WO2020153855A1 - Composition for use in the treatment of skin wound - Google Patents
Composition for use in the treatment of skin wound Download PDFInfo
- Publication number
- WO2020153855A1 WO2020153855A1 PCT/PL2019/000008 PL2019000008W WO2020153855A1 WO 2020153855 A1 WO2020153855 A1 WO 2020153855A1 PL 2019000008 W PL2019000008 W PL 2019000008W WO 2020153855 A1 WO2020153855 A1 WO 2020153855A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- quercetin
- skin
- ascorbic acid
- trans
- retinoic acid
- Prior art date
Links
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/203—Retinoic acids ; Salts thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
- A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
Definitions
- the present invention relates to pharmaceutical compositions and its medical use in skin wound treatment by healing - for use in enhancing skin wound healing.
- Quercetin is a polyphenolic flavonoid, known as one of the most abundant in human diet with the estimated daily intake of 3-40 mg [Andres et al., Molecular nutrition & food research, 2018, 62:1700447]. It is found in onions, leafy green vegetables, berries, red grapes, citruses, tomatoes, wine and tea. Quercetin glycosides, but not the quercetin aglycone, are normal dietary components. Quercetin displays antioxidant, anti inflammatory, and immunoprotective effects and its use as a supplement has been discussed in cardiovascular diseases, diabetes, and cancer prevention.
- Topically applied quercetin exerts anti-aging and anti-inflammatory actions on skin, though no transdermal penetration of quercetin has been determined [Hatahet et al., European Journal of Pharmaceutics and Biopharmaceutics, 2016, 108:41-53].
- quercetin may act as an epigenetic inhibitor. Quercetin has been reported to inhibit DNA methyltransferases and histone deacetylases [Lee et al., Molecular pharmacology, 2005, Alvarez et al., Clinical epigenetics, 2018, 10:139].
- vitamin C L- Ascorbic acid, known as vitamin C, and retinoic acid, a metabolite of vitamin A, are involved in multiple biological functions. Both vitamin A and vitamin C, are recognized for their importance in maintaining proper skin condition. The deficits of vitamin A and C have detrimental effects on skin condition, including dry and scaly skin in the case of the first and scurvy in the case of the latter one. Nevertheless, neither vitamin A nor C is used as a drug for wound treatment.
- Vitamin C is used to prevent and treat skin photodamage and wrinkling. In oral therapies along with vitamin E and zinc, it has been tried to treat pressure ulcers [Desneves et al., Clinical nutrition, 2005, 24:979-987] and burns [Barbosa et al., Journal of Burn Care & Research, 2009, 30:859-866].
- L-ascorbic acid The levels of L-ascorbic acid have been found to decrease rapidly at a wound site [Shukla et al., Free Radical Research, 1997, 26:93-101]. In human dermal fibroblasts, L-ascorbic acid induces a number of genes associated with DNA repair, proliferation and cell motility. [Duarte et al, Free Radical Biology and Medicine, 2009, 46:78-87]
- All trans-retinoic acid administered topically is used to treat acne and to reverse skin aging caused by ultraviolet radiation.
- All-trans retinoic is used topically in the form of creams, typically in the concentrations ranging from 0.01 to 0.1%. The concentration of all-trans retinoic acid of 0.001% has not been found effective [Bhawan et al., Archives of dermatology, 1991, 127:666-672].
- Retinoic acid-mediated signalling pathways are involved in immune homeostasis [Erkelens and Mebius, Trends in immunology, 2017, 38: 168-180] and skin physiology. Retinoids stimulate angiogenesis, collagen synthesis, fibroplasia and epithelialization [Hunt, Journal of the American Academy of Dermatology, 1986, 15:817-821, Elson, Journal of the American Academy of Dermatology, 1998, 39:S79-S81].
- Topical retinoids promote proliferation and differentiation of keratinocytes [Griffiths et al., New England Journal of Medicine, 1993, 329:530-535] Retinoids are known to regulate cellular differentiation and organism development [Cunningham and Duester, Nature reviews Molecular cell biology, 2015, 16:110] Their regenerative properties are connected with the activation of homeotic genes [Marshall et al., The FASEB Journal, 1996, 10:969-978]
- L-ascorbic acid and all-trans-retinoic acid are not listed as epigenetic modifiers, however, both have been demonstrated to promote DNA demethylation.
- L-Ascorbic acid and all-trans-retinoic acid stimulate active DNA demethylation; the first through the upregulation of TET dioxygenases activity by reducing cellular Fe 3+ to Fe 2+ [Hore et al., Proceedings of the National Academy of Sciences, 2016, 113:12202-12207], the latter, through RAR-mediated recruitment of TET/TDG complex [Hassan et al., Cell reports, 2017, 19:1685-1697]
- Epigenetic drugs act on the processes of DNA methylation, histone modifications and chromatin remodelling. Most epigenetic drugs are the inhibitors of DNA methyltransferases, histone methyltransferases, histone demethylases, histone acetyltransferases, and histone deacetylases. Presently, only two epigenetic drugs, the inhibitors of DNA methyltransferase, 5-azactidine and 5 -aza-2’ -deoxycytidine are in medical use for the treatment of myeloid malignancies.
