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WO2004057965A1 - Reduction d'especes reactives d'oxygene dans le traitement de plaies chroniques - Google Patents

Reduction d'especes reactives d'oxygene dans le traitement de plaies chroniques Download PDF

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Publication number
WO2004057965A1
WO2004057965A1 PCT/US2003/041038 US0341038W WO2004057965A1 WO 2004057965 A1 WO2004057965 A1 WO 2004057965A1 US 0341038 W US0341038 W US 0341038W WO 2004057965 A1 WO2004057965 A1 WO 2004057965A1
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WO
WIPO (PCT)
Prior art keywords
solution
ions
wound
weight
parts
Prior art date
Application number
PCT/US2003/041038
Other languages
English (en)
Inventor
Stephen H. Monroe
Hans Hoekstra
A. J. J. Van Den Berg
Original Assignee
Greystone Medical Group, Inc.
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.)
Filing date
Publication date
Application filed by Greystone Medical Group, Inc. filed Critical Greystone Medical Group, Inc.
Priority to JP2004563981A priority Critical patent/JP2006511585A/ja
Priority to AU2003303335A priority patent/AU2003303335A1/en
Priority to EP03808544A priority patent/EP1575359A4/fr
Priority to CA002511440A priority patent/CA2511440A1/fr
Publication of WO2004057965A1 publication Critical patent/WO2004057965A1/fr

