WO2018015976A1 - Nouvelle formule pour la cicatrisation rapide des plaies et le contrôle de l'infection - Google Patents

Nouvelle formule pour la cicatrisation rapide des plaies et le contrôle de l'infection Download PDF

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Publication number
WO2018015976A1
WO2018015976A1 PCT/IN2017/050301 IN2017050301W WO2018015976A1 WO 2018015976 A1 WO2018015976 A1 WO 2018015976A1 IN 2017050301 W IN2017050301 W IN 2017050301W WO 2018015976 A1 WO2018015976 A1 WO 2018015976A1
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WO
WIPO (PCT)
Prior art keywords
formulation
wound healing
wound
control
gel
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Application number
PCT/IN2017/050301
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English (en)
Inventor
Milind CHOUDHARI
Original Assignee
Weinnovate Biosolutions Pvt. Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Weinnovate Biosolutions Pvt. Ltd. filed Critical Weinnovate Biosolutions Pvt. Ltd.
Priority to GB1900381.3A priority Critical patent/GB2566235B/en
Publication of WO2018015976A1 publication Critical patent/WO2018015976A1/fr

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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
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/38—Silver; Compounds thereof

Definitions

  • Present invention relates to a novel organic and inorganic nano-formulation for antimicrobial activity and wound healing. More particularly the invention relates to a formulation of, antibacterial agent, antioxidant agents and an inducer of endothelial cell proliferation to be used in gel, cream, spray, liquid solution and bandages for wound care.
  • Wound care is one of the major factors in controlling the infection.
  • Such wound dressings are manufactured with a sole goal of preventing infections.
  • the management of wound varies according to the category of the wounds, which includes diabetic wounds, pressure wounds/ ulcers; venous ulcers, skin burns wounds, post traumatic ulcers, and general surgery.
  • diabetic foot ulcers pressure wounds and venous ulcers are considered as critical wounds and result into necrosis, impaired vascularization, improper fibroblast formation and alleviated MMP2 levels.
  • US 8686051 B2 discloses a pharmaceutical composition for treatment of diabetic complications by administration of beta-blockers.
  • the invention further discloses employing a topical formulation of a beta-blocker, having substantially no antibacterial activity, to improve the process of diabetic wound healing.
  • the composition as disclosed in the cited patent takes care of the diabetic wound and is responsible only for wound healing.
  • the formulation of the present invention is a mixture of organic and inorganic components and provides antibacterial effect, safeguard from reactive oxygen species (ROS) as well as rapid wound healing properties.
  • ROS reactive oxygen species
  • the primary object of the invention is to develop an organic and inorganic nano- formulation for rapid wound healing and antimicrobial activity.
  • Another object of the present invention is to develop an organic and inorganic nano-formulation having a synergistic effect of Gallic Acid, Sphingosine-1- Phosphate, Silver nanoparticles
  • Yet another object of the present invention is to eradicate the need of amputation in turn minimizing the disability.
  • Another object invention is to reduce the toxicity of silver, achieved by addition of Gallic acid which masks the silver toxicity.
  • Still another object of the invention is to develop an organic and inorganic nano- formulation is to make it into liquid solution, cream/gel and biodegradable scaffolds.
  • Another object of invention is the formulation using antibacterial, antioxidant and cell proliferating molecules for rapid healing of wounds.
  • the formulation of the present invention provides a holistic solution to issues associated with wound healing and consists of 3 components organic (Sphingosine 1 phosphate), inorganic (Silver nanoparticles) and an antioxidant agent (Gallic Acid) and provides a nano-formulation for rapid wound healing and control of infection.
  • the formulation is developed in such a way that the mixing the components in appropriate proportions gives an antimicrobial and wound healing effect in a synergistic way. Additionally the composition also helps in faster wound healing by the way of inducing the cell proliferation, blood vessel formation and collagen matrix formation.
  • Silver nanoparticles act as effective antibacterial agent at the wound site.
  • Sphingosine-l-Phosphate is a lipid molecule, which helps in proliferation of the endothelial cells required at the wound area.
