JP2006511585A - Reduction of reactive oxygen species in chronic wound management - Google Patents

Abstract

The reactive oxygen species associated with the wound are adjusted in the treatment of the wound with a pH of about 5 to about 7 of metal ions selected from the group consisting of potassium ions, zinc ions, calcium ions, and rubidium ions. Preferably, citric acid is used to adjust the pH. It has been found that by applying an extract to the superoxide anion appearing in the wound, the superoxide anion concentration is reduced, thereby being effective in treating the wound. Furthermore, it has been found that the treatment of partially thick cuts and contact burns using this composition improves the epithelialization of these wounds. In addition to the antioxidant activity of the present invention, treatment of the wound using the composition produces an inhibitory effect on ROS production and human complement activity by human PMN and is therefore further beneficial for chronic wound management. is there.

Description

Detailed Description of the Invention

(Related application)
This application is the present application filed on the basis of provisional patent application No. 60/436197 filed on December 23, 2002, claiming priority.
(Statement on federal research and development)
Not applicable.

(Technical field)
The present invention relates to wound management and in particular to chronic (non-responsive) wounds with properties such as decubitus ulcers, burns and the like.

(Background technology)
In recent years, it has also become clear that free radicals play an important role in wound healing disorders. In local and chronic wounds, free radicals are known to cause cell damage and can function as inhibitors in the healing process. In chronic wounds, ischemic symptoms can convert the enzyme xanthine dehydrogenase to xanthine oxidase, which catalyzes the conversion of oxygen to the superoxide anion. Superoxide anions are also produced in wound beds upon stimulation of polymorphonuclear neutrophils (PMN). Superoxide anions are free radicals that are toxic to tissues and, by their generation, other reactive oxygen species (ROS), including more toxic hydroxyl radicals and hypochlorous acid, a powerful and non-radical oxidant. ) Also occurs. The superoxide anion reacts readily with nitric oxide, a radical generated in macrophages of other inflammatory cells in the wound bed, to produce peroxynitrite, which has the most harmful effect on surrounding tissues. Ultimately, the superoxide anion can also cause cross-linking of the substrate molecules fibrin and fibronectin, which occur in converted substrates that are not well suited for epithelial growth.

(Summary of Invention)
In one embodiment of the invention, the reactive oxygen species is associated with a wound that is regulated by treatment with a synthesized composition of metal ions. Application of the composition to wounds that exhibit signs of superoxide anion has been found to be effective in the treatment and healing of these wounds through a reduction in the level of superoxide anion associated with the wound. The present invention is particularly effective in the treatment and healing of chronic wounds. Furthermore, it has been found that the treatment of partially thick cuts and contact burns using this composition improves the epithelialization of these wounds.

  In addition to the antioxidant activity of the present invention, the treatment of wounds using this composition results in an inhibitory effect on the production of ROS and human complement activation by human PMN and is therefore beneficial in further chronic wound management.

(Detailed description of the invention)
Techniques and methods used in the invention
Materials Preferred compositions useful in the present invention include 10-80 parts by weight of potassium ions, 0.00001-20 parts by weight of zinc ions, 0.01-10 parts by weight of calcium ions, and up to 40 parts by weight of rubidium ions. A volume of solution between about 5 and about 7 is included. In one embodiment, the metal ions are derived from their respective salts, including, for example, chloride, sulfate, citrate, hydroxide, and the like. The pH of the composition is preferably adjusted by adding citric acid to the solution as necessary. For this purpose, this composition is sometimes referred to herein as PHI5 (polyhydroxylated ionogen adjusted to pH 5 with citric acid).

  Therapeutic value has been found using potassium, zinc and rubidium ions without calcium. However, calcium is useful for the treatment of certain types of wounds, even if it is pharmaceutically ineffective for a particular wound, even if included in the solution of the present invention It is not detrimental to the effectiveness of a suitable solution when treating wounds.

Assay for inhibition of ROS production by human neutrophils (chemiluminescence assay)
Polymorphonuclear neutrophils (PMN) were isolated from venous blood of healthy volunteers (Bloedbank Midden-Nederland, Utrecht, The Netherlands). Test samples were diluted serially and finally to 50 μL in white 96 well flat bottom microtiter plates (Costar, Badhoevedorp, The Netherlands). To each well, 50 μL of PMN suspension (1 · 10 ⁷ cells / mL) and 50 μL of luminol (120 μM) were added. PMN was induced by adding opsonized zymosan A (OPZ; final concentration: 200 μg / mL). Chemiluminescence was monitored every 2 minutes for 0.5 seconds at 30 minute intervals using a Titertek Luminoskan luminometer (TechGen International, Zellik, Belgium).

