Patent Application
20170231949
The present invention relates to a cell repair agent and a disinfection system.
The risk of infectious diseases increases with age. For age groups of 60 or older in Japan, an infectious disease, pneumonia, is the fourth leading cause of death. Infectious diseases include various pathological conditions ranging from life-threatening diseases such as pneumonia and septicemia to skin infections such as dermatophytosis (vesicular eczema) and dental diseases such as periodontal disease, which differ in causative microorganisms or infected sites, for example. Removal of pathogenic microbes is the primary objective of treatment for all of these infectious diseases, and a drug such as an antibiotic is administered for treatment in many cases. However, the widespread use of antibiotics results in the advent of drug-resistant organisms, and this may soon lead to severe problems such as the development of hospital-acquired infections due to multidrug-resistant bacteria. Therefore, the establishment of sterilizing therapy as an alternative to antibiotic administration is desired.
Acidic electrolyzed water that is obtained by electrolysis of sodium chloride or potassium chloride solution contains hypochlorous acid as the main contributor to the activity (for example, see Non-patent Document 1). When hypochlorous acid enters cells, hydroxyl radicals, which are a type of active oxygen, are generated and exert a sterilizing effect because of the strong oxidizing power (for example, see Non-patent Document 2). Moreover, a disinfection technology based on photolysis of hydrogen peroxide involves the application of the oxidizing power of hydroxyl radicals generated by homolytic cleavage of hydrogen peroxide resulting from irradiation of hydrogen peroxide with near-ultraviolet visible light (for example, see Non-patent Document 3).
A big advantage of sterilization technology using hydroxyl radicals, such as acidic electrolyzed water or disinfection technology based on photolysis of hydrogen peroxide over conventional sterilizing therapies involving antibiotic administration is to allow no development of resistant bacteria (for example, see Non-patent Document 4). Acidic electrolyzed water is used not only in the field of foods, but also in the medical field because of this advantage. In particular, the use of acidic electrolyzed water is being attempted for disinfection required for oral infectious diseases or as sterilization for surgeries (for example, see Non-patent Document 4 or 5). Applying the photosterilization technology using hydrogen peroxide to periodontal diseases is being explored (for example, see Non-patent Document 6). However, there is a concern that both acidic electrolyzed water and the disinfection technology based on photolysis of hydrogen peroxide can damage not only pathogenic microbes, but also cells at the disease site, because of the strong oxidizing power (for example, see Non-patent Documents 7 to 9). Furthermore, some existing non-hydroxy-radical-mediated disinfectants possess strong cytotoxicity and there is a similar concern that such disinfectants damage cells at the disease site (for example, see Non-patent Document 10).
On the other hand, polyphenols have an antioxidant effect and are known to have an effect of inhibiting oxidative injuries of local tissues due to active oxygen produced by inflammatory cells such as neutrophils at inflamed sites. Polyphenols are used for skin preparations, oral agents, and the like (for example, see Patent Documents 1 to 6, Non-patent Document 11 or 12).
As described in Non-patent Documents 7 to 10, there is a concern that acidic electrolyzed water, the disinfection technology based on photolysis of hydrogen peroxide, and existing disinfectants may damage not only pathogenic microbes, but also cells at disease sites because of the strong oxidizing power, and the development of technology for repairing the resulting damage has been expected. Furthermore, as described in Patent Documents 1 to 6 and Non-patent Documents 11 and 12, polyphenols are known to have an effect of inhibiting oxidative damage to local tissues due to active oxygen produced by inflammatory cells. However, it has not been confirmed that polyphenols have an effect of repairing cells severely damaged by acidic electrolyzed water or the like.
The present invention has been achieved, noting these problems. An objective of the present invention is to provide a cell repair agent capable of repairing cells damaged by the oxidizing power of acidic electrolyzed water or the like, and, a disinfection system capable of safely and efficiently accelerating the sterilization and cure of infected skin or intraoral wounds.
As a result of intensive studies to achieve the above objective, the present inventors have discovered that a specific polyphenol having a catechol group or a pyrogallol group and a polymer composition thereof have an effect of repairing cells severely damaged by acidic electrolyzed water, and thus have completed the present invention.
Specifically, the cell repair agent according to the present invention contains at least one type of a polyphenol represented by the following general formula (1), a polymer thereof, and a pharmaceutically acceptable salt thereof.