- epigenetic drugs are being developed to treat not only cancers but other conditions including neurodegenerative diseases, autoimmune disorders, and chronic obstructive pulmonary disease [Heerboth et al., Genetics & epigenetics, 2014, 6:GEG. SI 2270]
- Epigenetic aspects of skin wound healing have been noticed [Lewis et al., Advances in wound care, 2014, 3:468-475] but they have not been widely investigated.
- the role of epigenetic mechanisms in dermal repair is poorly recognized and the use of epigenetic inhibitors to promote skin wound healing has not been extensively explored.
- the approved epigenetic drugs, 5-azacytidine and 5 -aza-2’ -deoxycytidine are nucleoside inhibitors of DNA methyltransferases and they may cause DNA mutations. While nucleoside inhibitors of DNA methyltransferases are potential mutagens, there is a number of non-nucleoside inhibitors of DNA methyltransferases, including several flavonoids of plant origin [Busch et al., Clinical epigenetics, 2015, 7:64, Kanwal et al., PLoS One, 2016, l l :e0162956], the demethylating activities of which were determined experimentally [Lee, Shim et al., Molecular pharmacology, 2005, Alvarez, Maso et al., Clinical epigenetics, 2018, 10:139]. One of such flavonoids is 3,3',4',5,7-pentahydroxyflavone, known as quercetin.
- the subject of the invention is to provide the composition for wound healing - combination of active substances.
- quercetin with at least L-ascorbic acid and in preferably embodiment of the invention quercetin with all-trans retinoic acid and L-ascorbic acid lead to a synergistic effect in enhancing skin wound healing.
- combination of quercetin with L-ascorbic acid and in the embodiment with all-trans retinoic acid provides therapeutic action based on the synergism of wound healing mechanisms characteristic of each of these two or three compounds.
- the invention relates to compositions comprising 3,3',4',5,7-pentahydroxyflavone, known as quercetin, and/or a pharmaceutically acceptable salt thereof, L-ascorbic acid known, as vitamin C, and/or a pharmaceutically acceptable salt thereof.
- the invention relates to compositions comprising 3,3',4',5,7-pentahydroxyflavone, known as quercetin, and/or a pharmaceutically acceptable salt thereof, all-trans-retinoic acid, a metabolite of vitamin A, and/or a pharmaceutically acceptable salt thereof, L-ascorbic acid, known as vitamin C, and/or a pharmaceutically acceptable salt thereof.
- the compositions may comprise at least one pharmaceutically acceptable carrier and/or diluent.
- the invention furthermore relates to the medical use of the composition - for promoting skin wound healing.
- composition is in the form of a topical formulation e.g. ointments, creams, gels, foams, sprays, or dressings, oils, subcutaneous injections.
- a topical formulation e.g. ointments, creams, gels, foams, sprays, or dressings, oils, subcutaneous injections.
- the composition for use in wound healing therapy according to the invention is designed to couple epigenetic inhibition with transcriptional activation.
- the composition is thought to combine quercetin as a natural compound, acting as a non-nucleoside inhibitor of DNA methyltransferase with L-ascorbic acid or all-trans-retinoic acid acting as transcriptional regulators and enhancers of active DNA demethylation. Epigenetic de-repression at different levels is expected to facilitate the activation of silenced genes responsible for healing processes.
- quercetin L-ascorbic acid and all-trans-retinoic acid to act synergistically has not been proposed as a wound healing composition.
- Retinoic acid acts as a signalling molecule, regulating a number of pathways involved in inflammatory responses and skin functioning. It plays a role in skin wound healing by stimulating angiogenesis, collagen production, fibrous tissue formation and keratinocyte proliferation and differentiation.
- L-ascorbic acid acts as an anti-oxidant limiting free radical damage and decreasing inflammatory responses. As a factor necessary for collagen hydroxylation, it stimulates collagen synthesis.
- Quercetin is as a potent anti-oxidant.
- the mechanism of quercetin action on skin wound healing has been associated with the induction of matrix metalloproteinase- land the inhibition of fibroblast proliferation leading to decreased deposition of extracellular matrix resulting in reduced scarring.
- Quercetin, L-ascorbic acid and all-trans-retinoic acid are known to have beneficial actions on skin condition. Quercetin, L-ascorbic acid and all-trans-retinoic acid were associated with different biological properties, different mechanism of action on skin and different effects.
- the beneficial effects of quercetin, all-trans-retinoic acid and L-ascorbic acid on skin condition are observed either after oral supplementation or topical application, prior to injury or after injury.
- One of the beneficial effects is improved or accelerated skin wound healing.
- quercetin, or all-trans-retinoic acid and or L- ascorbic acid delivered separately has a very limited usefulness for skin wound healing.
- the present invention is characterized in that quercetin, all-trans-retinoic acid and or L- ascorbic acid are delivered in combinations in order to integrate different healing actions so as to exert a synergistic effect on skin wound repair.
- the present invention is characterized in that quercetin, all-trans-retinoic acid and or L-ascorbic acid are delivered topically to enhance skin wound healing.
- quercetin all-trans-retinoic acid and or L-ascorbic acid is for use in enhancing skin wound healing after injury.
