US20070196434A1 - Methods of preventing or treating sinusitis with oxidative reductive potential water solution - Google Patents

Methods of preventing or treating sinusitis with oxidative reductive potential water solution Download PDF

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US20070196434A1
US20070196434A1 US11/656,088 US65608807A US2007196434A1 US 20070196434 A1 US20070196434 A1 US 20070196434A1 US 65608807 A US65608807 A US 65608807A US 2007196434 A1 US2007196434 A1 US 2007196434A1
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water solution
orp water
reductive potential
oxidative reductive
potential water
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Hojabr Alimi
Andres Gutierrez
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Sonoma Pharmaceuticals Inc
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Oculus Innovative Sciences Inc
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    • A61K33/20—Elemental chlorine; Inorganic compounds releasing chlorine
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    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the cranial sinuses are air chambers within the bone of the cheeks, eyebrows and jaw. These chambers include the frontal sinuses in the eyebrow area, maxillary sinuses inside each cheekbone, ethmoid sinuses just behind the bridge of the nose and between the eyes, and sphenoid sinuses located behind the ethmoids in the upper region of the nose and behind the eyes.
  • the sinuses are lined by a respiratory type epithelium with an underlining subepithelial layer rich in mucus glands and small blood vessels.
  • Sinusitis is a condition in which the lining of the sinuses becomes inflamed.
  • Sinusitis can be acute or chronic.
  • Viruses are a frequent cause of acute sinusitis, which produces significant inflammation. This inflammation results in increased mucus production and congestion of the nasal passages. When there is swelling of the mucous membranes of the sinuses, air and mucus are trapped behind the narrowed openings of the sinuses. This congestion predisposes the individual to bacterial sinusitis.
  • Chronic inflammation of the nasal passages such as allergic rhinitis (hay fever) also predisposes the individual to episodes of acute sinusitis.
  • Vasomotor rhinitis which can be caused by, e.g., humidity, cold air, alcohol, perfumes, and other environmental conditions, also can predispose the individual to sinus infection.
  • Chronic sinusitis is an inflammatory disease that often occurs in patients with asthma. It can be caused by infectious agents, although airborne allergens, such as dust, mold, and pollen, which trigger allergic rhinitis, may contribute to or cause chronic sinusitis.
  • An immune response to antigens in fungi also can be responsible for at least some cases of chronic sinusitis.
  • the ORP water solution can be administered by any suitable route including, for example, intranasally, through the mouth or both.
  • the ORP water solution can be administered in any suitable form such as, e.g., a liquid, spray, mist or aerosol, and can be delivered by any suitable method, e.g., aerosolization, nebulization and atomization.
  • the ORP water solution is administered in the form of droplets having a diameter in the range of from about 0.1 micron to about 100 microns, preferably 1 micron to about 10 microns.
  • FIG. 14 is a graphical comparison of IL-13 secretion by antigen-activated mast cells treated with various concentrations of an exemplary ORP water solution (MCN).
  • MCN ORP water solution
  • the cranial sinuses can include, e.g., the frontal sinuses, maxillary sinuses, ethmoid sinuses, and sphenoid sinuses.
  • the ORP water solution is administered to one or more of the patient's ethmoid sinuses, e.g., so as to contact one or more tissues residing in the ethmoid sinuses with the ORP water solution.
  • chronic sinusitis is more common in patients with immune deficiency or abnormalities of mucus secretion or movement (e.g., immune deficiency, HIV infection, cystic fibrosis, Kartagener's syndrome).
  • immune deficiency HIV infection
  • cystic fibrosis Kartagener's syndrome
  • some patients have severe asthma, nasal polyps, and severe asthmatic responses to aspirin and aspirin-like medications (so-called non-steroidal anti-inflammatory drugs, or NSAIDs). These latter patients have a high frequency of chronic sinusitis.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • a doctor can diagnose sinusitis by medical history, physical examination, X-rays, and if necessary, MRIs or CT scans (magnetic resonance imaging and computed tomography). After diagnosing sinusitis and identifying a possible cause, a doctor can prescribe a course of treatment that will reduce the inflammation and relieve the symptoms. Treating acute sinusitis typically requires re-establishing drainage of the nasal passages, controlling or eliminating the source of the inflammation, and relieving the pain. Doctors generally recommend decongestants to reduce the congestion, antibiotics to control a bacterial infection, if present, and pain relievers to reduce the pain.
  • the ORP water solution administered in accordance with the invention is a highly effective inhibitor of mast cell degranulation, one of the primary inflammation-causing biological cascades.
  • the ORP water solution administered in accordance with the invention inhibits degranulation of mast cells regardless of whether they are activated with an antigen or a calcium ionophore.
  • the ORP water solution administered in accordance with the present invention non-selectively inhibits the secretion of pro-inflammatory cytokines in mast cells.
  • the ORP water solution of the present invention can inhibit the secretion of, e.g., TNF- ⁇ and MIP 1- ⁇ in mast cells.
  • ORP water solution administered in accordance with the invention also can inhibit the secretion of pro-inflammatory cytokines in other cytokine-secreting cells.
  • the ORP water solution administered in accordance with the invention preferably inhibits mast cell degranulation by more than about 50% relative to untreated mast cells, more preferably by more than about 80% relative to untreated mast cells, still more preferably by more than about 90% relative to untreated mast cells, and even more preferably by more than about 95% relative to untreated mast cells, when contacted with the ORP water solution for up to about 30 minutes, more preferably for up to about 15 minutes, and still more preferably for up to about 5 minutes.
  • histamine secretion e.g., from degranulation
  • a diluent e.g., water or saline solution
  • histamine secretion can be therapeutically inhibited by administering compositions in which the ORP water solution is diluted, e.g., by a ratio of up to about 50% (vol./vol.) ORP water solution/diluent, by a ratio of up to about 25% (vol./vol.) ORP water solution/diluent, by a ratio of up to about 10% (vol./vol.) ORP water solution/diluent, by a ratio of up to about 5% (vol./vol.) ORP water solution/diluent, or even by a ratio of up to about 1% (vol./vol.) ORP water solution/diluent.
  • the ORP water solution is diluted, e.g., by a ratio of up to about 50% (vol./vol.) ORP water solution/diluent, by a ratio of up to about 25% (vol./vol.) ORP water solution/diluent, by a ratio of up to about 10% (vol./vol.)
  • the ORP water solution administered in accordance with the invention also preferably inhibits the secretion of TNF- ⁇ by more than about 50%, more preferably by more than about 60%, still more preferably by more than about 70%, and even more preferably by more than about 85%.
  • the ORP water solution administered in accordance with the invention also preferably inhibits the secretion of MIP 1- ⁇ by more than 25%, more preferably by more than about 50%, and still more preferably by more than about 60%.
  • the ORP water solution administered in accordance with the invention also preferably inhibits the secretion of IL-6 and/or IL-13 by more than 25%, more preferably by more than about 50%, and still more preferably by more than about 60%.
  • the sinusitis treatable or preventable in accordance with the present invention also can include sinusitis that results from an infection.
  • the present invention provides a method of treating or preventing sinusitis, wherein the sinusitis results from infection caused by, e.g., one or more microorganisms selected from the group consisting of viruses, bacteria, and fungi.
  • the present invention provides a method of treating or preventing a viral sinusitis, wherein the sinusitis is associated with infection by one or more viruses, which are preferably susceptible to the ORP water solution administered to the patient.
  • Susceptible viruses can include, e.g., one or more viruses selected from the group consisting of HIV, coxsackie viruses, adenoviruses, rhinoviruses, herpes viruses, influenza viruses, and combinations thereof.
  • the present invention also provides a method of treating or preventing a bacterial sinusitis, wherein the sinusitis is associated with infection by one or more bacteria, which are preferably susceptible to the ORP water solution administered to the patient.
  • Susceptible bacteria can include, e.g., one or more bacteria selected from the group consisting staphylococci, streptococci, corynebacterium , anaerobes, and, particularily, Streptococcus pneumoniae and Haemophilus influenzae .
  • the present invention further provides a method of treating or preventing a fungal sinusitis, wherein the sinusitis is associated with infection by one or more fungi, which are preferably susceptible to the ORP water solution administered to the patient.
  • Susceptible fungi can include, e.g., one or more fungi selected from the group consisting of zygomycetes, aspergillosis , and candida.
  • the invention also provides methods for killing bacteria in biofilms, e.g., Pseudomonas aeruginosa in biofilms.
  • the invention further provides methods for killing of Moraexlla catarrhalis and antibotic resistant bacteria, e.g., penicillin resistant Streptococcus .
  • the methods disclosed herein can be used in accordance with the invention for killing bacteria using ORP water solutions faster than with using Bacitracin.
  • Suitable antihistamines can include, e.g., diphenhydramine, chlorpheniramine, brompheniramine, loratadine, clemastine, fexofenadine, derivatives thereof, and combinations thereof.
  • Suitable decongestants can include, e.g., phenylephrine, pseudoephedrine, other ⁇ - and ⁇ -adrenergic agonists, derivatives thereof, and combinations thereof.
  • Suitable antibacterial agents can include, e.g., penicillins, cephalosporins or other ⁇ -lactams, macrolides (e.g., erythromycin, 6-0-methylerythromycin, and azithromycin), fluoroquinolones, sulfonamides, tetracyclines, aminoglycosides, clindamycin, quinolones, metronidazole, vancomycin, chloramphenicol, antibacterially effective derivatives thereof, and combinations thereof.
