50g, or preferably from 5 to 10 g per dose and PQQ in an amount comprised from 0.1 to 200 mg, from 1 to 100 mg, or from 5 to 20 mg per dosage unit for administration one or more times per day, for example two times per day.

[0151] According to some embodiments, the composition of the invention is a topical preparation adapted to regenerate or heal cutaneous injuries or lesions.

[0152] The biologically active ingredients described previously contained in the composition of the present invention can be combined or mixed with avehicle or physiologically acceptable excipient by means of conventional techniques for the production of preparations for topical application.

[0153] The topical preparation can be in a solid physical state in any form suitable for external use, for example it can be in solid form, preferably cream, or semisolid, fluid, or semifluid, serum or gel.

[0154] Suitable topical preparations for cosmetic use in the treatment of skin blemishes or for medical use in the regeneration of skin are in solid form for example creams, ointments, pastes, lotions, salves, masks, paste, strips or patches.

[0155] In cosmetic use, the topical preparation preferably is in the form of cream, gel or serum for topical application on the skin, for example of the face, neck and neckline.

[0156] The topical preparation, for example in the form of a cream, can contain excipients that are compatible with the skin, for example oils, glycerin, moisturizers, emulsifiers, dispersants in suitable amounts for the formulation of creams.

[0157] In some embodiments the topical preparation for the cosmetic uses described herein can contain lipophilic substances or excipients, for example oils and butters, such as shea butter or coconut oil.

[0158] According to some embodiments, the topical preparations for cosmetic or medical use described herein can comprise one or more of thickeners, solubilizing agents, preservatives, water, alcohols, glycerin, stabilizers, antioxidants and physiologically acceptable antibacterial agents in amounts in line with those envisaged for common formulations for external use.

[0159] The following examples are provided for the purpose of illustrating the present invention.

[0160] EXAMPLE 1

[0161] A food supplement for treating skin blemishes or for promoting and accelerating the cutaneous healing of skin injuries or lacerations, containingthe following active ingredients:

[0162] PQQ: 5-20 mg per dosage unit;

[0163] Hydrolyzed collagen with average MW of 2kDa: 5-10g per dosage unit Excipients 0.5-3 g

[0164] EXAMPLE 2

[0165] Nutraceutical water with anti-aging activity, containing hydrolyzed collagen having one of the following formulations

[0166] FORMULA 1 FORMULA 2 FORMULA 3 FORMULA 4 FORMULA 5 INGREDIENT liquid liquid liquid liquid liquid liquid 500 liquid 500 mL 500 mL 500 mL mL 500 mL COLLAGEN 96% 5,000 mg

[0167] COLLAGEN 50% — 10,000 mg ii—

[0168] COLLAGEN + HYALURONIC —

[0169] ACID ~ 5,000 mg 5,000 mg 5,000 mg HYALURONIC ACID 30 mg 30 mg 30 mg VITAMIN C 80 mg 80 mg 80 mg 80 mg 80 mg M ETHYLSU LFONYLM ETHANE — __::....

[0170] (MSM) 1,500 mg

[0171] ZINC GLUCONATE __ — 10 mg SODIUM BENZOATE 2,500 mg 2,500 mg 2, SOO mg 2,500 mg 2,500 mg POTASSIUM SORBATE 2,500 mg 2,500 mg 2,500 mg 2,500 mg 2,500 mg

[0172] __

[0173]

[0174] CITRIC ACID q.s. — —

[0175] EXAMPLE 3

[0176] Hydrolyzed collagen-based formulation having one of the following compositions:

[0177] INGREDIENT 1 (%) 2 (%) 4 (%) 5 (%) 6 (%) DEMINERALIZED WATER 96.99 87.69 96.499 87.699 96.99 87.6 COLLAGEN 50% 0.5 10

[0178] COLLAGEN 96% - 0.5 10 - COLLAGEN (EYE)...... 0.5 10 WQ 0.01 0.01 0.001 0.001 0.01 0.1 PROPANEDIOL 1 1 1 1 1 1 TAMARINDUS INDICA SEED GUM 0.5 0.3 1 0.3 0.5 0.3 PRESERVATIVES 1 1 1 1 1 1

[0179]

[0180] TOTAL 100 100 100 100 100 100EXAMPLE 4

[0181] Human dermal fibroblasts (HDF) (ATCC, Manassas, VA, USA) were cultivated in a basal culture medium for fibroblasts integrated with the serum-free fibroblast growth kit (ATCC).

[0182] Fibroblasts are the most abundant type of cell in the dermal layer of the skin; they are also accessory cells in inflammatory response. Fibroblasts can produce or respond to a wide variety of cytokines and these mediators enable fibroblasts and leukocytes to cooperate during complex processes like the healing of injuries. Investigation into the effect of the compounds of the present invention on HDF cells provides information on how they can have an impact on skin aging and on the healing of injuries.