- the present invention is characterized in that quercetin, all-trans-retinoic acid and or L-ascorbic acid are delivered at different time-points after injury and during different phases of skin wound healing.
- quercetin, all-trans-retinoic acid and or L-ascorbic acid are natural compounds that are applied in small or moderate amounts, topically, directly onto the wound, during the limited time of skin wound healing, and therefore, the proposed way of use is safe for animals and humans.
- the present invention is characterized in that all-trans retinoic acid was applied at unexpectedly low dose of even 0.0001% (3.33 mM), and, unexpectedly, not in a hydrophobic formulation but in a water solution. At least this concentration of said active substance plays biological role in combination according to the invention.
- compositions according to the invention enhances healing of skin wounds resulting from mechanical injuries, chemical injuries, radiation injuries, burns, surgical operations or pathological conditions including diabetes complications such as diabetic foot ulcers, impaired circulation caused by prolonged pressure and resulting in pressure ulcers or peripheral artery disease.
- the pharmaceutical compositions is for used in healing of chronic and non-healing wounds, especially in the case of pressure ulcers, wounds resulting from diabetic complications and / or complications of peripheral arterial disease.
- Use of the combination of quercetin, all-trans-retinoic acid and L-ascorbic acid enables to enhance healing of wounds resulting from mechanical, chemical, thermal, radiation, surgical or pathological conditions.
- skin wound encompasses any injury of dermal tissue caused by mechanical factors including impact and pressure, radiation, chemical agents, heat, cold, ischemia, diabetes and other pathological conditions.
- wound healing refers to the process of repair following lesion which involves the debridement of foreign bodies and necrotic tissues within the wound area, secretion of acellular factors within the wound area, cell proliferation and migration, tissue remodelling that leads to the restoration of tissue continuity with or without scar formation.
- chronic wound or“non-healing wound” refers to a wound that does not show complete healing after a lapse of time considerably exceeding the usual time of normal healing. Typically, wounds that do not heal completely within 3 months are considered chronic. Most often, chronic wounds or non-healing wounds are diabetic and ischemic complications, such as diabetic foot ulcers and pressure ulcers, respectively, however, delayed wound healing may occur from other reasons.
- FIG. 1 Time line of skin wound healing in mice treated topically with quercetin compositions.
- FIG. 1 Representative photographs of dorsal skin wounds in mice treated topically with wound healing compositions
- Figure 6 Effects of topically applied composition of quercetin combined with all-trans- retinoic acid and L-ascorbic acid on the transcriptional activity of genes responsible for cell pluripotency, DNA methylation and DNA demethylation in the healing skin.
- the effect of topically delivered quercetin on skin wound healing was determined using the dorsal skin excision model.
- Female 2-month-old mice of the BALB/c strain were used for the experiment. The mice were anaesthetized, the dorsal skin was shaved and disinfected. Two full-thickness through-and-through symmetrical wounds were made in the dorsum using a 6 mm biopsy punch. Ten microliter of quercetin suspension (25 mM) in 1% L-ascorbic acid in saline was dropped onto each wound immediately after wounding. Promptly after quercetin application, the wounds were covered with a transparent dressing, and next, the dressing was fastened with adhesive plaster wrapped around the mouse.
- CpG methylation levels were determined in selected promoter regions of the Nanog and Pou5fl genes.
- the animals were sacrificed and skin samples were excised using 3 mm-biopsy punches from the central part of the wound. Methylation levels in selected genomic loci situated in promoter regions of the Nanog and Pou5fl genes were examined using methylation dependent restriction enzyme digestion.
- r l -(McrBC digested DNA/input DNA).
- composition containing quercetin, all-trans-retinoic acid and L-ascorbic on the expression of genes responsible for pluripotency and DNA methylation
- RNAlater Qiagen
- the tissue samples for the analysis were excised from the central part wound with a 3 -mm biopsy punch, following RNA extraction with an RNeasy Kit (Qiagen).
- the cDNA templates for PCR were synthesized in a reaction mix containing 200 ng of RNA, 100 pmoles of oligo dT2o, 200 units of Maxima Reverse Transcriptase (ThermoScientific Bio, Cat. No. EP0742) and 4 pL of 5 x concentrated reaction buffer in a final volume of 20 pL.
- Real-time PCR was performed in a final volume of 10 pL containing 1 pL of template cDNA, 5 pL of FastStart Essential DNA Green Master (Roche, Cat. No.
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Abstract
The invention relates to the composition comprising quercetin, all-trans-retinoic acid and L-ascorbic acid for use in skin wound treatment.
Description
Composition for use in the treatment of skin wound
The present invention relates to pharmaceutical compositions and its medical use in skin wound treatment by healing - for use in enhancing skin wound healing.