  • Suitable antifungal agents can include, e.g., amphotericin B, fluconazole, flucytosine, ketoconazole, miconazole, derivatives thereof, and combinations thereof.
  • Suitable antiviral agents can include, e.g., acyclovir, amantadine, didanosine, famciclovir, fortovase, gancicolvir, valacyclovir, zanamivir, interferons, derivatives thereof, and combinations thereof.
  • Suitable anti-inflammatory agents can include, e.g., one or more anti-inflammatory drugs, e.g., one or more anti-inflammatory steroids or one or more non-steroidal anti-inflammatory drugs (NSAIDs).
  • NSAIDs non-steroidal anti-inflammatory drugs
  • Exemplary anti-inflammatory drugs can include, e.g., leukotriene receptor antagonists, cyclophilins, FK binding proteins, steroids, and NSAIDs.
  • the ORP water solution can be administered topically, e.g., as a liquid,.spray, mist, aerosol or steam by any suitable process, e.g., by spraying, aerosolization, nebulization, atomization, and the like.
  • the ORP water solution is administered to the upper respiratory airway and/or one or more cranial sinuses as a spray, mist, or aerosol.
  • the ORP water solution is administered by aerosolization, nebulization or atomization.
  • the ORP water solution can be mixed with one or more suitable carriers (e.g., a diluent) using a multiple-chamber device, e.g., a dual-chamber device, in which the ORP water solution and carrier(s) reside in separate chambers and are combined and/or mixed when they exit the chambers so that the ORP water solution and carrier(s) are combined upon (e.g., immediately prior to or simultaneously upon) delivery to the patient.
  • suitable carriers e.g., a diluent
  • Medical nebulizers for example, have been used to deliver a metered dose of a physiologically active liquid into an inspiration gas stream for inhalation by a recipient. See, e.g., U.S. Pat. No. 6,598,602 (hereby incorporated by reference). Medical nebulizers can operate to generate liquid droplets, which form an aerosol with the inspiration gas.
  • medical nebulizers may be used to inject water droplets into an inspiration gas stream to provide gas with a suitable moisture content to a recipient, which is particularly useful where the inspiration gas stream is provided by a mechanical breathing aid such as a respirator, ventilator or anaesthetic delivery system.
  • a mechanical breathing aid such as a respirator, ventilator or anaesthetic delivery system.
  • nebulizer An exemplary nebulizer is described, for example, in WO 95/01137, which describes a hand held device that operates to eject droplets of a medical liquid into a passing air stream (inspiration gas stream), which is generated by a recipient's inhalation through a mouthpiece.
  • WO 95/01137 describes a hand held device that operates to eject droplets of a medical liquid into a passing air stream (inspiration gas stream), which is generated by a recipient's inhalation through a mouthpiece.
  • U.S. Pat. No. 5,388,571 which describes a positive-pressure ventilator system which provides control and augmentation of breathing for a patient with respiratory insufficiency and which includes a nebulizer for delivering particles of liquid medication into the airways and alveoli of the lungs of a patient.
  • U.S. Pat. No. 5,287,847 (hereby incorporated by reference) describes a pneumatic nebulizing apparatus with scalable flow rates and output volumes for delivering a medicinal aerosol to neonates, children and adults.
  • U.S. Pat. No. 5,063,922 (hereby incorporated by reference) describes an ultrasonic atomizer.
  • the ORP water solution also may be dispensed in aerosol form as part of an inhaler system for treatment of infections in the lungs and/or air passages or for the healing of wounds in such parts of the body.
  • the particular device used to disperse the ORP water solution may be incorporated into a ventilation system to provide for widespread application of the ORP water solution throughout an entire house or healthcare facility (e.g., hospital, nursing home, etc.).
  • the ORP water solution also can be administered to a patient in a chamber or tent, or can be administered through a mask or endoscopically.
  • the ORP water solution can be administered alone or in combination with one or more pharmaceutically acceptable carriers, which can include, e.g., vehicles, adjuvants, excipients, diluents, combinations thereof, and the like.
  • Such carriers are preferably compatible with one or more of the chemical species that exist in the ORP water solution.
  • One skilled in the art can easily determine the appropriate formulation and method for administering the ORP water solution administered in accordance with the present invention. Any necessary adjustments in dose can be readily made by a skilled practitioner to address the nature and/or severity of the condition being treated in view of one or more clinically relevant factors, such as, e.g., side effects, changes in the patient's overall condition, and the like.
  • the ORP water solution can be formulated by combining or diluting the ORP water solution with up to about 25% (wt./wt. or vol./vol.) of a suitable carrier, up to about 50% (wt./wt. or vol./vol.) of a suitable carrier, up to about 75% (wt./wt. or vol./vol.) of a suitable carrier, up to about 90% (wt./wt. or vol./vol.) of a suitable carrier, up to about 95% (wt./wt. or vol./vol.) of a suitable carrier, or even up to about 99% (wt./wt. or vol./vol.) or more of a suitable carrier.
  • a suitable carrier up to about 25% (wt./wt. or vol./vol.) of a suitable carrier, up to about 50% (wt./wt. or vol./vol.) of a suitable carrier, up to about 75% (wt./wt. or vol./vol.
  • Suitable carriers can include, e.g., water (e.g., distilled water, sterile water, e.g., sterile water for injection, sterile saline and the like). Suitable carriers also can include one or more carriers described in U.S. patent application No. 10/916,278 (hereby incorporated by reference). Exemplary formulations can include solutions in which the ORP water solution is diluted with sterile water, sterile saline, or a combination thereof.
  • the ORP water solution can be diluted by up to about 25% (vol./vol.), by up to about 50% (vol./vol.), by up to about 75% (vol./vol.), by up to about 90% (vol./vol.), by up to about 95% (vol./vol.), or by up to 99% (vol./vol.) or more with sterile water, sterile saline, or a combination thereof.
  • ORP water solution administered in accordance with the invention is virtually free of toxicity to normal tissues and normal mammalian cells.
  • the ORP water solution administered in accordance with the invention causes no significant decrease in the viability of eukaryotic cells, no significant increase in apoptosis, no significant acceleration of cell aging and/or no significant oxidative DNA damage in mammalian cells.
  • the non-toxicity is particularly advantageous, and perhaps even surprising, given that the disinfecting power of the ORP water solution administered in accordance with the invention is roughly equivalent to that of hydrogen peroxide, yet, unlike hydrogen peroxide, is virtually non-toxic to normal tissues and normal mammalian cells.
  • Factors which can be taken into account for a specific dosage can include, for example, bioavailability, metabolic profile, time of administration, route of administration, rate of excretion, the pharmacodynamics associated with a particular ORP water solution in a particular patient, and the like. Other factors can include, e.g., the potency or effectiveness of the ORP water solution with respect to the particular condition to be treated, the severity of the symptoms presented prior to or during the course of therapy, and the like.
  • the ORP water solution administered in accordance with the invention also can produce about a four log (10 4 ) reduction in the concentration of a spore suspension of Bacillus athrophaeus spores within about thirty (30) seconds of exposure when measured at least about two months after preparation of the ORP water solution.
  • the ORP water solution can achieve this reduction in the concentration of Bacillus athrophaeus spores when measured at least about six months after preparation, and more preferably when measured at least about one year after preparation.
  • the total chlorine content which includes both free chlorine and bound chlorine, is from about 50 parts per million (ppm) to about 400 ppm. More preferably, the total chlorine content is about 80 ppm to about 150 ppm.
  • the chlorine content may be measured by methods known in the art, such as the DPD colorimeter method (Lamotte Company, Chestertown, Md.) or other known methods such as, e.g., methods established by the Environmental Protection Agency.
  • DPD colorimeter method a yellow color is formed by the reaction of free chlorine with N,N-diethyl-p-phenylenediamine (DPD) and the intensity is measured with a calibrated calorimeter that provides the output in parts per million. Further addition of potassium iodide turns the solution a pink color to provide the total chlorine value. The amount of bound chlorine present is then determined by subtracting free chlorine from the total chlorine.
  • the total amount of free chlorine species is preferably from about 10 ppm to about 400 ppm, more preferably from about 50 ppm to about 200 ppm, and most preferably from about 50 ppm to about 80 ppm.
  • the amount of hypochlorous acid is preferably from about 15 ppm to about 35 ppm.
  • the amount of sodium hypochlorite is preferably in the range of from about 25 ppm to about 50 ppm.
  • the ORP water solution includes one or more chlorine species or one and, optionally, or more precursors thereof, and is stable for at least about 24 hours, preferably for at least about one week, more preferably for at about least two months, and still more preferably for at least about six months after its preparation. Even more preferably, such ORP water solution is stable for at least about one year, and most preferably for more than about one year, e.g., at least about two years or at least about three years.
  • the ORP water solution which includes one or more chlorine species, one or more additional superoxidized water species (e.g., one or more oxygen species, dissolved oxygen) or one or more precursors thereof and has a pH of from about 6 to about 8. More preferably, the pH of such ORP water solution is from about 6.2 to about 7.8, and most preferably from about 7.4 to about 7.6.