[0183] Collagen production

[0184] To monitor the collagen produced in supernatants of cellular cultures under different conditions, the Sircol Assay (Biocolor, Northern Ireland, UK) was conducted. Briefly, supernatants were prepared from HDF cells, unstimulated cells and cells treated with 10 pM of PQQ, 1 mg / ml of HcoL, PQQ plus HCoL or with PBS as a control. Cell-free supernatants were collected after 48 and 72 hours of incubation and evaluated for the total production of soluble collagen in accordance with the manufacturer's protocol.

[0185] The collagen synthesis was measured in the cells of human dermal fibroblasts. Total collagen production and secretion were significantly upregulated in the treated HDF cells compared to the cells in the untreated control.

[0186] These data show that the treatment with PQQ + HCOL has induced an increase of 4.8 times at 48 h and 4.4 times at 72 hours in collagen production compared to the cells in the untreated control (Figure 1).

[0187] Migration of fibroblast cells and closure of injuries

[0188] The actuation, proliferation and migration of fibroblasts are theprincipal stages in the healing of injuries, which involve many types of cells and other environmental micro-factors. The scratch assay is an in vitro technique used widely to understand the injury healing capacity of major compounds in medicine.

[0189] HDF cells were seeded in plates with 6 wells and cultured overnight. The cells were then washed with Delbucco’s Phosphate Buffered Saline (DPBS) and a scratch was made using 200μL of sterile pipette tip. Detached cells and other cellular debris were removed by washing the cells with DPBS. The cells were treated with 10 pM of PQQ, HCOL 1 mg / ml or PQQ + HCOL and incubated for 24 hours. The untreated cells were a negative control. The cellular migration and the morphological changes in the cells were observed in images taken with an inverted microscope, equipped with a digital camera.

[0190] The experiments were conducted in triplicate (n = 3). The width of the scratch and the closure of the injury at different time intervals (0, 12, 24 and 48 hours) were analyzed using the Image J software program.

[0191] The results indicated that the combination of PQQ+HCOL closed the gap created by the scratch by 94% in 48 hours. The percentage closure of the wound at different time intervals with PQQ alone, HCOL alone, or untreated cells is shown in Figure 2. The combination of PQQ+HCOL induced the migration of HDF cells with consequent closure of the wound.

[0192] ATP production

[0193] The ATP content was measured using a test based on luminometry (CellTiter-Glo® Luminescent Cell Viability Assay for ATP). The reagents of the kit were prepared in accordance with the manufacturer’s instructions. Identical volumes (50μl) of sample (which reaches a concentration of 100,000 cells / well) and working reagent were combined in a plate of 96 wells. Luminescence was measured using a Tecan Infinite 200 approximately 10 minutes after initial mixing, in accordance with the manufacturer's instructions. To rale out any possible interaction betweenPQQ and / or HCOL and the ATP working reagent, the test was conducted on solutions of PQQ and / or HCOL diluted in HBSS without cell lysates as a positive control, showing that: no variation in luminescence occurred with the increase in the concentrations of PQQ and / or HCOL. Before evaluating the ATP, the cells were treated for 2 hours with PQQ 1 p. M, PQQ 10 pM, HCOL 0.1 mg / ml, HCOL 1 mg / ml or PQQ 10 M + HCOL 1 mg / ml. The data show that the treatment with PQQ + HCOL induced an increase by 77% of ATP compared to the untreated control cells. PQQ alone, with the same dose, was much less effective (44%) while PQQ 1 1M and both concentrations of HCOL were not capable of increasing ATP production. EXAMPLE 5

[0194] Preliminary remarks

[0195] A key phase in the healing process is neovascularization, or angiogenesis, starting from existing blood vessels.

[0196] An evaluation was conducted of the formation of new tubules and the ramification of human aortic endothelial cells (HAEC) promoted by the combination composition according to an embodiment.

[0197] The data are expressed as average ± standard deviation (SD) and analyzed by means of one-way analysis of variance ([one-way] ANOVA). All the samples were compared with the controls and the experiments were conducted up to three times in duplicate or triplicate. A p value of less than 0.05 was considered significant in all cases.

[0198] The HAEC cells were seeded (0.3×10^5 / well) on a layer of Matrigel® (Coming, New York, New York) in a plate with 96 wells. After cellular adhesion, the HAEC cells were treated with 10 n PQQ, HCOL 1 mg / mL or PQQ + HCOL for 18 hours at 37 °C with 5% CO2; a negative control and a positive control with a vascular endothelial growth factor (VEGF, 30 ng / mL) were conducted in parallel.