The process of skin wound healing is a natural response to injury. Skin injuries occur many times in lifetime. Effective wound repair is vital to normal body functioning. In most cases, small lesions heal fast. As a rule, the repair of incisional injuries is rapid and effective. However, if a substantial area of skin is lost or damaged, skin restoration in the affected area is more challenging and depends on the extent of lesion. Also, the type of injury impacts healing - burns and radiation injuries are usually more severe than the mechanical ones. A number of other conditions complicate dermal healing. Prolonged pressure on the skin, peripheral vascular disease and diabetes are the main causes of delayed and complicated wound healing. Pressure ulcers and diabetic ulcers are chronic wounds and a serious medical problem. Proper wound care and dressings help but is in many cases are insufficient to heal complicated wounds. While skin grafts or transplantation of dermal cells are demanding and expensive therapies, pharmacological solutions are cost-effective and simple to use. Unfortunately, there is a lack of effective medicaments enhancing skin wound healing.
Quercetin is a polyphenolic flavonoid, known as one of the most abundant in human diet with the estimated daily intake of 3-40 mg [Andres et al., Molecular nutrition & food research, 2018, 62:1700447]. It is found in onions, leafy green vegetables, berries, red grapes, citruses, tomatoes, wine and tea. Quercetin glycosides, but not the quercetin aglycone, are normal dietary components. Quercetin displays antioxidant, anti inflammatory, and immunoprotective effects and its use as a supplement has been discussed in cardiovascular diseases, diabetes, and cancer prevention. Topically applied quercetin exerts anti-aging and anti-inflammatory actions on skin, though no transdermal penetration of quercetin has been determined [Hatahet et al., European Journal of Pharmaceutics and Biopharmaceutics, 2016, 108:41-53]. Several studies report improved skin wound healing in normal rats [Gomathi et al., Biomaterials, 2003, 24:2767-2772, Gopalakrishnan et al., 2016, Ahmad et al., Pharmacognosy magazine, 2017, 13:S633, Jangde et al., International journal of biological macromolecules, 2018, 115:1211-1217], streptozocin-induced diabetic rats [Ahmad, Sultana et al., Pharmacognosy magazine, 2017, 13 :S633], and in pressure-ulcer lesion model in mice [Yin et al., Experimental
dermatology, 2018, 27:779-786]after topical delivery of quercetin in different formulations and concentrations ranging from 1 mM in phosphate buffer saline buffer to 33 mM in ointment. In addition, the effects of orally administered quercetin on skin wound healing in rats have been reported [Ahmad, Sultana et ah, Pharmacognosy magazine, 2017, 13:S633, Ahmed et al., Biomedicine & Pharmacotherapy, 2018, 101 :58-73] However, the experimental models used in these studies do not prevent wound contraction, therefore, the observed wound closure was mainly the result of wound shrinking. In addition, quercetin has been implicated to have a potential for reducing scars [Phan et al., Journal of Trauma and Acute Care Surgery, 2004, 57:1032-1037]. The mechanism of anti-scarring action of quercetin may be connected with induction of matrix metalloproteinase- 1 [Cho et al., International journal of molecular medicine, 2010, 25:347-352] leading to decreased deposition of extracellular matrix resulting in reduced scarring as well as with the antiproliferative effects of quercetin on fibroblasts [Pawlikowska-Pawlega and Gawron, Polish journal of pharmacology, 1995, 47:531-535] and keratinocytes [Fotsis et al., Cancer research, 1997, 57:2916-2921] As a number of other flavonoids, quercetin may act as an epigenetic inhibitor. Quercetin has been reported to inhibit DNA methyltransferases and histone deacetylases [Lee et al., Molecular pharmacology, 2005, Alvarez et al., Clinical epigenetics, 2018, 10:139].
L- Ascorbic acid, known as vitamin C, and retinoic acid, a metabolite of vitamin A, are involved in multiple biological functions. Both vitamin A and vitamin C, are recognized for their importance in maintaining proper skin condition. The deficits of vitamin A and C have detrimental effects on skin condition, including dry and scaly skin in the case of the first and scurvy in the case of the latter one. Nevertheless, neither vitamin A nor C is used as a drug for wound treatment.