  • An exemplary ORP water solution administered in accordance with the present invention can comprise, e.g., from about 15 ppm to about 35 ppm hypochlorous acid, from about 25 ppm to about 50 ppm sodium hypochlorite, with these components at a pH of from about 6.2 to about 7.8, and can be stable for at least about one week, e.g., at least about two months, at least about six months, at least about one year, or more than about one year, e.g., at least about two years or at least about three years.
  • the ORP water solutions administer in accordance with the invention preferably comprises one or more oxidized water species which can yield free radicals (such as, e.g., hydroxyl radicals) on exposure to iron.
  • the ORP water can optionally include one or more chemical compounds generated during the production thereof such as, e.g., sodium hydroxide (NaOH), chlorine dioxide (ClO 2 ), peroxides (e.g., hydrogen peroxide (H 2 O 2 ), and ozone (O 3 ) although, it has been reported that sodium hydroxide, chlorine dioxide, hydrogen peroxide, and ozone may react with hypochlorite resulting in their consumption and the production of other chemical species.
  • sodium hydroxide, chlorine dioxide, hydrogen peroxide, and ozone may react with hypochlorite resulting in their consumption and the production of other chemical species.
  • anode water typically contains oxidized species produced from the electrolytic reaction while cathode water typically contains reduced species from the reaction.
  • Anode water generally has a low pH, typically of from about 1 to about 6.8.
  • the anode water preferably contains chlorine in various forms including, for example, chlorine gas, chloride ions, hydrochloric acid and/or hypochlorous acid, or one or more precursors thereof.
  • Oxygen in various forms is also preferably present including, for example, oxygen gas and, optionally, peroxides, and/or ozone, or one or more precursors thereof.
  • Cathode water generally has a high pH, typically from about 7.2 to about 11.
  • Cathode water can contain hydrogen gas, hydroxyl radicals, and/or sodium ions.
  • the ORP water solution administered in accordance with the invention can include a mixture of anode water (e.g., water produced in the anode chamber of an electrolytic cell) and cathode water (e.g., water produced in the cathode chamber of an electrolysis cell).
  • the ORP water solution administered in accordance with the present invention contains cathode water, e.g., in an amount of from about 10% by volume to about 90% by volume of the solution. More preferably, cathode water is present in the ORP water solution in an amount of from about 10% by volume to about 50% by volume, and still more preferably in an amount of from about 20% by volume to about 40% by volume of the solution, e.g., from about 20% by volume to about 30% by volume of the solution.
  • anode water can be present in the ORP water solution, e.g., in an amount of from about 50% by volume to about 90% by volume of the solution.
  • Exemplary ORP water solutions can contain from about 10% by volume to about 50% by volume of cathode water and from about 50% by volume to about 90% by volume of anode water.
  • the anode and cathode water can be produced using the three-chambered electrolysis cell shown in FIG. 1 .
  • the electrolysis cell 100 has an anode chamber 102 , cathode chamber 104 and salt solution chamber 106 .
  • the salt solution chamber is located between the anode chamber 102 and cathode chamber 104 .
  • the anode chamber 102 has an inlet 108 and outlet 110 to permit the flow of water through the anode chamber 100 .
  • the cathode chamber 104 similarly has an inlet 112 and outlet 114 to permit the flow of water through the cathode chamber 104 .
  • the salt solution chamber 106 has an inlet 116 and outlet 118 .
  • the electrolysis cell 100 preferably includes a housing to hold all of the components together.
  • the anode chamber 102 is separated from the salt solution chamber by an anode electrode 120 and an anion ion exchange membrane 122 .
  • the anode electrode 120 may be positioned adjacent to the anode chamber 102 with the membrane 122 located between the anode electrode 120 and the salt solution chamber 106 .
  • the membrane 122 may be positioned adjacent to the anode chamber 102 with the anode electrode 120 located between the membrane 122 and the salt solution chamber 106 .
  • the cathode chamber 104 is separated from the salt solution chamber by a cathode electrode 124 and a cathode ion exchange membrane 126 .
  • the cathode electrode 124 may be positioned adjacent to the cathode chamber 104 with the membrane 126 located between the cathode electrode 124 and the salt solution chamber 106 .
  • the membrane 126 may be positioned adjacent to the cathode chamber 104 with the cathode electrode 124 located between the membrane 126 and the salt solution chamber 106 .
  • a source of electrical potential is connected to the anode electrode 120 and cathode electrode 124 so as to induce an oxidation reaction in the anode chamber 102 and a reduction reaction in the cathode chamber 104 .
  • the ion exchange membranes 122 and 126 used in the electrolysis cell 100 may be constructed of any suitable material to permit the exchange of ions between the salt solution chamber 106 and the anode chamber 102 such as, e.g., chloride ions (Cl) and between the salt solution salt solution chamber 106 and the cathode chamber 104 such as, e.g., sodium ions (Na + ).
  • the anode ion exchange membrane 122 and cathode ion exchange membrane 126 may be made of the same or different material of construction.
  • the anode ion exchange membrane comprises a fluorinated polymer.
  • Suitable fluorinated polymers include, for example, perfluorosulfonic acid polymers and copolymers such as perfluorosulfonic acid/PTFE copolymers and perfluorosulfonic acid/TFE copolymers.
  • the ion exchange membrane may be constructed of a single layer of material or multiple layers. Suitable ion exchange membrane polymers can include one or more ion exchange membrane polymers marketed under the trademark Nafion®.
  • the source of the water for the anode chamber 102 and cathode chamber 104 of the electrolysis cell 100 may be any suitable water supply.
  • the water may be from a municipal water supply or alternatively pretreated prior to use in the electrolysis cell.
  • the water is pretreated and is selected from the group consisting of softened water, purified water, distilled water, and deionized water. More preferably, the pretreated water source is ultrapure water obtained using reverse osmosis purification equipment.
  • the salt solution can have any suitable concentration.
  • the salt solution can be saturated or concentrated.
  • the salt solution is a saturated sodium chloride solution.
  • FIG. 2 illustrates what are believed to be various ionic species produced in the three chambered electrolysis cell useful in connection with the invention.
  • the three chambered electrolysis cell 200 includes an anode chamber 202 , cathode chamber 204 , and a salt solution chamber 206 .
  • the ions present in the salt solution flowing through the salt solution chamber 206 migrate through the anode ion exchange membrane 212 and cathode ion exchange membrane 214 into the water flowing through the anode chamber 202 and cathode chamber 204 , respectively.
  • Positive ions migrate from the salt solution 216 flowing through the salt solution chamber 206 to the cathode water 218 flowing through the cathode chamber 204 .
  • Negative ions migrate from the salt solution 216 flowing through the salt solution chamber 206 to the anode water 220 flowing through the anode chamber 202 .
  • the salt solution 216 is aqueous sodium chloride (NaCl), which contains both sodium ions (Na + ) and chloride ions (Cl ⁇ ) ions.
  • Na + ions migrate from the salt solution 216 to the cathode water 218 .
  • Negative Cl ⁇ ions migrate from the salt solution 216 to the anode water 220 .
  • the sodium ions and chloride ions may undergo further reaction in the anode chamber 202 and cathode chamber 204 .
  • chloride ions can react with various oxygen ions and other species (e.g., oxygen containing free radicals, O 2 , O 3 ) present in the anode water 220 to produce ClOn- and ClO ⁇ .
  • Other reactions may also take place in the anode chamber 202 including the formation of oxygen free radicals, hydrogen ions (H + ), oxygen (e.g., as O 2 )and, optionally, ozone (O 3 ) and peroxides.
  • hydrogen gas (H 2 ), hydroxide ions (OH ⁇ ), and other radicals, and, optionally, sodium hydroxide (NaOH) may be formed.
  • the apparatus for producing the ORP water solution also can be constructed to include at least two three chambered electrolysis cells.
  • Each of the electrolytic cells includes an anode chamber, cathode chamber, and salt solution chamber separating the anode and cathode chambers.
  • the apparatus includes a mixing tank for collecting the anode water produced by the electrolytic cells and a portion of the cathode water produced by one or more of the electrolytic cells.
  • the apparatus further includes a salt recirculation system to permit recycling of the salt solution supplied to the salt solution chambers of the electrolytic cells.
  • FIG. 3 A diagram of an exemplary process for producing an ORP water solution using two electrolysis cells is shown in FIG. 3 .
  • the process 300 includes two three-chambered electrolytic cells, specifically a first electrolytic cell 302 and second electrolytic cell 304 .
  • Water is transferred, pumped or otherwise dispensed from the water source 305 to anode chamber 306 and cathode chamber 308 of the first electrolytic cell 302 and to anode chamber 310 and cathode chamber 312 of the second electrolytic cell 304 .
  • this process can produce from about 1 liter/minute to about 50 liters/minute of ORP water solution.
  • the production capacity may be increased by using additional electrolytic cells. For example, three, four, five, six, seven, eight, nine, ten or more three-chambered electrolytic cells may be used to increase the output of the ORP water solution administered in accordance with the invention.
  • the mixing tank 314 may optionally be connected to a recirculation pump 315 to permit homogenous mixing of the anode water and portion of cathode water from electrolysis cells 302 and 304 . Further, the mixing tank 314 may optionally include suitable devices for monitoring the level and pH of the ORP water solution.