[0199] Three images per well were acquired and evaluated for the formation of tubules and the ramification points using an inverted microscope(Olympus 1X50; Olympus, Tokyo, Japan). The group data were obtained by averaging three images per well and with three wells per condition.

[0200] The results indicated that the combination PQQ + HCOL increased both the ramification points and the tubules in the TIAEC cells, experimental evidence indicative of angiogenesis. The angiogenic response was comparable to that of the cells stimulated with VEGF, a potent angiogenic factor. The capacity of the formulation to induce angiogenesis is consistent with the clinical resolution of latent and persistent inflammation and with the buildup of collagen observed in the example presented.

[0201] An important aspect of pyrroloquinoline quinone (PQQ) is its capacity to support the cellular energy metabolism, at least partially through mitochondrial adjustment of the intracellular availability of NAD+

[0202] To investigate the mechanistic basis of this effect, we evaluated the modulatory interaction of PQQ with nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+salvage pathway in mammals, and a primary determiner of NAD+biosynthesis.

[0203] Analyses of molecular docking in silico show a show a high affinity interaction between PQQ and NAMPT, with an estimated binding energy of -9.4 kcal / mol. The envisaged bond shape structure has shown that several amino acid residues in NAMPT stabilize hydrogen-bonded and hydrophobic interactions with the PQQ, so stabilizing the ligand in the binding pocket. It is noteworthy that the identified pocket overlaps or lies adjacent to a previously reported allosteric region known to mediate enzymatic activation by known modulators of NAMPT. Consistent with this observation, the computational data support the hypothesis that PQQ can positively modulate NAMPT activity, thereby enhancing NAD+biosynthesis and downstream ATP producing pathways.

[0204] As a consequence, the formulation can comprise further precursors of NAD+and / or sublayers and cofactors of the NAD+pathway, including, by way of non-limiting example, nicotinamide adenine dinucleotide (NAD Q,nicotinamide riboside (ND), nicotinamide mononucleotide (NMN), niacin (nicotinic acid) and / or nicotinamide. In some embodiments, the presence of such NAD+precursors supports the maintenance of adequate NAD+pools, maximizing the bioenergetic effects associated with PQQ-mediated modulation of NAD metabolism.

[0205] The disclosures in Italian Patent Application No. 102025000002175 from which this application claims priority are incorporated herein by reference.

Claims

CLAIMS1. A composition comprising or consisting of low-molecular- weight hydrolyzed collagen in combination with pyrroloquinoline quinone in free form or in a physiologically acceptable salt or ester thereof, for use in the treatment of skin lesions, lacerations or wounds and / or for healing or repairing damaged skin.

2. The composition for use according to claim 1 wherein the hydrolyzed collagen has an average molecular weight from 0.5 to 6 kDa.

3. The composition for use according to claim 1 or 2 wherein the hydrolyzed collagen has an average molecular weight comprised from 1 to 4.5 kDa, preferably from 1.5 to 2.5 kDa.

4. The composition for use according to any one of claims 1 -3, further comprising nicotinamide adenine dinucleotide and / or a precursor and / or derivative thereof selected from the group consisting of nicotinamide riboside, nicotinamide mononucleotide, niacin, nicotinamide and mixtures thereof.

5. The composition for use according to any one of claims 1-4, further comprising hyaluronic acid.

6. A food supplement for oral administration for use in the treatment of skin lesions, lacerations or wounds and / or for healing or repairing damages skin, comprising a composition for use according to any one of claims 1-5 and a physiologically acceptable excipient.

7. The food supplement for use according to claim 6, further comprising one or more additional biologically active ingredients or biomolecules selected from the group consisting of vitamins, preferably B group vitamins, folic acid, niacin, vitamin A, vitamin C, vitamin PP and mixtures thereof, and / or one or more trace elements, preferably selected from the group consisting of salts of Mg, K, Na, Zn, Fe, Cr, Se, Mn and mixtures thereof.

8. A nutritional water for use in the treatment of skin lesions,lacerations or wounds and / or for healing or repairing damaged skin, comprising a composition for use according to any one of claims 1-5.

9. A topical preparation for use in the treatment of skin lesions, lacerations or wounds and / or for healing or repairing damaged skin, comprising a composition for use according to any one of claims 1-5 and a physiologically acceptable excipient.

10. The topical preparation for use according to claim 9 in the form of a cream, ointment, salve, topical dressing, patch or topical solution.

11. The topical preparation for use according to claim 9 or 10, for application to damaged skin.

12. The topical preparation for use according to any one of claims 9- 11, for accelerating the process of wound healing and closure of skin lesions and / or damaged skin.

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