  • Gallic acid is used to reduce the oxidative stress at the wound area.
  • the present invention discloses an organic and inorganic nano-formulation for rapid wound healing and control of infection.
  • the said compositions are designed to offer improved advantages with respect to wound healing along with control of infection.
  • the nano-formulation is prepared by adding sphingosine-1- phosphate (10 ⁇ - 1 mM/ g of gel), Silver Nanoparticles (0.8 mg/g - 1.5 mg/g of gel), Gallic acid (1 mg/g - 5 mg/g of gel)
  • Ki67 expression in wound skin sections formalin-fixed paraffin- embedded tissue are cut in sections of 5-7 ⁇ thickness. The sections are deparaffinized, blocked, and incubated with monoclonal Ki-67 primary antibody (1:50 dilution) for 1 h. The sections are then incubated with HRP-lgG (1:200 dilution) for 1 h, followed by incubation with 3,3'-diaminobenzidine hydrochloride substrate for 30 min in the dark. Cells were counterstained using hematoxylin (1:50 dilution in distilled water) for 1 h. Slides were then imaged with an optical microscope. For semi quantitation, the Ki67 cells were counted from three different fields across all groups.
  • Histopathological analysis of the skin at wound site is performed for healing process with presence of inflammatory cells, neutrophils, macrophages, proliferation of granulation tissue comprised of fibroblasts and neovascularization process and collagen deposition in the healing process.
  • the inflammatory cells neutrophils and macrophages were more in diseased control as compared with the standard control and the current formulation.
  • the presence of inflammatory cells confirms prolong inflammation in control group which delays wound healing.
  • the presence of these cells shows an early stage of wound healing in diseased control rats while in the rats treated with the current formulation the inflammation had reduced indicating the progress of wound healing.
  • Granulation tissue, proliferation of fibroblast cells and Collagen deposition was more in the current formulation than in control and standard gel.
  • the Collagen deposition was more compact in the group of rats treated with the current formulation. In the control group the deposition is in patches and not in a compact manner.
  • Collagen is an important protein produced by fibroblast cells and helps in healing of the wound. Further confirmation of collagen deposition is done by using
  • Masson's trichrome stain which stains specifically the collagen. It is observed that rats treated with the current formulation shows good deposition of Collagen as opposed to Standard and diseased control. This staining confirms the histology results.
  • Sphingosine-l-Phosphate (1 mM), Gallic acid (5 mg) and were added to 100 ml of Silver nanoparticles (SNP) 1100 ppm .
  • Carbopol (0.5 %) added to the solution with continuous stirring on a magnetic stirrer. The solution was neutralized by Triethanolamine.
  • base gel preparation 0.5 % Carbopol-934 was added to 100 ml deionized water with continuous stirring, the solution was neutralized with Triethanolamine. The gels were stored at 4°C till their further use.
  • Sphingosine-l-Phosphate 100 ⁇
  • Gallic acid 2.5 mg
  • SNP Silver nanoparticles 1500 ppm
  • Carbopol 0.5 % added to the solution with continuous stirring on a magnetic stirrer.
  • the solution was neutralized by Triethanolamine.
  • base gel preparation 0.5 % Carbopol-934 was added to 100 ml deionized water with continuous stirring, the solution was neutralized with Triethanolamine. The gels were stored at 4°C till their further use.
  • Antimicrobial activity is checked against seven common wound pathogens by performing well plate method (Fig. 1).
  • the data shows larger zone of inhibition when the formulation of the present invention applied as compared to silver nano-particles, Gallic acid, sphingosine-l-phosphate and collagen hydrolysates applied alone.
  • fibroblast cells are grown in a well and a scarring is performed onto the cells followed by treatment with novel formulation solution.
  • Fig 3 represents that with novel formulation gap is filling fast as compared to control.
  • the antioxidant used in the formulation is Gallic acid.
  • the release of Gallic acid is measured by standard micro well plate assay (Modified McDonald method) and the plate was read at 690 nm.
  • the gel formulation showed release of Gallic acid till 3 rd week. Release of Gallic acid on day 1 increased with increase in time. (Fig. 5) Excision wound model:
  • Rats were anesthetized with Xyalzine (22-24 mg/kg) and Ketamine (10-12 mg/kg, ip) and an area of about 330 ⁇ 10mm 2 was marked on the dorsal side of the rat. The marked skin was cut carefully. During the study the wound areas were measured on 1mm 2 gra ph paper on days 0, 3, 6, 9, 12 ,15 and 18 for all groups.
  • Percent wound contraction (Healed area)/ (Initial wound area) x 100
  • Fig. 1 is a graphical representation of antimicrobial activity of the novel formulation against the common infection causing bacteria
  • Fig. 2 is a representative of the toxicity of each component in the novel formulation on endothelial cells
  • Fig.3 shows the fibroblast migration when the wound is administered with just lipid formulation, Sphingosine-l-phosphate ( ⁇ ) and Sphingosine-l-phosphate ( ⁇ . ⁇ )
  • Fig. 4 shows the quantitative data of fibroblast migration against different concentrations of Sphingosine-l-phossphate and lipid formulation
  • Fig. 5 shows antioxidant assay of Gallic acid used in the formulation
  • Fig. 6 shows wound healing in natural conditions i.e. control
  • Fig.7 shows wound healing after application of novel formulation
  • Fig. 8 shows wound closure percentage in vivo in excision wound model in rats
  • Fig. 9 and 10 represents levels of Lipid peroxidation and Catalase in the wound area.
  • Fig. 11 is Photomicrographs of histology from wound site Detailed description of the drawings:
  • Fig. 1 further explains the antimicrobial activity of each of the components in the novel formulation along with novel formulation, Iodine Tincture and Ethanol against the common infection causing bacteria such as E.coli, E. cloacae, P. mirabilis, S. aureus, S. marcescens, P. aeruginosa, S. neumoniae ⁇ using well plate method which shows larger zone of inhibition when treated with novel formulation as compared to single component such as to silver nano-particles, Gallic acid, sphingosine-l-phosphate and collagen hydrolysates
  • Fig 2 further explains the toxicity of Silver nanoparticles on Human cell lines (Human umbilical cord endothelial cells) after adding individual components such as silver nanoparticles, Gallic acid and sphingosine-l-phoshpate and the novel formulation
  • Fig.3 explains fibroblast migration in the wound healing process after regular intervals of 24 hours to 36 hours after the onset of wound.
  • the gap fills fast i.e. the fibroblast migration happens fast
  • Fig. 4 explains the quantitative data represented in a graph showing the difference between the novel formulation and control was significant when SIP is added at ⁇ concentration which is responsible for fibroblast migration and also for proliferation
  • Fig.5 further explains the release of Gallic acid as an indicator of stability of the gel over time.
  • the release pattern of Gallic acid is not changing with time over 3 weeks.
  • the Gallic acid release over the period of 300 min is increasing indicating a sustained release from the gel
  • Fig.6 represents the control rat wound healing, excision wound was formed and representative images were taken on 0, 6, 12 and 18 days. Wound healing is observed, however on the 16 th day a little scar observed.
  • Fig.7 represents wound closure in the test rat Fig.8 Current formulation showed a significant increase in wound healing (**p ⁇ 0.01) as compared to control.
  • Fig. 9 shows the levels of LPO in rats treated with current formulation was lesser than the rats treated with standard gel. This justifies that the current formulation is effective against the oxidative stress generated during the wound healing procedure.
  • Fig.10 show that the levels of catalase were increased significantly for current formulation treated groups
  • Fig. 11 shows that the inflammatory cells neutrophils and macrophages were more in diseased control as compared with the standard control and current formulation.
  • the presence of inflammatory cells confirms prolong inflammation in control which delays wound healing.
  • the presence of these cells shows an early stage of wound healing in diseased control rats while in the rats treated with the current formulation, inflammation has reduced indicating the progress of wound healing.
  • Granulation tissue, proliferation of fibroblast cells and Collagen deposition is more in current formulation than in control and standard gel used (Panel 1 horizontal).
  • the Collagen deposition is more compact in the group of rats treated with current formulation.
  • the deposition is in patches (Panel 2 horizontal). Further confirmation of collagen deposition is done by using Masson's trichrome stain which stains specifically the collagen.
  • LPO Lipid Peroxidase
  • ROS Reactive oxygen species
  • the novel formulation helps the wound to heal holistically faster by addressing bacterial infection, ROS at wound site and angiogenesis