  • the ORP water solution may be transferred from the mixing tank 314 via pump 317 for application in disinfection or sterilization at or near the location of the mixing tank. Alternatively, the ORP water solution may be dispensed into one or more suitable containers for shipment to a remote site (e.g., warehouse, hospital, etc.).
  • the process 300 further includes a salt solution recirculation system to provide the salt solution to salt solution chamber 322 of the first electrolytic cell 302 and the salt solution chamber 324 of the second electrolytic cell 304 .
  • the salt solution is prepared in the salt tank 320 .
  • the salt is transferred via pump 321 to the salt solution chambers 322 and 324 .
  • the salt solution flows in series through salt solution chamber 322 first followed by salt solution chamber 324 .
  • the salt solution may be pumped to both salt solution chambers simultaneously.
  • the salt solution may flow through a heat exchanger 326 in the mixing tank 314 to control the temperature of the ORP water solution as needed.
  • the ions present in the salt solution are depleted over time in the first electrolytic cell 302 and second electrolytic cell 304 .
  • An additional source of ions periodically can be added to the mixing tank 320 to replace the ions that are transferred to the anode water and cathode water.
  • the additional source of ions may be used, e.g., to maintain a constant pH of the salt solution, which can to drop (i.e., become acidic) over time.
  • the source of additional ions may be any suitable compound including, for example, salts such as, e.g., sodium chloride.
  • sodium hydroxide is added to the mixing tank 320 to replace the sodium ions (Na + ) that are transferred to the anode water and cathode water.
  • the ORP water solution can be transferred to one or more suitable containers, e.g., a sealed container for distribution and sale to end users such as, e.g., health care facilities including, e.g., hospitals, nursing homes, doctor offices, outpatient surgical centers, dental offices, and the like.
  • suitable containers can include, e.g., a sealed container that maintains the sterility and stability of the ORP water solution held by the container.
  • the container can be constructed of any material that is compatible with the ORP water solution.
  • the container is generally non-reactive with one or more ions or other species present in the ORP water solution.
  • the container is constructed of plastic or glass.
  • the plastic can be rigid so that the container is capable of being stored on a shelf.
  • the container can be flexible, e.g., a container made of flexible plastic such as, e.g., a flexible bag.
  • the headspace gas of the sealed container can be air or any other suitable gas, which preferably does not react with one or more species in the ORP water solution.
  • Suitable headspace gases can include, e.g., nitrogen, oxygen, and mixtures thereof.
  • the ORP water solution administered in accordance with the present invention also can be used for treating or preventing cell-mediated inflammation and inflammation, which results from an autoimmune reaction, including, but not limited to, SLE, autoimmune thyroiditis, sarcoidosis, inflammatory bowel disease, rheumatoid arthritis, and rheumatic fever.
  • the ORP water solution administered in accordance with the present invention can be used for treating or preventing inflammation, which results from infection, e.g., from an infection by one or more microorganisms selected from the group consisting of viruses, bacteria, and fungi, including hypersensitivity and autoiimmune-mediated inflammation resulting from infection.
  • the ORP water solution administered in accordance with the present invention also can be used for preventing or treating inflammation affecting one or more upper respiratory airway tissues (e.g., nasal tissue as described herein) or lung tissues.
  • upper respiratory airway tissues e.g., nasal tissue as described herein
  • lung tissues e.g., pharyngitis, asthma, and the like, which are preventable or treatable with the ORP solution administered in accordance with the invention.
  • Type II hypersensitivity is mediated by antibodies directed to antigens on the surfaces of cells and in the extracellular space. These antibodies can direct cell lysis or result in opsonization of the target molecules (preparation for phagocytosis by other cells). Alternatively, the antibodies can be directed to and activate cell surface receptors. Conditions resulting from Type II reactions include transfusion reactions, Graves disease (thyrotoxicosis), drug reactions, pernicious anemia, and acute rheumatic fever. In rheumatic fever the antibodies are formed against Streptococcal antigens but, cross-react with human tissues such as heat valves.
  • the ORP water solution administered in accordance with the invention also can be used for the prevention or treatment of an infection, e.g., by one or more infectious pathogens such as, for example, infectious microorganisms.
  • infectious pathogens such as, for example, infectious microorganisms.
  • microorganisms can include, for example, viruses, bacteria, and fungi.
  • the viruses can include, e.g., one or more viruses selected from the group consisting of adenoviruses, herpes viruses, coxsackie viruses, HIV, rhinoviruses, coronaviruses, and flu viruses.
  • administering the ORP water solution for the prevention or treatment of infection also can serve to prevent or treat sinusitis associated with the infection (or the affected tissues) as described herein.
  • the ORP water solution administered in accordance with the invention can be used for treating tissues, which have been impaired or damaged by one or more bums, cuts, abrasions, scrapes, rashes, ulcers, puncture wounds, combinations thereof, and the like, which are not necessarily caused by surgery.
  • the ORP water solution administered in accordance with the invention can be used for treating impaired or damaged tissue, which is infected, or tissue impaired or damaged due to infection.
  • infection can be caused by one or more infectious pathogens, such as, e.g., one or more microorganisms selected from the group consisting of viruses, bacteria, and fungi, as described herein.
  • the ORP water solution administered in accordance with the invention also can be used as a disinfectant to eradicate microorganisms, including bacteria, viruses and spores, in a variety of settings, e.g., in the healthcare and medical device fields, to disinfect surfaces and medical equipment, and also can be applied in wound care, medical device sterilization, food sterilization, hand disinfection in medical personnel, hospitals, consumer households and anti-bioterrorism.
  • the ORP water solution can be used for disinfecting a surface, e.g., by contacting the surface with an anti-infective amount of the ORP water solution.
  • the surface can be contacted using any suitable method. For example, the surface can be contacted by irrigating the surface with the ORP water solution, so as to disinfect the surface.
  • the surface(s) can include one or more biological surfaces, one or more inanimate surfaces, and combinations thereof.
  • Biological surfaces can include, for example, tissues within one or more body cavities such as, for example, the oral cavity, the sinus cavity, the cranial cavity, the abdominal cavity, and the thoracic cavity. Tissues within the oral cavity include, e.g., mouth tissue, gum tissue, tongue tissue, and throat tissue.
  • the biological tissue also can include muscle tissue, bone tissue, organ tissue, mucosal tissue, vascular tissue, neurological tissue, and combinations thereof.
  • Inanimate surfaces include, for example, surgically implantable devices, prosthetic devices, and medical devices.
  • the surfaces of internal organs, viscera, muscle, and the like, which may be exposed during surgery can be disinfected, e.g., to maintain sterility of the surgical environment.
  • the ORP water solution may also be applied to humans and/or animals to treat various conditions, including inflammation, associated with one or more of the following: surgical/open wound cleansing agent; skin pathogen disinfection (e.g., for bacteria, mycoplasmas, virus, fungi, prions); battle wound disinfection; wound healing promotion; burn healing promotion; treatment of stomach ulcers; wound irrigation; skin fungi; psoriasis; athlete's foot; pinkeye and other eye infections; ear infections (e.g., swimmer's ear); lung/nasal/sinus infections; and other medical applications on or in the human or animal body.
  • skin pathogen disinfection e.g., for bacteria, mycoplasmas, virus, fungi, prions
  • battle wound disinfection e.g., for bacteria, mycoplasmas, virus, fungi, prions
  • wound healing promotion e.g., burn healing promotion
  • treatment of stomach ulcers e.g., stomach ulcers
  • wound irrigation e
  • Organisms that can be controlled, reduced, killed or eradicated by treatment with the ORP water solution used in accordance with the invention include, e.g., Pseudomonas aeruginosa, Escherichia coli, Enterococcus hirae, Acinetobacter baumannii, Acinetobacter species, Bacteroides fragilis, Enterobacter aerogenes, Enterococcus faecalis , Vancomycin resistant- Enterococcus faecium (VRE, MDR), Vancomycin resistant- Escherichia coli, Haemophilus influenzae, Klebsiella oxytoca, Klebsiella pneumoniae, Micrococcus luteus, Proteus mirabilis, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus,
  • the ORP water solution can also be used in accordance with the invention to controll, reduce, kill or eradicate viruses including, for example, adenovirus, human immunodeficiency virus (HIV), rhinovirus, influenza (e.g., influenza A), hepatitis (e.g., hepatitis A), coronavirus (responsible for Severe Acute Respiratory Syndrome (SARS)), rotavirus, respiratory syncytial virus, herpes simplex virus, varicella zoster virus, rubella virus, and other susceptible viruses.
  • viruses including, for example, adenovirus, human immunodeficiency virus (HIV), rhinovirus, influenza (e.g., influenza A), hepatitis (e.g., hepatitis A), coronavirus (responsible for Severe Acute Respiratory Syndrome (SARS)), rotavirus, respiratory syncytial virus, herpes simplex virus, varicella zoster virus, rubella virus
  • allergens typically include any substance other than bacteria, fungi, yeasts, or viruses that can trigger an adverse immune response, or allergy, in susceptible people or animals. Asthma is a common physiological response following exposure to one or more of such allergens. Allergens can be either viable (i.e., from living or dead organisms) or non-viable (e.g., non-living such as textiles), and may be present in the environment, for example, in households and/or workplaces.