(wherein R1 to R3 are each selected from a group consisting of a substituted or unsubstituted hydrogen atom, a hydroxyl group, a halogen atom, a nitro group, a cyano group, a thio group, an alkoxy group, an aryloxy group, a heteroaryloxy group, a carbonyl group, an amino group, a C1-10 alkylamino group, a sulfonamide group, an imino group, a sulfonyl group, a sulfinyl group, a C1-10 alkyl group, a C3-12 cycloalkyl group, a C3-12 heterocycloalkyl group, a C9-12 bicycloalkyl group, a C3-12 heterobicycloalkyl group, a C1-10 arylalkyl group, a C1-5 heteroarylalkyl group, a C1-10 parhaloalkyl group, a C1-3 carbonylalkyl group, a C1-3 thiocarbonylalkyl group, a C1-3 sulfonylalkyl group, a C1-3 sulfinylalkyl group, a C1-10 aminoalkyl group, a C1-3 iminoalkyl group, an aryl group, a heteroaryl group, a C9-12 bicycloaryl group, and a C4-12 hetero bicycloaryl, are each independent, or, any two of R1, R2, and R3 together form a substituted or unsubstituted ring, and X is a substituted or unsubstituted C1-20 aliphatic hydrocarbon group, a C3-20 aromatic hydrocarbon group, or a C3-20 polycyclic aromatic hydrocarbon group.)
Specifically, the cell repair agent according to the present invention contains at least one type of a polyphenol represented by the following general formula (1), a polymer thereof, and a pharmaceutically acceptable salt thereof, so as to be able to repair cells damaged by the oxidizing power of acidic electrolyzed water or the like to be used for disinfection. The cell repair agent can also be expected to have an effect of inhibiting the oxidative damage to local tissues due to active oxygen produced by inflammatory cells such as neutrophils at inflamed sites.
In the cell repair agent according to the present invention, the above R2 and the above R3 are preferably hydrogen atoms. In addition, the above R1 is preferably a hydrogen atom or a hydroxyl group. Especially in such a case, the cell repair agent has a high effect of repairing severely damaged cells.
In the cell repair agent according to the present invention, the above polyphenol is preferably selected from a group consisting of proanthocyanidin, (+)-catechin, chlorogenic acid, caffeic acid and gallic acid. In this case, the cell repair agent can also repair cells damaged by the oxidizing power of acidic electrolyzed water or the like to be used for disinfection.
The disinfection system according to the present invention comprises a disinfection means for performing sterilization and/or disinfection for a living body, and a post-treatment means mounted to be able to bring the cell repair agent according to the present invention into contact with a part of said living body to be treated by the above disinfection means.
The disinfection system according to the present invention is capable of repairing cells damaged by the oxidizing power of acidic electrolyzed water or the like used for disinfection by treating the cells with the cell repair agent according to the present invention after disinfection. Therefore, the disinfection system according to the present invention can safely and efficiently accelerate the sterilization and cure of infected skin or intraoral wounds. Moreover, the disinfection system according to the present invention is easy to handle and capable of safely and efficiently performing disinfection of infected sites, surgeries, and the like even via short-term treatment. Examples of a disinfection means include an apparatus for injecting, spraying, immersing, draining, or applying a liquid microbicide and/or disinfectant, and an apparatus for filling with or injecting a gas microbicide and/or disinfectant. Examples of a post-treatment means include an apparatus for injecting, spraying, immersing, draining, or applying a solution or a dispersed solution of a cell repair agent. Such a post-treatment means may be designed to operate independently from a disinfection means, or designed to operate in conjunction with a disinfection means for contacting with a cell repair agent, when the disinfection means is operated.
According to the present invention, a cell repair agent capable of repairing cells damaged by the oxidizing power of acidic electrolyzed water or the like, and, a disinfection system capable of safely and efficiently accelerating the sterilization and cure of infected skin or intraoral wounds can be provided.
The cell repair agent of each embodiment of the present invention contains at least one type of a polyphenol represented by the above general formula (1), a polymer thereof, and a pharmaceutically acceptable salt thereof.
The cell repair agents of the embodiments of the present invention will be explained as follows based on the results of various tests. In addition, the present invention is not limited by these test examples (Examples).