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Inorganic Chemistry (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

La présente invention est une formule de composants organiques et non organiques pour la cicatrisation rapide des plaies et le contrôle de l'infection. Les composants de la présente invention, c'est-à-dire la formule, sont un agent antimicrobien, un antioxydant, un facteur induisant la prolifération cellulaire et un composant organique responsable de la formation de la matrice. En ajoutant ces quatre composants dans des proportions prédéterminées, il est possible d'obtenir des formules pour des gels, une solution liquide et des échafaudages biodégradables qui sont sûrs et donnent un effet synergique.
PCT/IN2017/050301 2016-07-22 2017-07-22 Nouvelle formule pour la cicatrisation rapide des plaies et le contrôle de l'infection WO2018015976A1 (fr)

Priority Applications (1)

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GB1900381.3A GB2566235B (en) 2016-07-22 2017-07-22 A novel formulation for rapid wound healing and control of infection

Applications Claiming Priority (2)

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IN201621025112 2016-07-22
IN201621025112 2016-07-22

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019152766A1 (fr) * 2018-02-01 2019-08-08 North Carolina State University Particules de type plaquettes antimicrobiennes
CN111588837A (zh) * 2020-05-14 2020-08-28 中南大学湘雅三医院 一种蛋白-纳米银复合物的制备方法及制备的复合物和用途
CN116327684A (zh) * 2023-03-20 2023-06-27 南京农业大学 一种常山酮-银纳米温敏凝胶及其制备方法与应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7678393B1 (en) * 2006-04-23 2010-03-16 DB Laboratories LLC Mixture composition and method useful for topical and internal application
WO2014147638A1 (fr) * 2013-03-22 2014-09-25 Datt Mediproducts Limited Matrice de cicatrisation de plaie naturelle multifonctionnelle

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Publication number Priority date Publication date Assignee Title
EP2218447B1 (fr) * 2008-11-04 2017-04-19 PharmaSol GmbH Compositions contenant des micro ou nanoparticules lipides pour l'amélioration de l'action dermique de particules solides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7678393B1 (en) * 2006-04-23 2010-03-16 DB Laboratories LLC Mixture composition and method useful for topical and internal application
WO2014147638A1 (fr) * 2013-03-22 2014-09-25 Datt Mediproducts Limited Matrice de cicatrisation de plaie naturelle multifonctionnelle

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HSINYU LEE ET AL.: "Lysophosphatidic acid and sphingosine 1-phosphate stimulate endothelial cell wound healing", AM. J. PHYSIOL. CELL PHYSIOL., vol. 278, 2000, pages C612 - C618, XP055455290 *
THIRUMURUGAN GUNASEKARAN ET AL.: "Silver nanoparticles as real topical bullets for wound healing", JOURNAL OF THE AMERICAN COLLEGE OF CLINICAL WOUND SPECIALISTS, vol. 3, 2012, pages 82 - 96 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019152766A1 (fr) * 2018-02-01 2019-08-08 North Carolina State University Particules de type plaquettes antimicrobiennes
US11419892B2 (en) 2018-02-01 2022-08-23 North Carolina State University Antimicrobial platelet-like particles
CN111588837A (zh) * 2020-05-14 2020-08-28 中南大学湘雅三医院 一种蛋白-纳米银复合物的制备方法及制备的复合物和用途
CN111588837B (zh) * 2020-05-14 2023-06-06 中南大学湘雅三医院 一种蛋白-纳米银复合物的制备方法及制备的复合物和用途
CN116327684A (zh) * 2023-03-20 2023-06-27 南京农业大学 一种常山酮-银纳米温敏凝胶及其制备方法与应用

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GB201900381D0 (en) 2019-02-27
GB2566235B (en) 2022-02-16

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