Peak levels are used to calculate the activity of the test sample in correlation with their corresponding controls (no test sample, identical incubation). The experiment was performed using Hank balanced salt solution (I-IBSS), buffered with NaHCO ₃ at pH 7.35 and supplemented with 0.1% (w / v) gelatin (HBSS-gel) to avoid cell aggregation. ). OPZ was obtained by incubating washed commercial zymosan A and human pooled serum (HPS) diluted 1:10 at 37 ° C. for 30 minutes. After washing, the opsonized product was resuspended with HBSS (final concentration: 0.8 mg / mL).

Superoxide anion removal assay In a white 96 well flat bottom microtiter plate, test samples were serially diluted with phosphate buffered saline (PBS; pH 7.4) to a final volume of 50 μL. Subsequently, hypoxanthine (50 μL; final concentration 1 mM) and buffer or superoxide dismutase (SOD, 25 μL; 10 U / mL) were added. Superoxide anion (.O ₂ ) radical generation is initiated by the addition of 25 μL xanthine oxidase (10 mU / mL) and chemiluminescence is performed for 15 minutes using a Fluoroskan Ascent FL luminometer (Labsystems, Breda, The Netherlands). , Monitored every mm for 0.5 seconds. The activity of the test compound was calculated from the chemiluminescent signal of the SOD-inhibitable moiety. To eliminate the direct effect of the test sample on xanthine oxidase activity, uric acid formation was determined by spectrophotometer at 290 nm.

Hemolytic assay for human complement activity (classical and alternative pathways)
The inhibitory activity of the test sample against the classical and alternative pathways of human complement (CP and AP, respectively) is shown in Klerx et al. (Klerx, JPAM, Beukelman, CJ, Van Dijk, H. et al., Microassay for colorimetric. J. Immunlol Methods 1983, 63: 215-220) was quantified by a modified version of the microassay described in estimation of complement activity in guinea pig, human and mouse serum. In a U-well microtiter plate (Greiner Labortechnik, Nortingen, Germany), the final amount of the test sample is (1) VSB-CP (Veronal saline, 5 mM Veronal, 150 mM saline; adjusted to pH 7.35). 50 μL (CP) supplemented with 0.15 mM Ca ²⁺ and 0.5 mM Mg ²⁺ , or (2) the above veronal buffered saline with 0.5 mM Mg ²⁺ and It was diluted with VSB-AP prepared to a final volume of 100 μL (AP) supplemented with 0.8 mM EGTA. Subsequently, 50 μL (CP) or 25 μL (AP) of a dilution of the appropriate human pool serum (HPS; obtained from a healthy donor) was added and the plates were incubated at 37 ° C. for 30 minutes. After adding 50 μL of sensitized sheep red blood cells (CP) or 25 μL of rabbit red blood cells (see below), the plates were again incubated at 37 ° C. for 1 hour. Sheep or rabbit blood in Alsever solution is supplied as a source of red blood cells. Prior to use, erythrocytes were washed 3 times with saline. Sheep red blood cells were sensitized by incubation with diluted (1: 800) amboceptor for 10 minutes; after washing, the sensitized red blood cells were resuspended in VSB-CP (4 × 10 ⁸ cells / mL). . Rabbit erythrocytes were suspended in VSB-AP (3 × 10 ⁸ cells / mL). Finally, the microtiter plate is centrifuged (900 × g for 5 minutes), the intact cells and the residue are allowed to settle, and 50 μL of the supernatant is added to the well and a 96-well flat bottom containing 200 μL of water. Transfer to a microtiter plate. In the latter plate, the amount of hemoglobin released by lysis of red blood cells was measured with a spectrophotometer operating at 405 nm using the automatic ELISA reader described above. Controls are water (100% hemolysis), or buffer (VSB-CP or VSB-AP; 0% hemolysis), and HPS inactivated by heat (56 ° C., 30 min; test sample background) Incubation of the incubated red blood cells with an incubation replaced with a color correction) is carried out in the same way.

Quantification of cytotoxicity An acetone stock solution of 5-carboxyfluorescein diacetate (CFDA; 10 mg / mL) was prepared and stored at -20 ° C. Prior to use, this stock solution was diluted 1: 1000 with buffer. Propidium iodide (PI; 1.5 mg), 10 mL phosphate buffered saline (PBS) containing 2.5% quenching ink, 5% w / v EDTA, and 8 mg bovine serum albumin (BSA). ). PMN is labeled with vital staining CFDA (10 μg / mL) for 15 minutes at 20 ° C., washed and resuspended in buffer until a concentration of 10 ⁷ cells / mL. 100 μL of this cell suspension was incubated with the same diluted sample at 37 ° C. for 15 minutes. Subsequently, the cells were washed and stained with 25 μL of PI / ink solution to distinguish between viable (light emitting green) and dead (light emitting red) cells. The percentage of dead cells was quantified using a fluorescence microscope (Fluovert, Leitz, Wetzlar, Germany).