  • Protein-based household allergens that may be treated with the ORP water solution can include, for example, animal fur, skin, and feces, household dust, weeds, grasses, trees, mites, and pollens.
  • Animal allergens can include, for example, cat epithelium, dog epithelium, horse dander, cow dander, dog dander, guinea pig epithelium, goose feathers, mouse epithelium, mouse urine, rat epithelium and rat urine.
  • the ORP water solution may be applied to disinfect and sterilize in any suitable manner.
  • the equipment can be maintained in contact with the ORP water solution for a sufficient period of time to reduce the level of organisms present on the equipment to a desired level.
  • the ORP water solution can be applied to the hard surface directly from a container in which the ORP water solution is stored.
  • the ORP water solution can be poured, sprayed or otherwise directly applied to the hard surface.
  • the ORP water solution can then be distributed over the hard surface using a suitable substrate such as, for example, cloth, fabric or paper towel. In hospital applications, the substrate is preferably sterile.
  • the ORP water solution can first be applied to a substrate such as cloth, fabric or paper towel. The wetted substrate can then be contacted with the hard surface.
  • the ORP water solution can be applied to hard surfaces by dispersing the solution into the air as described herein.
  • the ORP water solution can be applied in a similar manner to humans and animals.
  • the substrate for the cleaning wipe can be any suitable water-insoluble absorbent or adsorbent material.
  • a wide variety of materials can be used as the substrate. It should have sufficient wet strength, abrasivity, loft and porosity. Further, the substrate should not adversely impact the stability of the ORP water solution. Examples include non woven substrates, woven substrates, hydroentangled substrates and sponges.
  • the substrate can have one or more layers. Each layer can have the same or different textures and abrasiveness. Differing textures can result from the use of different combinations of materials or from the use of different manufacturing processes or a combination thereof.
  • the substrate should not dissolve or break apart in water. The substrate can thereby provide a vehicle for delivering the ORP water solution to the surface to be treated.
  • nonwoven, water insoluble substrates examples include 100% cellulose Wadding Grade 1804 from Little Rapids Corporation, 100% polypropylene needlepunch material NB 701-2.8-W/R from American Non-wovens Corporation, a blend of cellulosic and synthetic fibres-Hydraspun 8579 from Ahlstrom Fibre Composites, and 70% Viscose/30% PES Code 9881 from PGI Nonwovens Polymer Corp. Additional examples of nonwoven substrates suitable for use in the cleaning wipes are described in U.S. Pat. Nos. 4,781,974, 4,615,937, 4,666,621, and 5,908,707, and International Patent Application Publications WO 98/03713, WO 97/40814, and WO 96/14835 (which are hereby incorporated by reference).
  • the liquid loading capacity of the substrate should be at least about 50%-1000% of the dry weight thereof, most preferably at least about 200%-800%. This is expressed as loading 1 ⁇ 2 to 10 times the weight of the substrate.
  • the weight of the substrate varies without limitation from about 0.01 to about 1,000 grams per square meter, most preferably 25 to 120 grams/m 2 (referred to as “basis weight”) and typically is produced as a sheet or web which is cut, die-cut, or otherwise sized into the appropriate shape and size.
  • the cleaning wipes will preferably have a certain wet tensile strength which is without limitation about 25 to about 250 Newtons/m, more preferably about 75-170 Newtons/m.
  • the ORP water solution can be dispensed, impregnated, coated, covered or otherwise applied to the substrate by any suitable method.
  • individual portions of substrate can be treated with a discrete amount of the ORP water solution.
  • a mass treatment of a continuous web of substrate material with the ORP water solution is carried out.
  • the entire web of substrate material can be soaked in the ORP water solution.
  • the ORP water solution can be sprayed or metered onto the web.
  • a stack of individually cut and sized portions of substrate can be impregnated or coated with the ORP water solution in its container by the manufacturer.
  • the cleaning wipes optionally can contain additional components to improve the properties of the wipes.
  • the cleaning wipes can further comprise polymers, surfactants, polysaccharides, polycarboxylates, polyvinyl alcohols, solvents, chelating agents, buffers, thickeners, dyes, colorants, fragrances, and mixtures thereof to improve the properties of the wipes.
  • These optional components should not adversely impact the stability of the ORP water solution. Examples of various components that may optionally be included in the cleaning wipes are described in U.S. Pat. Nos. 6,340,663, 6,649,584 and 6,624,135 (which are hereby incorporated by reference).
  • the cleaning wipes can be individually sealed with a heat-sealable or glueable thermoplastic overwrap (such as polyethylene, Mylar, and the like).
  • the wipes can also be packaged as numerous, individual sheets for more economical dispensing.
  • the cleaning wipes can be prepared by first placing multiple sheets of the substrate in a dispenser and then contacting the substrate sheets with the ORP water solution administered in accordance with the invention.
  • the cleaning wipes can be formed as a continuous web by applying the ORP water solution to the substrate during the manufacturing process and then loading the wetted substrate into a dispenser.
  • the dispenser includes, but is not limited to, a canister with a closure, or a tub with closure.
  • the closure on the dispenser can be employed to seal the moist wipes from the external environment and to prevent premature volatilization of the liquid ingredients.
  • the dispenser can be made of any suitable material that is compatible with both the substrate and the ORP water solution.
  • the dispenser can be made of plastic, such as high density polyethylene, polypropylene, polycarbonate, polyethylene terephthalate (PET), polyvinyl chloride (PVC), or other rigid plastics.
  • the continuous web of wipes can be threaded through a thin opening in the top of the dispenser, most preferably, through the closure.
  • a means of sizing the desired length or size of the wipe from the web can then be desirable.
  • a knife blade, serrated edge, or other means of cutting the web to desired size can be provided on the top of the dispenser, for non-limiting example, with the thin opening actually doubling in duty as a cutting edge.
  • the continuous web of wipes can be scored, folded, segmented, perforated or partially cut into uniform or non-uniform sizes or lengths, which would then obviate the need for a sharp cutting edge. Further, the wipes can be interleaved, so that the removal of one wipe advances the next.
  • the ORP water solution administered in accordance with the invention alternatively can be dispersed into the environment through a gaseous medium, such as air.
  • the ORP water solution can be dispersed into the air by any suitable means.
  • the ORP water solution can be formed into droplets of any suitable size and dispersed into a room.
  • the ORP water solution optionally can contain a bleaching agent.
  • the bleaching agent can include, e.g., any suitable compound that lightens or whitens a substrate.
  • the ORP water solution containing a bleaching agent can be used in home laundering to disinfect and sterilize bacteria and germs as well as brighten clothing.
  • Suitable bleaching agents include, but are not limited to, chlorine-containing bleaching agents and, optionally, peroxide-containing bleaching agents. Mixtures of bleaching agents also can be added to the ORP water solution.
  • the bleaching agent is added in the form of an aqueous solution to the ORP water solution.
  • Suitable chlorine-containing bleaching agents can include, e.g., chlorine, hypochlorites, N-chloro compounds, and, optionally, chlorine dioxide.
  • the chlorine-containing bleaching agent added to the ORP water solution is sodium hypochlorite or hypochlorous acid.
  • Other suitable chlorine-containing bleaching agents include, e.g., chlorine, calcium hypochlorite, bleach liquor (e.g., aqueous solution of calcium hypochlorite and calcium chloride), bleaching powder (e.g., mixture of calcium hypochlorite, calcium hydroxide, calcium chloride, and hydrates thereof), dibasic magnesium hypochlorite, lithium hypochlorite, chlorinated trisodium phosphate and mixtures thereof.
  • the ORP water solution has suitable physical characteristics for use in, e.g., disinfection, sterilization, cleaning, and/or the prevention and/or treatment of inflammation, sinusitis, peritonitis, or infection.
  • Examples 4-10 were subjected to a high spore count test using Bacillus subtilis var. niger spores (ATCC #9372 obtained from SPS Medical of Rush, New York). Spore suspensions were concentrated (by evaporation in a sterile hood) to 4 ⁇ 10 6 spores per 100 microliters. A 100 microliter sample of the spore suspension were mixed with 900 microliters of each of the samples in Examples 4-10. The samples were incubated at room temperature for periods of 1 to 5 minutes as set forth in Table 3.
  • Microcyn 60 is a superoxidized solution of neutral pH with germicidal, sterilizing and wound antiseptic activity in accordance with certifications obtained from the Secretariat of Health of Mexico.
  • Microcyn 60 is prepared from pure water and salt (NaCl), has a small concentration of sodium ( ⁇ 55 ppm) and chlorine ( ⁇ 80 ppm), a pH in the range of 7.2 to 7.8, and oxidation-reduction potential in the range of 840 mV to 960 mV.
  • Microcyn 60 is produced in one concentration only, and need not be activated or diluted.
  • Microcyn 60 is produced in only one concentration, the dose of Microcyn 60 can be changed only by changes in the volume applied per unit area of the skin.
  • the doses of Microcyn 60 applied topically to the intact skin varied from about 0.05 to about 0.07 mL/cm 2 ; in the study of acute dermatological toxicity and in the investigation of skin irritation, Microcyn 60 can be applied in doses of up to 8.0 mL/cm 2 , and in those that investigated its application in deep wounds applied Microcyn 60 in a dose of about 0.09 mL/cm 2 .