L-Ascorbic acid has been reported to promote keratinocyte differentiation [Savini et al., Journal of investigative dermatology, 2002, 118:372-379] and to increase fibroblast proliferation [Phillips et al., Journal of investigative dermatology, 1994, 103]. Vitamin C is used to prevent and treat skin photodamage and wrinkling. In oral therapies along with vitamin E and zinc, it has been tried to treat pressure ulcers [Desneves et al., Clinical nutrition, 2005, 24:979-987] and burns [Barbosa et al., Journal of Burn Care & Research, 2009, 30:859-866]. The levels of L-ascorbic acid have been found to decrease rapidly at a wound site [Shukla et al., Free Radical Research, 1997, 26:93-101]. In human dermal
fibroblasts, L-ascorbic acid induces a number of genes associated with DNA repair, proliferation and cell motility. [Duarte et al, Free Radical Biology and Medicine, 2009, 46:78-87]
All trans-retinoic acid administered topically is used to treat acne and to reverse skin aging caused by ultraviolet radiation. All-trans retinoic is used topically in the form of creams, typically in the concentrations ranging from 0.01 to 0.1%. The concentration of all-trans retinoic acid of 0.001% has not been found effective [Bhawan et al., Archives of dermatology, 1991, 127:666-672]. Topical delivery of all-trans retinoic acid has been reported to accelerate contraction of dorsal kin wounds in rats [de Campos Peseta et al., Wound Repair and Regeneration, 2016, 24:411-417] Epidermal cells preferentially interact with all-trans retinoic acid [Fisher et al., Journal of investigative dermatology, 1998, 110:297-300] that could be either delivered by topical administration or synthesized in keratinocytes and fibroblasts from retinol (vitamin A) [Fisher et al., Archives of dermatology, 2002, 138: 1462-1470] supplied through dermal capillaries [Fisher and Voorhees, The FASEB Journal, 1996, 10:1002-1013] Retinoids, the metabolites of vitamin A, regulate a number of genes at the transcriptional level through interactions with their nuclear RAR receptors and cellular RXR receptors. Retinoic acid-mediated signalling pathways are involved in immune homeostasis [Erkelens and Mebius, Trends in immunology, 2017, 38: 168-180] and skin physiology. Retinoids stimulate angiogenesis, collagen synthesis, fibroplasia and epithelialization [Hunt, Journal of the American Academy of Dermatology, 1986, 15:817-821, Elson, Journal of the American Academy of Dermatology, 1998, 39:S79-S81]. Topical retinoids promote proliferation and differentiation of keratinocytes [Griffiths et al., New England Journal of Medicine, 1993, 329:530-535] Retinoids are known to regulate cellular differentiation and organism development [Cunningham and Duester, Nature reviews Molecular cell biology, 2015, 16:110] Their regenerative properties are connected with the activation of homeotic genes [Marshall et al., The FASEB Journal, 1996, 10:969-978]
L-ascorbic acid and all-trans-retinoic acid are not listed as epigenetic modifiers, however, both have been demonstrated to promote DNA demethylation. L-Ascorbic acid and all-trans-retinoic acid stimulate active DNA demethylation; the first through the upregulation of TET dioxygenases activity by reducing cellular Fe3+ to Fe2+ [Hore et al., Proceedings of the National Academy of Sciences, 2016, 113:12202-12207], the latter,
through RAR-mediated recruitment of TET/TDG complex [Hassan et al., Cell reports, 2017, 19:1685-1697]
Epigenetic drugs act on the processes of DNA methylation, histone modifications and chromatin remodelling. Most epigenetic drugs are the inhibitors of DNA methyltransferases, histone methyltransferases, histone demethylases, histone acetyltransferases, and histone deacetylases. Presently, only two epigenetic drugs, the inhibitors of DNA methyltransferase, 5-azactidine and 5 -aza-2’ -deoxycytidine are in medical use for the treatment of myeloid malignancies. A number of epigenetic drugs are being developed to treat not only cancers but other conditions including neurodegenerative diseases, autoimmune disorders, and chronic obstructive pulmonary disease [Heerboth et al., Genetics & epigenetics, 2014, 6:GEG. SI 2270] Epigenetic aspects of skin wound healing have been noticed [Lewis et al., Advances in wound care, 2014, 3:468-475] but they have not been widely investigated. The role of epigenetic mechanisms in dermal repair is poorly recognized and the use of epigenetic inhibitors to promote skin wound healing has not been extensively explored. The approved epigenetic drugs, 5-azacytidine and 5 -aza-2’ -deoxycytidine are nucleoside inhibitors of DNA methyltransferases and they may cause DNA mutations. While nucleoside inhibitors of DNA methyltransferases are potential mutagens, there is a number of non-nucleoside inhibitors of DNA methyltransferases, including several flavonoids of plant origin [Busch et al., Clinical epigenetics, 2015, 7:64, Kanwal et al., PLoS One, 2016, l l :e0162956], the demethylating activities of which were determined experimentally [Lee, Shim et al., Molecular pharmacology, 2005, Alvarez, Maso et al., Clinical epigenetics, 2018, 10:139]. One of such flavonoids is 3,3',4',5,7-pentahydroxyflavone, known as quercetin.
The subject of the invention is to provide the composition for wound healing - combination of active substances. Unexpectedly it was revealed that the combination of quercetin with at least L-ascorbic acid and in preferably embodiment of the invention quercetin with all-trans retinoic acid and L-ascorbic acid lead to a synergistic effect in enhancing skin wound healing. Unexpectedly, during the study it was shown that combination of quercetin with L-ascorbic acid and in the embodiment with all-trans retinoic acid provides therapeutic action based on the synergism of wound healing mechanisms characteristic of each of these two or three compounds.
The invention relates to compositions comprising 3,3',4',5,7-pentahydroxyflavone, known as quercetin, and/or a pharmaceutically acceptable salt thereof, L-ascorbic acid known, as vitamin C, and/or a pharmaceutically acceptable salt thereof. The invention relates to compositions comprising 3,3',4',5,7-pentahydroxyflavone, known as quercetin, and/or a pharmaceutically acceptable salt thereof, all-trans-retinoic acid, a metabolite of vitamin A, and/or a pharmaceutically acceptable salt thereof, L-ascorbic acid, known as vitamin C, and/or a pharmaceutically acceptable salt thereof. The compositions may comprise at least one pharmaceutically acceptable carrier and/or diluent.
The invention furthermore relates to the medical use of the composition - for promoting skin wound healing.
According to the embodiment the composition is in the form of a topical formulation e.g. ointments, creams, gels, foams, sprays, or dressings, oils, subcutaneous injections.