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Classifications

    • 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
    • A61K33/14—Alkali metal chlorides; Alkaline earth metal chlorides
    • 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
    • A61K33/30—Zinc; Compounds thereof
    • 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
    • A61K33/32—Manganese; Compounds 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to management of wounds, particulary chronic (non-responding) wounds in the nature of decubitus ulcers, burns, and the like.
  • ROS reactive oxygen species
  • nitric oxide a radical produced by macrophages - another inflammatory cell in the wound bed - superoxide anion easily reacts to form peroxynitrite that also exerts most detrimental effects on surrounding tissue.
  • superoxide anion may also induce cross-linking of the matrix molecules fibrin and fibronectin resulting in a transformed matrix less suitable for epithelial outgrowth.
  • Figure 1 is a graphic comparison of the IC50 values of a natural oak bark extract and the synthetic solution of the present invention as determined by superoxide anion scavenger assay;
  • Figure 2 is a graphic comparison of the IC50 values of a natural oak bark extract and the synthetic solution of the present invention as determined by chemiluminescence assay.
  • Figure 3 is a graphic comparison of the IC50 values of a natural oak bark extract and the synthetic solution of the present invention as determined by complement assay classical pathway.
  • reactive oxygen species associated with a wound are modulated upon treatment with a synethetic composition of metal ions.
  • Application of the composition to a wound exhibiting superoxide anions has been found to be effective in the treatment and healing of these wounds through the reduction of the level of superoxide anions associated with the wound.
  • the present invention is particularly effective in the treatment and healing of chronic wounds.
  • treatment of partial thickness excision wounds as well as contact burn wounds with the present composition has been found to improve epithelialization of these wounds.
  • treatment of the wound employing the present composition produces inhibitory effects on ROS production by human PMNs and on human complement activation, and therefore, is further beneficial in chronic wound management.
  • a preferred composition useful in the present invention comprises
  • the solution having a pH of between about 5 and about 7.
  • the metal ions are derived from respective salts thereof, including chlorides, sulfates, citrates, hydroxides, for example.
  • Adjustment of the pH of the composition preferably is accomplished by the addition of citric acid to the solution, as needed.
  • this composition is at times herein referred to as PHI5 (polyhydrated ionogen adjusted to a pH of 5 using citric acid).
  • Therapeutic value has been found using potassium, zinc and rubidium ions, without calcium. Calcium, however, may be useful in the treatment of certain types of wounds and its presence in a solution of the present invention, even if non-pharmaceutically effective for a particular wound, is not detrimental to the effectiveness of the preferred solution when treating such particular wound.
  • PMNs Polymorphonuclear neutrophils
  • Peak levels were used to calculate the activity of test samples in relation to their corresponding controls (identical incubations without test sample).
  • Experiments were performed in Hank's balanced salt solution (I-IBSS) buffered at pH 7.35 with NaHCO 3 and supplemented with 0.1% (w/v) gelatin to avoid cell aggregation (HBSS-gel).
  • OPZ was obtained by incubation of washed commercial zymosan A with 1: 10 diluted human pooled serum (HPS) at 37 °C for 30 min. After washing, the opsonized product was resuspended in HBSS (final concentration: 0.8 mg/mL).
  • test samples were serially diluted in phosphate- buffered saline (PBS; pH 7.4) to a final volume of 50 ⁇ L. Subsequently, hypoxant-hine (50 ⁇ L; final concentration 1 mM), and either buffer or superoxide dismutase (SOD; 25 ⁇ L; 10 U/mL) were added.
  • PBS phosphate- buffered saline
  • test samples were serially diluted in (1) VSB- CP (Veronal saline buffer, prepared with 5 mM veronal, 150mM saline; pH 7.35), supplemented with 0.15 mM Ca 2+ and 0.5 mM Mg 2+ to final volumes of 50 ⁇ l (CP) or (2) VSB-AP, prepared with veronal saline buffer as above, supplemented, however, with 0.5 mM Mg 2+ and 0.8 mM EGTA, to final volumes of 100 ⁇ l (AP).
  • VSB- CP Very saline buffer, prepared with 5 mM veronal, 150mM saline; pH 7.35
  • VSB-AP prepared with veronal saline buffer as above, supplemented, however, with 0.5 mM Mg 2+ and 0.8 mM EGTA, to final volumes of 100 ⁇ l (AP).
  • CP sensitized sheep erythrocytes
  • AP rabbit erythrocytes
  • the plates were incubated again at 37°C for lh. Sheep or rabbit blood in Alsever solution served as sources of erythrocytes. Before use, erythrocytes were washed three times with saline.
  • Sheep erythrocytes were sensitized by incubation with diluted amboceptor (1:800) for 10 min; after washing the sensitized erythrocytes were resuspended in VSB-CP (4 x 10 8 cells/ml). Rabbit erythrocytes were suspended in VSB-AP (3 x 10 8 cells/ml). Finally, the microtiter plates were centrifuged (900 x g, 5 min to spin down intact cells and debris, and 50 ⁇ l of the supenatants were transferred to 96-well flat-bottom microtiter plates containing 200 ⁇ l of water per well.
  • CFDA 5-carboxyfluorescein diacetate
  • acetone a stock solution of 5-carboxyfluorescein diacetate (CFDA; 10 mg/ml) in acetone was prepared and stored at -20 °C. Prior to use, this stock solution was diluted 1: 1000 in buffer. Propidium iodide (PI; 1.5 mg) was dissolved in 10 ml of phosphate-buffered saline (PBS) contaming 2.5% quenching ink, 5% w/v EDTA, and 8 mg of bovine serum albumin (BSA). PMNs were labeled with the vital stain CFDA (10 ⁇ g/ml) at 20 °C for 15 min, washed, and resuspended in buffer to a concentration of 10 7 cells/mL.
  • PBS phosphate-buffered saline
  • BSA bovine serum albumin