A test was conducted for examining the impact of acidic electrolyzed water on mouse fibroblasts in growth phase and mouse fibroblasts in stationary phase. Acidic electrolyzed water (AEW) to be used in this test was obtained by 15 minutes of electrolysis of a 0.06% to 0.08% aqueous sodium chloride solution as an electrolyte solution and an electrolysis apparatus (“ALTRON-MINI AL-700”, ALTEC Co., Ltd.), alternating voltage of 100 V, and rated current of 0.6 A. The thus obtained acidic electrolyzed water was measured for pH, oxidation-reduction potential (ORP) and residual chlorine concentration using a pH/ORP meter (“SG2”, Mettler-Toled) and a residual chlorine meter (“HI96771C”, HANNA Instruments Japan). As a result, it was confirmed for acidic electrolyzed water that the pH ranged from 2.2 to 2.7, the ORP was 1100 mV or more, and the residual chlorine concentration ranged from 30 mg/l to 60 mg/l.
The test was conducted as follows. First, cells (mouse-derived 3T3-L1 fibroblasts; purchased from DS Pharma Biomedical Co., Ltd.) cultured in a 25-cm2 flask under conditions of 37° C. and 5% CO2 were treated with a 0.25% Trypsin-EDTA solution (Life Technologies), and then cell suspensions were prepared according to a standard technique in medium to a cell density of 2×104 cells/ml. Media used herein as growth media were prepared by adding fetal bovine serum (Life Technologies) and a penicillin/streptomycin solution (10000 units/ml penicillin, and 10 mg/ml streptomycin, Wako Pure Chemical Industries, Ltd.) to Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) to 10% (v/v) and 1% (v/v), respectively.
The cell suspension was seeded, 100 μl each, to each well of a 96-well microplate. After cells in growth phase were cultured for about 25 hours and cells in stationary phase were cultured for 4 days under conditions of 37° C. and 5% CO2, the medium was removed. After washing with phosphate-buffered saline (PBS, pH7.4), 100 μl each of two-fold dilution series of acidic electrolyzed water obtained by dilution with pure water was added to each well. After 30 seconds, two-fold dilution series of acidic electrolyzed water was removed, followed by washing with PBS.
Fresh medium was added and culture of cells in growth phase was resumed under conditions of 37° C. and 5% CO2. After 24 hours of culture, the degree of cell proliferation was determined by a MTT (methyl thiazolyl tetrazorium) method. Specifically, after 22 hours of culture, 10 μl each of a MTT reagent (Trevigen) was added to each well, followed by another 2 hours of culture under conditions of 37° C. and 5% CO2. After culture, 100 μl each of a Detergent reagent (Trevigen) was added, cells were left to stand overnight with protection from light at room temperature. Subsequently, the absorbance was read at 595 nm using a microplate reader (“FilterMax™ F5 Multi-Mode Microplate Reader”, Molecular Devices). A group treated with sterilized physiological saline was designated as a control group (control). The proportion (%) of each treated group was calculated with the absorbance of the control group designated as 100%.
Cells in stationary phase were washed, 10 μl each of a MTT reagent (Trevigen) was added to each well, and then cells were further cultured for 2 hours under conditions of 37° C. and 5% CO2, followed by similar procedures. The proportion (%) of each treated group was calculated with the absorbance of a control group (control) designated as 100%. In addition, a significant difference to the average value of each control group was tested by Dunnett's multiple comparison test (multiple comparative analysis).
Test results are shown in
A test was conducted for examining the intracellular levels of reactive oxygen species (ROS) generated when acidic electrolyzed water is brought into contact with mouse fibroblasts in stationary phase. First, mouse fibroblasts in stationary phase prepared and cultured similarly to Test 1 were washed with phosphate-buffered saline, and then 100 μl each of four-fold dilution series of acidic electrolyzed water obtained by dilution with pure water was added to each well. 30 seconds later, two-fold dilution series of acidic electrolyzed water was removed. Immediately after washing with PBS, intracellular ROS levels were measured using a measurement kit (“Oxiselect™ Intracellular ROS Assay Kit”, Cell Biolabs).
Specifically, 100 μl of 1 mM 2′,7′-dichlorodihydrofluorescin Diacetate (DCFH-DA) dissolved in serum-free medium (DMEM only) was added to each well, and then cells were cultured for 1 hour under conditions of 37° C. and 5% CO2. After culture, washing with phosphate-buffered saline was performed, 100 μl of 2× Cell Lysis Buffer (Cell Biolabs) and 100 μl of serum-free DMEM were added, and then the resultants were left to stand for 5 minutes. 150 μl of the resultants was transferred from each well to a black 96-well microplate, fluorescence intensities were measured at an excitation wavelength of 450 nm and a fluorescence wavelength of 530 nm using a microplate reader (“FilterMax™ F5 Multi-Mode Microplate Reader”, Molecular Devices). A standard curve was prepared using 2′,7′-dichlorodihydrofluorescein (DCF), and intracellular ROS levels were represented by DCF equivalents (nM). In addition, a significant difference to the average value of each control group (control) was tested by Dunnett's multiple comparison test (multiple comparative analysis).