The composition for use in wound healing therapy according to the invention is designed to couple epigenetic inhibition with transcriptional activation. The composition is thought to combine quercetin as a natural compound, acting as a non-nucleoside inhibitor of DNA methyltransferase with L-ascorbic acid or all-trans-retinoic acid acting as transcriptional regulators and enhancers of active DNA demethylation. Epigenetic de-repression at different levels is expected to facilitate the activation of silenced genes responsible for healing processes. Each of these three compounds has already been reported to promote cutaneous repair through different mechanisms, but the combination of quercetin, L-ascorbic acid and all-trans-retinoic acid to act synergistically has not been proposed as a wound healing composition.
Retinoic acid acts as a signalling molecule, regulating a number of pathways involved in inflammatory responses and skin functioning. It plays a role in skin wound healing by stimulating angiogenesis, collagen production, fibrous tissue formation and keratinocyte proliferation and differentiation.
L-ascorbic acid acts as an anti-oxidant limiting free radical damage and decreasing inflammatory responses. As a factor necessary for collagen hydroxylation, it stimulates collagen synthesis.
Quercetin is as a potent anti-oxidant. The mechanism of quercetin action on skin wound healing has been associated with the induction of matrix metalloproteinase- land the
inhibition of fibroblast proliferation leading to decreased deposition of extracellular matrix resulting in reduced scarring.
Quercetin, L-ascorbic acid and all-trans-retinoic acid are known to have beneficial actions on skin condition. Quercetin, L-ascorbic acid and all-trans-retinoic acid were associated with different biological properties, different mechanism of action on skin and different effects. The beneficial effects of quercetin, all-trans-retinoic acid and L-ascorbic acid on skin condition are observed either after oral supplementation or topical application, prior to injury or after injury. One of the beneficial effects is improved or accelerated skin wound healing. Each of these three compound: quercetin, or all-trans-retinoic acid and or L- ascorbic acid delivered separately has a very limited usefulness for skin wound healing.
The present invention is characterized in that quercetin, all-trans-retinoic acid and or L- ascorbic acid are delivered in combinations in order to integrate different healing actions so as to exert a synergistic effect on skin wound repair.
Also, the present invention is characterized in that quercetin, all-trans-retinoic acid and or L-ascorbic acid are delivered topically to enhance skin wound healing.
Also, the present invention is characterized in that quercetin, all-trans-retinoic acid and or L-ascorbic acid is for use in enhancing skin wound healing after injury.
Also, the present invention is characterized in that quercetin, all-trans-retinoic acid and or L-ascorbic acid are delivered at different time-points after injury and during different phases of skin wound healing.
Also, the present invention is characterized in that quercetin, all-trans-retinoic acid and or L-ascorbic acid are natural compounds that are applied in small or moderate amounts, topically, directly onto the wound, during the limited time of skin wound healing, and therefore, the proposed way of use is safe for animals and humans.
Also, the present invention is characterized in that all-trans retinoic acid was applied at unexpectedly low dose of even 0.0001% (3.33 mM), and, unexpectedly, not in a hydrophobic formulation but in a water solution. At least this concentration of said active substance plays biological role in combination according to the invention.
Use of the compositions according to the invention enhances healing of skin wounds resulting from mechanical injuries, chemical injuries, radiation injuries, burns, surgical operations or pathological conditions including diabetes complications such as
diabetic foot ulcers, impaired circulation caused by prolonged pressure and resulting in pressure ulcers or peripheral artery disease. The pharmaceutical compositions is for used in healing of chronic and non-healing wounds, especially in the case of pressure ulcers, wounds resulting from diabetic complications and / or complications of peripheral arterial disease. Use of the combination of quercetin, all-trans-retinoic acid and L-ascorbic acid enables to enhance healing of wounds resulting from mechanical, chemical, thermal, radiation, surgical or pathological conditions.
Use of the combination of quercetin, all-trans-retinoic acid and L-ascorbic acid for the manufacture of medicaments in the form of ointments, creams, gels, foams, sprays, dressings, or injections.
The following terms used shall have the following meanings:
It is to be understood that the term“skin wound” encompasses any injury of dermal tissue caused by mechanical factors including impact and pressure, radiation, chemical agents, heat, cold, ischemia, diabetes and other pathological conditions.
As used herein the term“wound healing” refers to the process of repair following lesion which involves the debridement of foreign bodies and necrotic tissues within the wound area, secretion of acellular factors within the wound area, cell proliferation and migration, tissue remodelling that leads to the restoration of tissue continuity with or without scar formation.
As used herein the term“chronic wound” or“non-healing wound” refers to a wound that does not show complete healing after a lapse of time considerably exceeding the usual time of normal healing. Typically, wounds that do not heal completely within 3 months are considered chronic. Most often, chronic wounds or non-healing wounds are diabetic and ischemic complications, such as diabetic foot ulcers and pressure ulcers, respectively, however, delayed wound healing may occur from other reasons.
The invention is described in detail in the example and drawing as follows:
Figure 1. Time line of skin wound healing in mice treated topically with quercetin compositions.