  • Amounts of 100 ⁇ l of this cell suspension were incubated with equal volumes of diluted samples at 37 °C for 15 min. Subsequently, the cells were washed and stained with 25 ⁇ l of Pl/ink solution for discrimination between viable (green-fluorescent) and dead (red-fluorescent) cells. The percentage of dead cells was determined using a fluorescence microscope (Fluovert, Leitz, Wetzlar, Germany).
  • ROS reactive oxygen species
  • ROS in a wound are reaactive oxygen species generated by stimulated human polymorphonu clear neutrophils (PMNs). PMNs recruited for instance to the wound site and activated, consume increased amounts of oxygen that is converted into ROS. This process known as the respiratory burst is dependent on the enzyme NADPH oxidase that can be activated by both receptor-mediated and receptor-independent processes.
  • Typical receptor-dependent stimuli are e.g. complement components C5a, and C3b, the bacterium-derived chemotactic tripeptide fMLP, and opsonized zymosan; receptor-independent stimuli include long-chain unsaturated fatty acids.
  • the multi-component NADPH oxidase Upon activation of the PMN, the multi-component NADPH oxidase is assembled in the cell membrane. Subsequently, the oxidase transfers electrons from NADPH at the cytosolic side of the membrane to molecular oxygen at the other side of the membrane. This results in the generation of superoxide anions (O2" either in (intracellular) phagosomes containing ingested microorganisms, or extracellularly. Most of the superoxide anions formed are converted into hydrogen peroxide (H2O2). The latter is bactericidal only at high concentrations, whereas superoxide anions themselves do not kill bacteria because of their limited membrane permeability.
  • HOC1 hypochlorous acid
  • MPO myeloperoxidase
  • nitric oxide NO- by macrophages present at the wound site
  • the radical nitric oxide may easily react with superoxide anion, which results in the formation of peroxynitrite (ONOO), a very potent, relatively stable oxidant with properties similar to those of the hydroxyl radical (see above).
  • ONOO peroxynitrite
  • PMNs an IC50 value of 12 ⁇ 2 ml/ml was determined for PHI5, employing Chemiluminescence assay. ⁇ (The IC50 value is the sample concentration in the test system giving 50% inhibition; IC50 values represent the mean ⁇ SD (standard deviation) of determinations obtained with two batches of PMNs from two different donors)). Since inhibitory effects in the assay for ROS production may be caused by cell death, also cytotoxic effects of the test samples were investigated. Resting PMNs were labeled with the vital stain CFDA (5 - carboxyfluorescein diacetate) and incubated with PHI5. Subsequently, dead cells were stained with propidium iodide.
  • CFDA vital stain CFDA
  • antioxidant activity including scavenging of superoxide anions, either produced by the PMN or through xanthine oxidase is regarded beneficial in the treatment chronic wounds.
  • PHI5 was shown to be a significant scavenger of superoxide anions mainly due to the presence of citric acid.
  • Inhibition as found in the assay for ROS production may also be caused by a specific scavenging of superoxide anions.
  • Oak bark extract (OBE) has been reported to have a direct effect on PMN functioning.
  • the increase in activity as observed in superoxide anion scavenger assays for employing PHI5 (IC5O 12 ⁇ l/ml) is most probably due to additional scavenging of superoxide anions by the citric acid component of PHI5.
  • PHI5 provides both superoxide anion-scavenging and inhibition of ROS production thereby enhancing the usefulness of the present invention in wound management, particularly management of chronic wounds.
  • PHI5 also was tested in the hemolytic assays for modulation of complement activity.
  • the complement system is part of the non-adaptive humoral immune system and plays an important role in the human defense mechanism.
  • the complement system comprises over twenty proteins, including complement components Cl to C9. Activation of complement via either the classical, alternative, or lectin pathway results in proteolytic cleavage of the successive complement proteins in a cascade-like manner which eventually leads to the formation of the high-molecular membrane attack complex (MAC) that causes death of bacteria (or foreign red blood cells through lysis). In addition, small split products are generated which mediate many immunoregulatory effects.
  • MAC high-molecular membrane attack complex
  • complement factor C3b has a major biological function since (pathogenic) microorganisms and foreign cells (zymosan) are covered with C3b (opsonization), which enables phagocytes with receptors for C3b on their membrane (e.g. PMNs) to recognize, and ingest these invaders and to destroy them by producing ROS.
  • Fragment C5a is another activating agent for PMNs; in addition it is a major chemotactic factor for these phagocytes.
  • Inhibition of complement activation limits the generation of complement split products such as C5a. As outlined above, this will result in less influx and decreased stimulation of PMNs in the wound bed, and thus reduced extracellular formation of ROS as well as peroxynitrite, and therefore reduced tissue damage.
  • PHI5 inhibits human complement activation via the classical pathway and inhibits production of ROS by activated PMNs.
  • citric acid associated with PHI5 has been found to be a contributor to the scavenging of superoxide anions. Such reduction of levels of ROS contribute to the beneficial effects observed in wound management, especially chronic wound management, with preparations containing the metal ions and citric acid of the composition of the present invention.
  • FIGs 1- 3 are graphs depicted to IC50 values of these two compositions as determined in superoxide anion scavenger assay (Figure 1), chemHuminexcence assay ( Figure 2), and complement assay classical pathway ( Figure 3). Review of these graphs shows that PHI5 is more effective than a natural oak bark extract (OBE) with respect to superoxide scavenging and PMN inhibition (chemiluminescence assay), and is only slightly less effective with respect to modulation of complement activity.
  • OBE natural oak bark extract
  • PMN inhibition chemiluminescence assay