  • Microcyn 60 was applied topically to the intact skin, using a single application with exposure of 4 to 24 h. Multiple applications of Microcyn 60, one or two times a day, during a period of 7 days were assessed for deep wounds in rats.
  • the systemic toxicity of Microcyn 60 was also evaluated by means of an intraperitoneal injection in mice. For this, five mice were injected with a single dose (50 mL/kg) of Microcyn 60 by the intraperitoneal route. In the same way, five control mice were injected with a single dose (50 mL/kg) of saline solution (sodium chloride at 0.9%). In this investigation, neither mortality nor any evidence of systemic toxicity was observed in any of the animals that received the single intraperitoneal dose of Microcyn 60, indicating that the LD 50 is above 50 mL/kg.
  • Microcyn 60 was administered by the oral route to rats to allow its absorption and to characterize any inherent toxic effect of the product.
  • a single dose (4.98 mL/kg) was administered by esophageal tube to three albino rats of the Sprague-Dawley strain. There was no mortality, nor were there clinical signs or abnormalities in the autopsies of any of the animals exposed to the single oral dose of Microcyn 60.
  • Microcyn 60 was applied by the inhalatory route to rats to determine potential acute toxicity by inhalation. All the animals showed a very slight or slight reduction in activity and piloerection after the exposure, but they were all asymptomatic on the following day. Mortality or abnormalities were not observed at autopsy of the animals exposed to Microcyn 60 by inhalation.
  • Microcyn 60 when it has been applied to the intact skin, deep open dermal wounds, in the conjunctival sac, by oral and inhalation routes or by means of intraperitoneal injection, Microcyn 60 has not shown adverse effects related to the product. There is also experience in having treated more than 500 patients with wounds of very diverse nature in the skin and mucosae, with excellent antiseptic and cosmetic results. Accordingly, topically applied Microcyn 60 should be effective and well-tolerated in this clinical trial.
  • Microcyn 60 is packaged in transparent 240 mL PET sealed bottles. This product is stored at ambient temperature and remains stable for up to 2 years in such bottles. From its profile of high biological safety, Microcyn 60 can be safely disposed of, e.g., emptied into the sink without risk of contamination or corrosion.
  • the sporicidal activity trial was carried out in accordance with the PAHO [Pan-American Health Organization]/WHO protocol.
  • Microcyn 60 when it is administered as recommended, can eradicate bacteria, fungi, viruses and spores from one to fifteen minutes of exposure.
  • the exclusion criteria are as follows: fever >38° C.; bronchospasm (excluded by the clinic); severe cough; sinusitis-rhinitis (excluded by the clinic); esophageal reflux (excluded by the clinic); use of antibiotics in the two weeks prior to the study; patients who have taken part in another clinical study in the last 8 weeks; rheumatic fever; poststreptococcal glomerulonephritis; severe chronic cardiopathy; severe renal, hepatic or pulmonary insufficiencies; and pregnancy or lactation.
  • patients may use such concomitant medicines as antipyretics and analgesics, including paracetamol and acetylsalicylics but not anti-inflammatories such as ibuprofen, Mesulid, COX-2 inhibitors, or steroids.
  • antipyretics and analgesics including paracetamol and acetylsalicylics but not anti-inflammatories such as ibuprofen, Mesulid, COX-2 inhibitors, or steroids.
  • anti-inflammatories such as ibuprofen, Mesulid, COX-2 inhibitors, or steroids.
  • the patients are evaluated in three visits.
  • the patient clinically presents acute pharyngitis/tonsillitis, and the clinical history is taken, and a medical examination, rapid immunoassay for Streptococcus , and taking of a pharyngeal exudate is carried out.
  • the patient After being declared eligible and after having signed the letter of informed consent, the patient is prescribed two oropharyngeal cleansings of 30 sec and 5 mL Microcyn 60 each. These rinsings are done every 3 h for a total of four times a day for 3 days.
  • the second is made 72 h after having been treated with Microcyn 60.
  • the clinical evolution and side effects of Microcyn 60 are evaluated.
  • a new pharyngeal exudate is taken, and it will be decided, in accordance with the clinical evolution, if the continuing treatment will be with antibiotics or a palliative.
  • a third visit is done after 10 days to discharge the patient.
  • each patient must present A ⁇ -hemolytic Streptococcus pharyngitis/tonsillitis confirmed by culture. All the patients must comply with 18 rinsings of 30 sec and 5 mL of Microcyn 60 each, or a maximum of 24 rinsings in the space of 72 h.
  • the primary parameter of efficacy is a reduction by 3 orders of magnitude in the bacterial load of the initial culture compared to the culture taken after the administration of Microcyn 60. This bacteriological evaluation is realized 72 h after treatment with Microcyn 60. Secondary parameters of efficacy are the improvement reported clinically, with particular emphasis on the reduction of pharyngeal pain and dysphagia. Clinical symptoms are reported in visits 1, 2 and 3.
  • the results of bacteriological efficacy are issued by a bacteriologist independently of the clinical symptoms.
  • the tests for the group A Streptococcus antigen and the initial pharyngeal exudate culture are done in the first visit (Visit 1), in accordance with the Schedule of Evaluations and before the administration of Microcyn 60.
  • the second taking and culture of pharyngeal exudate is carried out 72 h after the administration of Microcyn 60 (Visit 2).
  • An antibiogram is done on all the cultures to determine the bacterial resistance to penicillin, erythromycin, clarithromycin and lincomycin by means of the standard diffusion disc test.
  • Bacteriological efficacy is defined as the reduction by three orders of magnitude of the bacterial count between the initial culture and the culture taken 72 h after administering Microcyn 60.
  • Bacteriological failure is indicated by a reduction of less than three orders of magnitude of the bacterial count in the culture at 72 h posttreatment. Indeterminate responses are documented in those cases in which the transport of the sample has been delayed for more than 48 h, in those cases in which the swab has not been immersed in the transport medium, or in those cases in which the sample has been lost. These cases are outside the analysis of the study and are replaced by new cases until those of forty eligible patients have been completed.
  • the follow-up and reporting phase begins when the patient finishes the administration of Microcyn 60, and from the second visit.
  • the patients are categorized as follows:
  • an oral antibiotic such as procaine penicillin, clarithromycin or azithromycin at the dose and for the time that the treating doctor indicates, and they are evaluated in one week.
  • the statistical analysis used in this clinical study takes into account all the patients who have received at least 18 rinsings of Microcyn 60 of 30 sec each in a period of 72 h. This same criterion is considered to include any patient in the analysis of tolerance.
  • the principal criterion for analysis of efficacy is the reduction of the bacterial count of ⁇ -hemolytic Streptococcus by three orders of magnitude in the culture carried out at 72 h posttreatment with Microcyn 60.
  • the statistical analysis is realized by means of a Wilcoxon paired samples test. Statistical analysis of the clinical variables is realized using the ANOVA test for quantitative variables. The minimal evaluable number of patients is 30 patients.
  • An adverse event is any contrary medical occurrence in a patient or subject of clinical investigation to whom a pharmaceutical product is administered and that does not necessarily have a causal relationship with that medicine.
  • An adverse event can, therefore, be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom or illness temporarily associated with the use of a medical product, whether it is considered to be related to this use or not. Preexisting conditions that deteriorate during a study are reported as adverse events.
  • the treatment is suspended at any time during the 72 h of duration in case of adverse events that are moderate to severe in intensity. Subsequent treatment is determined by the treating doctor. In accordance with this example, the effectiveness of an ORP water solution of the present invention for treating sinusitis is thus demonstrated.
  • This example demonstrates the virucidal activity of an exemplary ORP water solution against Adenovirus-serotype 5.
  • Adenoviral (Ad) vectors based on human adenovirus type 5 which are E1a-, partially E1-b, and partially E3-deleted were used.
  • a shuttle plasmid containing the Green Fluorescent Protein (GFP) reporter gene under the transcriptional control of pCMV was prepared (pAd-Track ). Homologous recombination of this pShuttle plasmid with AdEasy 1 plasmid was carried out in electrocompetent bacteria. Clones that had inserts were tested by restriction endonuclease digestions.
  • supercoiled plasmid DMA was transformed into DH10B cells for large scale amplification.
  • 293 cells ATCC 1573 were cultured in serum-free medium (OptiMEM®-GIBCO) and transfected with recombinant plasmid digested with Pad. Infected cells were monitored for cytopathic effect, collected and lysed with three cycles of freezing and thawing.
  • the resultant viruses (AdGFP) were purified with AdenoPure columns (BD Clontech) according to the manufacturer's instructions. Viruses were quantitated by OD 260/280. Final yield was 1.52 ⁇ 10 11 pfu/mL.
  • AdGFP green fluorescence protein gene
  • ORP water solution-treated virus for 1, 5, and 10 min could only express GFP in 2.8%, 0.13%, and 0.09% of HeLa cell cultures, respectively.
  • the infectious titer was 6.6 ⁇ 10 7 pfu in the control AdGFP-HeLa group.
  • the infectious titers were 2.0 ⁇ 10 6 , 5.2 ⁇ 10 4 and 2.2 ⁇ 10 4 at one, five and ten minutes of virus exposure to the ORP water solution, respectively.