Figure 2. Representative photographs of dorsal skin wounds in mice treated topically with wound healing compositions
Figure 3. Compared effects of the tested compositions on skin wound healing
Figure 4. Effect of quercetin concentration on skin wound healing in the mouse.
Figure 5. Effects of topically applied quercetin compositions on DNA methylation status in gene promoters in healing skin.
Figure 6. Effects of topically applied composition of quercetin combined with all-trans- retinoic acid and L-ascorbic acid on the transcriptional activity of genes responsible for cell pluripotency, DNA methylation and DNA demethylation in the healing skin.
The invention is further illustrated by the following non- limiting examples.
Example 1
The effect of topically delivered quercetin on skin wound healing
The effect of topically delivered quercetin on skin wound healing was determined using the dorsal skin excision model. Female 2-month-old mice of the BALB/c strain were used for the experiment. The mice were anaesthetized, the dorsal skin was shaved and disinfected. Two full-thickness through-and-through symmetrical wounds were made in the dorsum using a 6 mm biopsy punch. Ten microliter of quercetin suspension (25 mM) in 1% L-ascorbic acid in saline was dropped onto each wound immediately after wounding. Promptly after quercetin application, the wounds were covered with a transparent dressing, and next, the dressing was fastened with adhesive plaster wrapped around the mouse. The procedure was repeated daily for the next four days. The experiment involved 6 mice. The animal study protocol was approved by the Local Ethics Committee for Animal Experimentation at the University of Science and Technology in Bydgoszcz, Poland (Permit No. 49/2016). Wound closure was photographically documented. The photographs were taken at eight time points: on the day of injury (dO) and on the day 2, 4, 7, 9, 11, 14, and 18 after injury. Wound areas were determined using computer-assisted image analysis. As demonstrated in Figure la, quercetin combined with L-ascorbic acid accelerated skin wound closure.
Example 2
Synergistic potentiation of quercetin (QR) effect on skin wound healing with all-trans retinoic acid (RA) and L-ascorbic acid (AA).
Analogous experiments as that described in Example 1 were performed for other compositions: L-ascorbic acid 1% alone, all-trans retinoic acid 3.33 mM with L-ascorbic acid 1%, and the combination of L-ascorbic acid 1%, all trans-retinoic acid 3.33 mM and quercetin 25 mM. Saline was used as vehiculum. While L-ascorbic acid either alone (Figure lb) or combined with all-trans-retinoic acid (Figure lc) or with quercetin (Figure la) moderately accelerated skin wound closure, the composition containing, quercetin combined with L-ascorbic acid and all trans-retinoic acid showed a synergistic effect resulting in significant healing enhancement (Figure Id). Representative photographs of skin wound healing effects are demonstrated in Figure 2. To compare wound closure effects for the tested compositions, the results were presented as the percentage differences in wound area relative to the controls receiving vehiculum only (Figure 3). This combined action of quercetin, all-trans-retinoic acid and L-ascorbic acid was the most accentuated on the day of 9 and 11 after injury, when the mean wound area was decreased relative to the controls by 39 and 43%, respectively.
Example 3
The effect of quercetin concentration on skin wound healing and the synergism of quercetin and L-ascorbic acid in combination - composition according to the invention.
An analogous experiment as that described in Example 1 was performed using a higher concentration of quercetin of 250 mM in 1% L-ascorbic acid. Saline was used as vehiculum. The effects on mouse wound closure were compared with those using 25 mM quercetin in 1% L-ascorbic acid. The application of a tenfold higher quercetin concentration improved significantly the results of skin wound closure (Figure 4). In order to compare healing effects for the tested quercetin concentrations, wound closure data were presented as the percentage differences in wound area relative to the controls receiving vehiculum only (Figure 4c). Both 25 mM and 250 mM quercetin significantly improved skin wound healing, yet the latter one was more effective. The use of a tenfold higher concentration of quercetin resulted in an over twofold decrease in wound area on the day 11 and 14 after injury as compared to 25 mM quercetin. The application of 250 mM quercetin with L-ascorbic acid showed the synergistic effect of these two compounds as demonstrated by the comparison with the results for L-ascorbic acid administered alone (Figure 4c). The combined use of 250 mM quercetin with 1% L-ascorbic acid resulted in an
over threefold decrease in wound area on the day 14 after injury as compared to 1% L- ascorbic acid applied alone. The combination of the two active compound in the composition has stronger treatment affect than both compound administered separately.
Example 4
The effects of quercetin, all-trans-retinoic acid and L-ascorbic on DNA methylation levels in selected gene promoter regions
In order to examine the effects of the topically applied compositions of quercetin, all-trans-retinoic acid and L-ascorbic acid described in Example 1 on DNA methylation status in healing skin in mice, CpG methylation levels were determined in selected promoter regions of the Nanog and Pou5fl genes. On the day 18 after injury described in Example 1, the animals were sacrificed and skin samples were excised using 3 mm-biopsy punches from the central part of the wound. Methylation levels in selected genomic loci situated in promoter regions of the Nanog and Pou5fl genes were examined using methylation dependent restriction enzyme digestion. A 100-ng aliquot of DNA extracted with a DNeasy kit (Qiagene) was treated with McrBC CpG-methylation dependent enzyme (New England Biolabs, Cat. No. M0272S) in a final volume of 10 pL. After digestion, the DNA samples were 10-fold diluted and used as the templates for PCR quantitation. Real time PCR was carried out in a final volume of 10 pL containing 5 pL of FastStart Essential DNA Green Master (Roche, Cat. No. 06402712001), 2 pL of DNA template, and 0.25 pL each of forward and reverse primers (10 pM) on a LightCycler LC96 (Roche). Gene methylation levels were determined using the 2~&Ct method. The relative CpG methylation (r) was calculated according to the following formula:
r=l -(McrBC digested DNA/input DNA).