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  • Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Inorganic Chemistry (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Dermatology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

L'invention concerne des espèces réactives d'oxygène associées à une plaie, lesquelles sont modulées par traitement de ladite plaie à l'aide d'une solution métallique choisie dans le groupe constitué d'ions potassium, d'ions zinc, d'ions calcium et d'ions rubidium, à un pH compris entre environ 5 et 7. De préférence, on utilise un acide citrique en vue de réguler le pH de la solution. L'application de l'extrait sur une plaie présentant des anions superoxydes s'est révélée efficace dans le traitement de ces plaies par réduction du niveau d'anions superoxydes. En outre, le traitement de plaies et d'excisions à épaisseur partielle et de brûlures de contact au moyen de la présente composition a révélé une amélioration de l'épithélialisation de ces plaies. Outre l'activité antioxydante de cette invention, le traitement de plaies avec cette composition produit des effets inhibiteurs sur la production d'espèces réactives d'oxygène (ROS) par des neutrophiles polymorphonucléaires humains et sur l'activation du complément humain, c'est pourquoi celui-ci présente des avantages supplémentaires dans le traitement de plaies chroniques.
PCT/US2003/041038 2002-12-23 2003-12-23 Reduction d'especes reactives d'oxygene dans le traitement de plaies chroniques WO2004057965A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2004563981A JP2006511585A (ja) 2002-12-23 2003-12-23 慢性創傷管理における活性酸素種の減少
AU2003303335A AU2003303335A1 (en) 2002-12-23 2003-12-23 Reduction of reactive oxygen species in chronic wound management
EP03808544A EP1575359A4 (fr) 2002-12-23 2003-12-23 Reduction d'especes reactives d'oxygene dans le traitement de plaies chroniques
CA002511440A CA2511440A1 (fr) 2002-12-23 2003-12-23 Reduction d'especes reactives d'oxygene dans le traitement de plaies chroniques

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US43619702P 2002-12-23 2002-12-23
US60/436,197 2002-12-23

Publications (1)

Publication Number Publication Date
WO2004057965A1 true WO2004057965A1 (fr) 2004-07-15

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PCT/US2003/041038 WO2004057965A1 (fr) 2002-12-23 2003-12-23 Reduction d'especes reactives d'oxygene dans le traitement de plaies chroniques

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EP (1) EP1575359A4 (fr)
JP (1) JP2006511585A (fr)
CN (1) CN1744819A (fr)
AU (1) AU2003303335A1 (fr)
CA (1) CA2511440A1 (fr)
WO (1) WO2004057965A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011022757A1 (fr) * 2009-08-24 2011-03-03 Queensland University Of Technology Diagnostic et thérapie des plaies ciblant les purines
WO2013016255A1 (fr) * 2011-07-28 2013-01-31 3M Innovative Properties Company Compositions cicatrisantes et leur méthode d'utilisation

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2009251711B2 (en) * 2008-04-01 2014-07-17 Antipodean Pharmaceuticals, Inc. Compositions and methods for skin care

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6149947A (en) * 1992-11-06 2000-11-21 Greystone Medical Group, Inc. Compositions of oak bark extract related synthetic compositions and method of using same

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3416777C2 (de) * 1984-05-07 1986-11-20 Gödecke AG, 1000 Berlin Pharmazeutische topische Zubereitungen
NZ533252A (en) * 2001-11-29 2006-03-31 Greystone Medical Group Inc Treatment of wounds and compositions employed

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6149947A (en) * 1992-11-06 2000-11-21 Greystone Medical Group, Inc. Compositions of oak bark extract related synthetic compositions and method of using same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1575359A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011022757A1 (fr) * 2009-08-24 2011-03-03 Queensland University Of Technology Diagnostic et thérapie des plaies ciblant les purines
WO2013016255A1 (fr) * 2011-07-28 2013-01-31 3M Innovative Properties Company Compositions cicatrisantes et leur méthode d'utilisation

Also Published As

Publication number Publication date
AU2003303335A1 (en) 2004-07-22
EP1575359A4 (fr) 2009-08-05
EP1575359A1 (fr) 2005-09-21
CA2511440A1 (fr) 2004-07-15
JP2006511585A (ja) 2006-04-06
CN1744819A (zh) 2006-03-08

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