  • the log-10 reduction factor was 1.5, 3.1, and 3.5 at one, five and ten minutes of viral exposure to the ORP water solution. Taken together, these results demonstrate that the virus exposure to the ORP water solution for 5 minutes achieves a log-10 reduction in the viral load of >3.
  • the control film was exposed to 2 ml HUT media for five minutes. The virus was then scraped and diluted. Separate dried films were exposed to 2 ml each of the ORP water solution for five minutes at room temperature. Following the exposure time, the plates were scraped and their contents were resuspended. The virus-ORP water solution mixture was immediately diluted (10:1) in HUT medium. Serial log dilutions of this resulting suspension were assayed for infectivity. (To control for a possible direct cytotoxic effect of ORP water solution on MT-2 cells, a 2 ml aliquot of ORP water solution was diluted serially (10:1 to 10:5) in medium and inoculated into MT-2 cell cultures.)
  • the MT-2 cell line was used as the indicator cell line in the infectivity assays. This line shows a cytopathic effect consisting of sincitia formation when infected with HIV-1.
  • Four microwells were inoculated with 0.2 ml of each dilution of the reconstituted virus suspension from test (reconstituted in ORP water) and control (reconsituted with control medium) groups. Uninfected cell controls were inoculated with test medium only. Cultures were incubated at 37° C. and 5% CO 2 .
  • the cultures were scored periodically every two days for the presence or absence of cytopathic effect as well as presence of p24-HIV-1 antigen by ELISA.
  • Experimental infection with control HIV-1 exerted a cytopathic effect and Ag p24 protein release into the supernatant in infected MT-2 cultures.
  • treatment of HIV-1 with the ORP water solution for five minutes achieved a log reduction factor >3 in the viral load as measured in MT-2 cultures by both assays.
  • HDFs human diploid fibroblasts
  • HP hydrogen peroxide
  • HP is known to be toxic to eukaryotic cells, increasing apoptosis and necrosis and reducing cellular viability.
  • cell viability, apoptosis and necrosis were measured in HDFs exposed to pure ORP water solution and 880 mM HP (a concentration employed for antiseptic uses of HP) for 5 and 30 minutes.
  • This example demonstrates the effect of an exemplary ORP water solution relative to hydrogen peroxide (HP) on oxidative DNA damage and formation of the DNA adduct 8-hydroxy-2′-deoxiguanosine (8-OHdG) in HDFs. It is known that the production of 8-OHdG adducts in a cell is a marker of oxidative damage at specific residues of DNA. In addition, high cellular levels of this adduct correlate with mutagenesis, carcinogenesis and cellular aging.
  • HP hydrogen peroxide
  • FIG. 5 shows the levels of 8-OHdG adducts present in DNA samples from HDFs after control treatments, ORP water solution treatments and HP-treatments for 30 minutes.
  • the exposure to ORP water solution for 30 minutes did not increase the formation of adducts in the treated cells in comparison to control cells after incubation for 30 minutes.
  • the treatment with 500 ⁇ M HP for 30 minutes increased the number of 8-OHdG adducts by about 25 fold relative to the control-treated or ORP water solution-treated cells.
  • the ORP water solution-treated cells were able to decrease the levels of 8-OHdG adducts if left in supplemented DMEM for 3 hours after exposure to the ORP water solution. Despite being allowed the same 3 hour recovery period, HP-treated cells still presented about 5 times more adducts than control-treated or ORP water solution treated cells. Altogether, these results demonstrate that acute exposure to the ORP water solution does not induce significant DNA oxidative damage. These results also indicate that the ORP water solution will not likely induce mutagenesis or carcinogenesis in vitro or in vivo.
  • This example demonstrates the effects on HDFs of chronic exposure to low concentrations of an exemplary ORP water solution versus HP. It is known that chronic oxidative stress induces premature aging of cells. In order to mimic a prolonged oxidative stress, primary HDF cultures were chronically exposed to low concentrations of the ORP water solution (10%) or HP (5 ⁇ M) during 20 population doublings. The expression and activity of the SA- ⁇ -galactosidase enzyme has previously been associated with the senescence process in vivo and in vitro. In this example the expression of the SA- ⁇ -galactosidase enzyme was analyzed after one month of continuous exposure of HDF to the ORP water solution or HP. The results are depicted in FIG. 6 .
  • This example demonstrates the effectiveness of an exemplary ORP water solution (Mycrocyn) in inhibiting mast cell degranulation.
  • Mast cells have been recognized as principal players in type I hypersensitivity disorders. Multiple clinical symptoms observed in atopic dermatitis, allergic rhinitis, and atopic asthma are produced by IgE-antigen stimulation of mast cells located in distinct affected tissues.
  • the currently accepted view of the pathogenesis of atopic asthma is that allergens initiate the process by triggering IgE-bearing pulmonary mast cells (MCs) to release mediators such as histamine, leukotrienes, prostaglandins, kininis, platelet activating factor (PAF), etc. in the so-called early phase of the reaction.
  • MCs IgE-bearing pulmonary mast cells
  • these mediators induce bronchoconstriction and enhance vascular permeability and mucus production.
  • those cells secrete various cytokines, including tumor necrosis factor alpha (TNF- ⁇ ), IL-4, IL-5 and IL-6, which participate in the local recruitment and activation of other inflammatory cells such as eosinophils, basophils, T lymphocytes, platelets and mononuclear phagocytes.
  • TNF- ⁇ tumor necrosis factor alpha
  • IL-4 interleukin-4
  • IL-5 interleukin-6
  • IL-6 IL-6
  • These recruited cells contribute to the development of an inflammatory response that may then become autonomous and aggravate the asthmatic symptoms.
  • This late phase response constitutes a long term inflammation process which can induce plastic changes in surrounding tissues (see Kumar et al., pp. 193-268).
  • Antigenic stimulation of mast cells occurs via the activation of the high affinity receptor for IgE (the Fc ⁇ RI receptor), which is a multimeric protein that binds IgE and subsequently can be aggregated by the interaction of the receptor-bound IgE with a specific antigen.
  • IgE the high affinity receptor for IgE
  • Fc ⁇ RI receptor the Fc ⁇ RI receptor
  • Its structure comprises four polypeptides, an IgE binding ⁇ chain, a ⁇ chain that serves to amplify its signaling capacity, and two disulfide-linked ⁇ chains, which are the principal signal transducers via the encoded immunoreceptor tyrosine-based (ITAM) activation motif.
  • ITAM immunoreceptor tyrosine-based
  • BMMC bone marrow-derived mast cells
  • RBL 2H3 the rat leukemia cell line
  • MC-9 other mast cell lines
  • This example demonstrates the inhibitory activity of an exemplary ORP water solution on mast cell activation by a calcium ionophore.
  • Mast cells can be stimulated via the activation of calcium fluxes induced by a calcium ionophore.
  • Signaling pathways activated by calcium ionophores have been characterized using bone marrow-derived mast cells (BMMC), the rat leukemia cell line RBL 2H3, mouse and rat peritoneal mast cells, and other mast cell lines, such as MC-9.
  • BMMC bone marrow-derived mast cells
  • RBL 2H3 the rat leukemia cell line
  • MC-9 other mast cell lines
  • the calcium mobilization causes mast cell degranulation (e.g., histamine release), cytoskeletal re-arrangements, and activation of different transcription factors (e.g., NFAT, NF ⁇ B, AP-1, PU.1, SP1, Ets.) which activate cytokine gene transcription that culminate with cytokine production and secretion.
  • different transcription factors e.g., NFAT, NF ⁇ B, AP-1, PU.1, SP1,
  • Mature murine BMMC were loaded with a monoclonal anti-Dinitrophenol IgE (300 ng/million cell) during 4 hours at 37° C.
  • Culture media was removed and cells were resuspended in physiological buffer (Tyrode's Buffer/BSA). Cells were then treated for 15 minutes at 37° C. with distinct concentrations of the ORP water solution (Microcyn). Buffer was removed and cells were resuspended in fresh Tyrode's/BSA and stimulated with calcium ionophore (100 mM A23187) during a 30 minute incubation at 37° C.
  • Degranulation was measured by ⁇ -hexosaminidase activity determination in supernatants and pellets of the stimulated cells, using a colorimetric reaction based on the capacity of this enzyme to hydrolyze distinct carbohydrates. ( ⁇ -hexosaminidase has been shown to be located in the same granules that contain histamine in mast cells.) The results ( FIG. 9 ) demonstrate that degranulation is significantly reduced with increasing concentrations of the ORP water solution.
  • ORP water solution is a non-specific inhibitor of histamine release.
  • ORP water solution even at different concentrations—will inhibit the degranulation of mast cells independently of the stimulus (e.g. antigen or ionophore).
  • the stimulus e.g. antigen or ionophore
  • ORP water solution probably modifies the secretory pathway system at the level of the plasma membrane and/or cytoskeleton. Because the mechanism of action of ORP water solution is believed to be non-specific, it is believed that ORP water solution can have broad potential clinical applications.
  • This example demonstrates the effect of an exemplary ORP water solution on the activation of mast cell cytokine gene transcription.
  • FIGS. 10A and 10B is an RNAase protection assay from mast cells treated with ORP water solution at different concentrations for 15 minutes and further stimulated by antigen as described in Example 20.