Results and Discussion The generation of reactive oxygen species associated with wounds arises from several possible sources. The solution of the present invention exhibited a pharmaceutically effective inhibitory effect on the production of reactive oxygen species (ROS) associated with the wound.

One source of ROS in wounds is reactive oxygen species that are generated upon stimulation of human polymorphonuclear neutrophils (PMN). PMN, for example, strengthens and activates the wound, consuming an increasing amount of oxygen and converting it to ROS. This process, known as respiratory burst, depends on the enzyme NADPH oxidase being activated by both receptor-mediated and receptor-independent processes. Typical receptor-dependent stimuli include, for example, complement elements C5a and C3b, bacterially chemotactic tripeptides fMLP, and opsonized zymosan; receptor-dependent stimuli including long chain unsaturated fatty acids. Upon activation of the PMN, the multi-component NADPH oxidase assembles at the cell membrane. Subsequently, the oxidase transfers electrons from NADPH to molecular oxygen on the opposite side of the membrane on the cellular substrate side of the membrane. As a result, superoxide anion (.O ₂ ⁻ ) is generated in the phagosome (intracellular) containing the ingested microorganisms or extracellularly. Most of the superoxide anion formed is converted to hydrogen peroxide (H ₂ O ₂ ). The latter is bactericidal only at high concentrations, whereas the superoxide anion itself does not kill bacteria due to its limited membrane permeability. Any of the hydrogen peroxide is converted to extremely active hydroxyl radicals by the iron-catalyzed Fenton reaction. However, most of the hydrogen peroxide is converted to hypochlorous acid (HOCl), a bactericidal oxidant known to be produced by PMN. The latter conversion occurs in the presence of halide (chloride) ions and is catalyzed by activated PMN by myeloperoxide (MPO), which is also an enzyme released. In phagolysosomes, intracellular ROS [along with proteolysis and other cytotoxic enzymes released from lysosomes (granules)] kills ingested bacteria, prevents wound infections, and these oxygen metabolites The extracellular generation of has a deleterious effect around the tissue.

  In addition to the ROS described above, the production of nitric oxide (by NO. Macrophages present in the wound) is also noted. Nitric oxide radicals readily react with superoxide anions, resulting in the formation of peroxonitrite, a very strong and relatively stable oxidant with properties similar to hydroxyl radicals (see above).

  For inhibition of ROS production by stimulation of human PMN, an IC50 value of 12 ± 2 mL / mL was quantified by PHI5 using a chemiluminescence assay. The IC50 value is the sample concentration in the test system giving 50% inhibition; the IC50 value is expressed as the mean ± SD (standard deviation) of the quantitative values obtained with two batches of PMN from two different donors. Is the value to be Since the inhibitory effect in the assay of ROS generation can be caused by cell death, the cytotoxic effect of the test sample was also studied. Stationary PMNs were labeled with vital staining CFDA (5-carboxyfluorescein diacetate) and incubated with PHI5. Subsequently, dead cells were stained with propidium iodide. It was found that incubation with 100 μL / mL PHI5 does not show any cytotoxic effect on PMN compared to control cells. It was concluded that inhibition of ROS production by PHI5 was not due to cytotoxic effects on PMN.

  As mentioned above, in addition to the production of superoxide anions upon stimulation with PMN, these radicals convert ischemic conditions to xanthine oxidase, which catalyzes the conversion of the enzyme xanthine dehydrogenase from oxygen to the superoxide anion. Can also occur in chronic wounds. Thus, antioxidant activity, including removal of the generated superoxide anion via the PMN or xanthine oxidase, is considered beneficial in the treatment of chronic wounds. PHI5 has been shown to be an effective scavenger of the superoxide anion primarily due to the presence of citric acid.

  The inhibition found in the assay of ROS production (see above) can also be caused by the specific removal of the superoxide anion. It has been reported that oak bark extract (OBE) has a direct effect on PMN function. In the superoxide anion scavenger assay using PHI5 (IC50 12 μL / mL), the observed increase in activity is probably due largely to the additional scavenging of the superoxide anion by the citrate component of PHI5. Thus, PHI5 provides both superoxide anion removal and ROS production inhibitory action, enhancing the utility of the present invention in wound management, particularly chronic wound management.