Test results are shown in
Test 3: Impact of DMSO and Proanthocyanidin on ROS Levels within Mouse Fibroblasts in Stationary Phase Increased by Acidic Electrolyzed Water
Similarly to Test 2, mouse fibroblasts in stationary phase were treated with four-fold-diluted acidic electrolyzed water and undiluted acidic electrolyzed water for 30 seconds. After washing with PBS, 100 μl each of 140 mM DMSO (Wako Pure Chemical Industries, Ltd.) or 1 mM DCFH-DA containing 1 mg/ml proanthocyanidin (“Leucoselect (Registered Trademark)”, Indeda) as a hydroxyl radical scavenger was added to each well, followed by 1 hour of culture under conditions of 37° C. and 5% CO2. After culture, similarly to Test 2, intracellular ROS levels were determined. In addition, a significant difference between groups was tested by Tukey-Krammer's multiple comparison test (multiple comparative analysis).
Test results are shown in
Similarly to Test 1, mouse fibroblasts in growth phase were treated with four-fold-diluted acidic electrolyzed water and undiluted acidic electrolyzed water for 30 seconds. After washing with phosphate-buffered saline, 100 μl of 140 mM DMSO or 1 mg/ml proanthocyanidin prepared with serum-free DMEM was added to each well, and then cells were cultured for 30 minutes under conditions of 37° C. and 5% CO2. After washing with PBS, fresh medium was added, and then the degree of cell proliferation after 24 hours of culture under conditions of 37° C. and 5% CO2 was determined by the MTT method. In addition, a significant difference between groups was tested by Tukey-Krammer's multiple comparison test (multiple comparative analysis).
Test results are shown in
A test similar to Test 4 was conducted for human gingival fibroblasts in growth phase. First, human gingival fibroblasts (purchased from Primary Cell Co., Ltd.) were cultured in a 25 cm2 flask under conditions of 37° C. and 5% CO2, and then a cell suspension was prepared similarly to the case of mouse fibroblasts in medium to a cell density of 2×104 cells/ml. 100 μl each of the cell suspension was seeded to each well of a 96-well microplate. After 24 hours of culture under conditions of 37° C. and 5% CO2, similarly to Test 4, cells in growth phase were treated with four-fold-diluted acidic electrolyzed water or undiluted acidic electrolyzed water for 30 seconds. After washing with PBS, 100 μl of 140 mM DMSO or 1 mg/ml proanthocyanidin prepared with serum-free DMEM was added to each well, followed by 30 minutes of culture under conditions of 37° C. and 5% CO2. After washing with PBS, fresh medium was added, and then the degree of cell proliferation after 24 hours of culture under conditions of 37° C. and 5% CO2 was determined by the MTT method. In addition, a significant difference between groups was tested by Tukey-Krammer's multiple comparison test (multiple comparative analysis).
Test results are shown in
Similarly to Test 4, mouse fibroblasts in growth phase were treated with four-fold-diluted acidic electrolyzed water and undiluted acidic electrolyzed water for 30 seconds. After washing with PBS, 100 μl of 1 mg/ml (+)-catechin (Tokyo Chemical Industry Co., Ltd.) prepared with serum-free DMEM was added to each well, followed by 30 minutes of culture under conditions of 37° C. and 5% CO2. After washing with PBS, fresh medium was added, and then the degree of cell proliferation after 24 hours of culture under conditions of 37° C. and 5% CO2 was determined by the MTT method. In addition, a significant difference between groups was tested by Tukey-Krammer's multiple comparison test (multiple comparative analysis).