PCR was performed in triplicate. The primer sequences are listed in Table 1.
The results are presented in Figure 5. Quercetin, all-trans-retinoic acid and L-ascorbic are potentially DNA demethylating agents. However, no significant reductions in CpG methylation levels in the promoter regions of the Nanog and Pou5fl were determined in the healing mouse skin after topical administration of the tested compositions in comparison with that treated with vehiculum (saline). On the contrary, a moderate increase in CpG methylation levels in the Nanog promoter regions was found in the skin after treatment with the composition containing 25 pM quercetin and 1% L-
ascorbic acid. The increase was suppressed for the combination of quercetin with all-trans- retinoic acid. Although the changes in CpG methylation levels in the examined loci were not large, their occurrence indicated possible DNA methylation re-patterning in wound area after treatment with quercetin.
Example 5
The effects of composition containing quercetin, all-trans-retinoic acid and L-ascorbic on the expression of genes responsible for pluripotency and DNA methylation
The effects of topical delivery of composition containing the combination of quercetin 25 mM, all-trans-retinoic acid (3.3 mM) and L-ascorbic (1%) on the expression of key genes determining cell pluripotency (Myc, Klf4, PouSfl, Sox2, Nanog ), DNA methylation ( Dnmtl , Dnmt3a, Dnmt3b ), and DNA demethylation processes (Tell, Tet2, Tet3, Tdg ) in healing skin were examined using real-time PCR quantitation. On the day 18 after injury experiment as described in Example 1, the mice were sacrificed, skin samples were preserved in RNAlater (Qiagen), and stored at -80°C. The tissue samples for the analysis were excised from the central part wound with a 3 -mm biopsy punch, following RNA extraction with an RNeasy Kit (Qiagen). The cDNA templates for PCR were synthesized in a reaction mix containing 200 ng of RNA, 100 pmoles of oligo dT2o, 200 units of Maxima Reverse Transcriptase (ThermoScientific Bio, Cat. No. EP0742) and 4 pL of 5 x concentrated reaction buffer in a final volume of 20 pL. Real-time PCR was performed in a final volume of 10 pL containing 1 pL of template cDNA, 5 pL of FastStart Essential DNA Green Master (Roche, Cat. No. 06402712001), 0.25 pL of forward primer (10 pM) and 0.25 pL of reverse primer (10 pM) on a LightCycler LC96 (Roche). The transcript levels were calculated using the 2~ACt method relative to Actb and Tbp. The PCR primer sequences are listed in Table 1. PCR was performed in triplicate.
The results are presented in Figure 6. A remarkable increase in the transcriptional activity of pluripotency genes was observed in the wound area in the skin after topical administration of the composition containing the combination of quercetin 25 pM, all- trans-retinoic acid (3.3 pM) and L-ascorbic acid (1%) in comparison with that treated with vehiculum (saline). Significant enhancement was found for the Nanog and Sox2 genes (Figure 6a). This result strongly suggested that the applied composition activated regenerative processes in the skin. The use of the same composition had a marked impact
on the expression profiles of genes responsible on DNA methylation and DNA demethylation. While the first ( Dnmtl , Dnmt3a, Dnmt3b ) showed an increased activity, the latter ( Tetl , Tet2, Tet3 ) displayed a decrease in expression. These observations indicated that the topical application of quercetin combined with all-trans-retinoic acid L-ascorbic is promoted a different course of epigenetic re-patterning in healing skin.
Table 1. The sequences of PCR primers used in the experiments.
Claims
1. The pharmaceutical composition comprising as active component: quercetin, all- trans retinoic acid and L-ascorbic acid for use in the treatment of skin wound.
2. The pharmaceutical composition for use according to the claim 1, wherein it
comprises at least one pharmaceutically acceptable carrier and/or diluent.
3. The pharmaceutical composition for use according to the claim 1, wherein the active component is in water solution or saline.
4. The pharmaceutical composition for use according to the claim 1 or 2 or 3, wherein the composition is in topical formulation.
5. The pharmaceutical composition for use according to the claim 1 or 2 or 3 or 4, wherein the all-trans retinoic acid is in concentration of at least 3 mM.
6. The pharmaceutical composition comprising as active component: quercetin and L- ascorbic acid for use in the treatment of skin wound.
7. The pharmaceutical composition for use according to the claim 6, wherein it
comprises at least one pharmaceutically acceptable carrier and/or diluent.
8. The pharmaceutical composition for use according to the claim 6, wherein the active component is in water solution or saline.
9. The pharmaceutical composition for use according to the claim 6 or 7 or 8, wherein the composition is in topical formulation.
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