  PHI5 was also tested for a hemolytic assay for modulation of complement activity. The complement system is part of non-applied humoral immunity and plays an important role in the human defense mechanism. The complement system consists of more than 20 proteins, including C1 to C9 complement components. Complement activity via the classical, alternative, or lectin pathway ultimately leads to the proteolytic cleavage of complement proteins in a cascade-like fashion, leading to the formation of macromolecular membrane attack complexes (MAC) Occurs and causes the death of bacteria (or exogenous red blood cells via hemolysis). In addition, it produces subdivided products and promotes many immune control effects. In this regard, complement factor C3b is recognized by phagocytic cells that have C3b receptors on membranes (eg, PMN, etc.), primarily pathogenic microorganisms and foreign cells (zymosan) covered with C3b (opsonization). And have biological functions that can ingest these invaders and destroy them by generating ROS. Fragment C5a is another activator for PMN; moreover, it is a major chemotactic factor in these phagocytes.

  Inhibition of complement activity limits the production of complement split products such as C5a. As noted above, this resulted in a low influx and reduction of wound bed PMN stimulation, thus reducing extracellular formation of ROS and peroxynitrite, thereby reducing tissue damage.

  While wound healing dominated by other factors may also be important (eg, MMP), PHI5 inhibits human complement activity via the classical pathway, is activated by PMN, and ROS Inhibits the production of Furthermore, citric acid associated with PHI5 has been found to contribute to the removal of superoxide anions. The reduction in ROS levels contributes to the beneficial effects observed in wound management, particularly chronic wound management, with preparations containing the components of the present invention, metal ions and citric acid.

  A comparison of the IC50 values of the prior art oak bark extract and the synthetic component of the present invention is shown in FIGS. The figures are graphs of the IC50 values of these two compositions, measured with a superoxide anion scavenger assay (Figure 1), measured with a chemiluminescent assay (Figure 2), and the classical pathway. It is measured by a complement assay (FIG. 3). As can be seen from these graphs, PHI5 is more effective than natural oak bark extract (OBE) in removing superoxide and inhibiting PMN (chemiluminescence assay), and only in regulating complement activity. Slightly less effective.

FIG. 1 is a comparison of graphs of IC50 values in which natural oak bark extract and the synthetic solution of the present invention are quantified by superoxide anion scavenger assay. FIG. 2 is a comparison of graphs of IC50 values quantified by chemiluminescence assay of natural oak bark extract and the synthetic solution of the present invention. FIG. 3 is a comparison of graphs of IC50 values quantifying natural oak bark extract and the synthetic solution of the present invention by classical pathway complement assay.

Claims (15)

  For neutralizing reactive oxygen species associated with wounds using a pharmaceutically effective, substantially neutral aqueous solution of a metal ion selected from the group consisting of potassium ion, zinc ion, calcium ion and rubidium ion A method of enhancing wound healing comprising applying the solution to the wound for a time sufficient to exert an effect and enhancing wound healing by neutralizing the solution against reactive oxygen species associated with the wound.   The method of claim 1 comprising adjusting the pH of the solution with citric acid.   The method of claim 2, wherein the pH of the solution is from about 5 to about 7.   The method of claim 1, wherein the solution exhibits an IC50 value that removes about 33 / mL of superoxide.   Applying the solution to a wound using an aqueous solution having a pharmaceutically effective pH of a metal ion selected from the group consisting of potassium ion, zinc ion, calcium ion and rubidium ion of about 5 to about 7, thereby The solution inhibits the production of reactive oxygen species associated with the wound by stimulated polymorphonuclear neutrophils, removes superoxide anions, inhibits polymorphonuclear neutrophil attractant or stimulator A method of enhancing wound healing comprising healing a wound by exerting an effect or combining them.   6. The method of claim 5, wherein the pH of the solution is adjusted to about 5 using citric acid.   6. The method of claim 5, wherein the pH of the solution is adjusted to about 7 using hydrochloric acid.   The method of claim 1, wherein the solution comprises up to 10 to 20 parts by weight potassium ions, 0.00001 to 20 parts by weight zinc ions, and up to about 40 parts by weight rubidium ions.   9. The method of claim 8, wherein the solution comprises a sufficient amount of citric acid to adjust the pH to about 5-7.   The method of claim 8 comprising 0.01 to 10 parts by weight of calcium ions.   11. The method of claim 10, wherein the solution comprises a sufficient amount of citric acid to adjust the pH to about 5-7.   6. The method of claim 5, wherein the solution comprises up to 10-20 parts by weight potassium ions, 0.00001-20 parts by weight zinc ions, and up to about 40 parts by weight rubidium ions.   13. The method of claim 12, wherein the solution comprises a sufficient amount of citric acid to adjust the pH to about 5 to about 7.   6. The method of claim 5, comprising 0.01 to 10 parts by weight of calcium ions. 6. The method of claim 5, wherein the solution comprises a sufficient amount of citric acid to adjust the pH to about 5 to about 7.

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