Test results are shown in
Similarly to Test 4, mouse fibroblasts in growth phase were treated with undiluted acidic electrolyzed water for 30 seconds. After washing with PBS, 100 μl of 1 mg/ml chlorogenic acid (Sigma-Aldrich), caffeic acid (Tokyo Chemical Industry Co., Ltd.) or gallic acid (Tokyo Chemical Industry Co., Ltd.) prepared with serum-free DMEM was added to each well, followed by 5 or 30 minutes of culture under conditions of 37° C. and 5% CO2. After washing with PBS, fresh medium was added, and then the degree of cell proliferation after 24 hours of culture under conditions of 37° C., 5% CO2 was determined by the MTT method. In addition, a significant difference between groups was tested by Tukey-Krammer's multiple comparison test (multiple comparative analysis).
Test results are shown in
Test 8: Impact of the Time for Post-Treatment with Proanthocyanidin on the Cytotoxicity of Acidic Electrolyzed Water Against Mouse Fibroblasts in Growth Phase
Similarly to Test 4, mouse fibroblasts in growth phase were treated with undiluted acidic electrolyzed water for 30 seconds. After washing with PBS, 100 μl of 1 mg/ml proanthocyanidin prepared with serum-free DMEM was added to each well, and the resultants were each left to sit at room temperature for 1 minute and 5 minutes. After washing with PBS, fresh medium was added, and then the degree of cell proliferation after 24 hours of culture under conditions of 37° C. and 5% CO2 was determined by the MTT method. In addition, a significant difference between groups was tested by Tukey-Krammer's multiple comparison test (multiple comparative analysis).
Test results are shown in
Similarly to Test 4, mouse fibroblasts in growth phase were treated with an undiluted chlorhexidine solution (CHL, Xttrium Laboratories) or an iodine gargle solution 7% (ISO, Meiji Seika Pharma Co., Ltd.) diluted 15-fold with PBS for 30 seconds. After washing with PBS, 100 μl of 1 mg/ml proanthocyanidin prepared with serum-free DMEM was added to each well, and then the resultants were left to stand at room temperature for 5 minutes. After washing with PBS, fresh medium was added, and then the degree of cell proliferation after 24 hours of culture under conditions of 37° C., 5% CO2 was determined by the MTT method. In addition, a significant difference between groups was tested by Tukey-Krammer's multiple comparison test (multiple comparative analysis).
Test results are shown in
Human gingival fibroblasts in growth phase obtained similarly to Test 5 were washed with PBS, 100 μl of a 500 mM hydrogen peroxide solution was added, and then laser light irradiation with an output of 100 mW (irradiance: 310 mW/cm2) was performed at 405 nm for 30 seconds. Laser light irradiation was performed using a laser apparatus (“RV-1000”, RICOH Optical Corporation, currently, RICOH Industrial Solutions Inc.) equipped with indium gallium nitride as a laser diode.
After irradiation, 100 μl of 1000 U/ml catalase (diluted with serum-free DMEM, Wako Pure Chemical Industries, Ltd.) was injected. After completion of this treatment for all wells, washing was performed twice with serum-free DMEM, 100 μl of serum-free DMEM containing 1 mg/ml proanthocyanidin was added, and then cells were cultured for 30 minutes. After culture, medium was removed, washing with PBS was performed, 100 μl of fresh medium was added, and then the degree of cell proliferation was determined by the MTT method after 24 hours of culture under conditions of 37° C. and 5% CO2. In addition, a significant difference between groups was tested by Tukey-Krammer's multiple comparison test (multiple comparative analysis).
Test results are shown in
Each of the above test results reveal the following. Specifically, as shown in
However, as shown in
As shown in
This is also applicable as a technology for protecting from cytotoxic stimulation that is performed, in the field of regenerative medicine, upon proliferation of various stem cells. For example, bone marrow stem cells require washing treatment; however, cellular damage due to washing treatment can be reduced by adding a small amount of polyphenol during or after the washing treatment.
An example of a disinfection system utilizing the cell repair agent of an embodiment of the present invention is shown in
The disinfection system 10 is used as follows. Specifically, as shown in
Treatment with the polyphenol represented by the above general formula (1) following treatment with acidic electrolyzed water, sterilization based on photolysis of hydrogen peroxide, or anti-microbial chemotherapy using various disinfectants can be used as a medical agent because of its effect. Hence, the disinfection system according to the present invention is useful in the fields of medicine, particularly the fields of skin therapy and endodontic therapy. Moreover, the cell repair agent according to the present invention can be used for repairing cells not only after disinfection with acidic electrolyzed water or the like, but also after a surgery such as a cancer operation, heart operation, transplant surgery, and the like.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2014/071330 | 8/12/2014 | WO | 00 |