  • mRNA was extracted using affinity chromatography columns (RNAeasy kit, Qiagene) and the RNAse Protection Assay was performed using standard kit conditions (Clontech, Becton & Dickinson) in order to detect mRNA production of distinct cytokines after antigen challenge.
  • the cytokines included TNF- ⁇ , LIF, IL13, M-CSF, IL6, MIF and L32.
  • FIG. 10A and 10B show that the ORP solution water (Microcyn) did not modify cytokine mRNA levels after antigen challenge in mast cells irrespective of the concentrations of ORP water solution or antigen used for the experiment.
  • the level of transcripts (i.e., the RNA content of stimulated mast cells) of proinflammatory genes was not changed in ORP water solution-treated mast cells after being stimulated with various concentrations of antigen.
  • the ORP water solution inhibited the secretory pathway of these cytokines without affecting their transcription.
  • This example demonstrates the inhibitory activity of an exemplary ORP water solution on mast cell secretion of TNF- ⁇ .
  • Mast cells were treated with different concentrations of ORP water solution for 15 minutes and further stimulated by antigen as described in Example 20, Thereafter, the tissue culture medium was replaced and samples of the fresh medium were collected at various periods of time (2-8 hours) for measuring TNF- ⁇ levels. Samples were frozen and further analyzed with a commercial ELISA kit (Biosource) according to the manufacturer's instructions.
  • FIG. 11 shows that the level of secreted TNF- ⁇ to the medium from ORP water solution-treated cells after antigen stimulation is significantly decreased in comparison to the untreated cells.
  • ORP water solution inhibited TNF- ⁇ secretion of antigen-stimulated mast cells.
  • This example demonstrates the inhibitory activity of an exemplary ORP water solution on mast cell secretion of MIP 1- ⁇ .
  • Mast cells were treated with different concentrations of an exemplary ORP water solution (Microcyn) for 15 minutes and further stimulated by antigen as described in Example 20. Thereafter, the tissue culture medium was replaced and samples of the fresh medium were collected at various periods of time (2-8 hours) for measuring MIP 1- ⁇ levels. Samples were frozen and further analyzed with a commercial ELISA kit (Biosource) according to the manufacturer's instructions.
  • FIG. 12 shows that the level of secreted MIP 1- ⁇ to the medium from ORP water solution-treated cells after antigen stimulation was significantly decreased in comparison to the untreated cells.
  • ORP water solution inhibited MIP 1- ⁇ secretion of antigen-stimulated mast cells.
  • FIGS. 13 and 14 The results of analogous studies measuring IL-6 and IL-13 secretion are depicted in FIGS. 13 and 14 .
  • Examples 19-21 and this example further demonstrate that the ORP water solution is able to inhibit early and late phase allergic responses initiated by IgE receptor crosslinking.
  • This example demonstrates the safety of an exemplary ORP water solution (Microcyn) when sprayed into the nasal cavity of rabbits.
  • mice Forty-two rabbits were randomly assigned to four groups; Groups I, II, III, and IV (Table 6). Rabbits were treated as follows: on Day 0, sterile saline was administered to the Group I rabbits and benzalkonium chloride was administered to Group II rabbits. Also on Day 0, Microcyn was administered to Groups III and IV at 40 ppm and 80 ppm, respectively. All articles were dosed by nasal spray into the right nostril. On Day 7, following the 8th dose, one-third of the rabbits from each group were sacrificed and necropsied. The remaining animals were dosed daily through Day 14 when half of the remaining animals from each group were sacrificed and necropsied. On Day 21, after seven days without dosing, the remaining rabbits were sacrificed and necropsied.
  • the minimal focal epithelial hyperplasia seen in two sections at the time is considered to be a renewal/recovery change, as it was during the treatment phase of the study.
  • Some Microcyn-treated rabbits had either goblet cell hypocellularity or goblet cell hyperplasia at the 21 day period, but these changes were no different from those found in controls. Lymphocytic inflammatory infiltrates were the main findings at the end of the 21 day test period.
  • a single control rabbit had minimal focal epithelial necrosis at Day 21 which was considered to be an incidental finding.
  • This example demonstrates a comparison of the antimicrobial activity of an exemplary oxidative reductive potential water, Microcyn, used in accordance with the invention versus HIBICLENS® chlorhexidine gluconate solution 4.0% (w/v) and 0.9% sodium chloride irrigation (USP).
  • Example 26 An In-Vitro Time-Kill evaluation was performed as described in Example 26 using HIBICLENS® chlorhexidine gluconate solution 4.0% (w/v) and a sterile 0.9% sodium chloride irrigation solution (USP) as reference products. Each reference product was evaluated versus suspensions of the ten American Type Culture Collection (ATCC) strains specifically denoted in the Tentative Final Monograph. The data collected was then analyzed against the Microcyn microbial reduction activity recorded in Example 26.
  • ATCC American Type Culture Collection
  • Microcyn oxidative reductive potential water reduced microbial populations of five of the challenge strains to a level comparable to that observed for the HIBICLENS® chlorhexidine gluconate solution.
  • Both Microcyn and HIBICLENS® provided a microbial reduction of more than 5.0 Log 10 following a thirty second exposure to the following species: Escherichia coli (ATCC #11229 and ATCC #25922), Pseudomonas aeruginosa (ATCC #15442 and ATCC #27853), and Serratia marcescens (ATCC #14756).
  • Microcyn demonstrated excellent antimicrobial activity against Micrococcus luteus (ATCC #7468) by providing a 5.8420 Log 10 reduction after a thirty second exposure.
  • ATCC #7468 a direct Micrococcus luteus (ATCC #7468) activity comparison to HIBICLENS® was not possible because after a thirty second exposure, HIBICLENS® reduced the population by the detection limit of the test (in this specific case, by more than 4.8 Log 10 ). It is noted that the sterile 0.9% sodium chloride irrigation solution reduced microbial populations of each of the six challenge strains discussed above by less than 0.3 Log 10 following a full twenty minute exposure.
  • Microcyn oxidative reductive potential water provided greater antimicrobial activity than both HIBICLENS® and the sodium chloride irrigation for four of the challenge strains tested: Enterococcus faecalis (ATCC #29212), Staphylococcus aureus (ATCC #6538 and ATCC #29213), and Staphylococcus epidermidis (ATCC #12228).
  • a culture of Streptococcus pneumoniae was prepared by using a frozen culture to inoculate multiple BAP plates and incubating for 2-3 days at 35-37° C. with CO2. Following incubation 3-7 mL of sterile diluent/medium was transferred to each agar plate and swabbed to suspend the organism. The suspensions from all plates were collected and transferred to a sterile tube and compared to a 4.0 McFarland Standard. The suspension was filtered through sterile gauze and vortex mixed prior to use in the testing procedure.
  • test mixture An inoculum of 0.1 ml of the organism suspension was added to 49.9 ml of the Microcyn or control substance. At each exposure period, the test mixture was mixed by swirling. The test mixture was exposed for 15 seconds, 30 seconds, 60 seconds, 120 seconds, 5 minutes, and 15 minutes at 25.0° C.
  • the bacterial subculture plates were incubated for 48 ⁇ 4 hours at 35-37° C. in C02. Subculture plates were refrigerated for two days at 2-8° C. prior to examination. Following incubation and storage, the agar plates were observed visually for the presence of growth. The colony forming units were enumerated and the number of survivors at each exposure time was determined. Representative subcultures demonstrating growth were appropriately examined for confirmation of the test organisms.
  • the exemplary ORP water solution, Microcyn demonstrated a >99.93197279% reduction of Penicillin Resistant Streptococcus pneumoniae (ATCC 51915) after 15 second, 30 second, 60 second, 120 second, 5 minute, and 15 minute contact times at 25.0° C.
  • the objective of this Example is to determine the microbial activity of an exemplary ORP water solution (Dermacyn) versus Bacitracin using a bacterial suspension assay.
  • Dermacyn is a ready to use product, therefore performing dilutions during testing was not required.
  • Bacitracin is a concentrated re-hydrated solution requiring a dilution to 33 Units/ml.
  • fresh suspensions of Pseudomonas aeruginosa , and Staphylococcus aureus were prepared and measured using a spectrophotometer to ensure the titer was acceptable
  • test substance was added to 100 ul of microbe suspension.
  • the test mixture was held at 20° C. for the contact times of 20 seconds, 5 minutes, and 20 minutes.
  • 1.0 ml of the test mixture (entire mixture) was added to 9,0 ml of neutralizer for 20 minutes (this is the original neutralization tube or ONT)
  • 1.0 ml of the neutralized test mixture was plated on Tryptic Soy Agar in duplicate for the 5 minute and 20 minute contact times. Additional dilutions and spread plates were used for the 20 second time point, to achieve countable plates.
  • Dermacyn when challenged with the test organisms showed total eradication (>4 log reduction) of the vegetative bacteria at all time points and for spores at the 5, and 20 minute time points. Bacitracin only produced approximately 1 log reduction. Microcyn at the 20 second time point showed some reduction in spores. Bacitracin showed no evidence of lowering the bacterial or spore populations over the time periods tested.
  • the parental strain for all studies is P. aeruginosa PAO1.
  • All planktonic strains were grown aerobically in minimal medium (2.56 g Na 2 HPO 4 , 2.08 g KH 2 PO 4 , 1.0 g NH 4 Cl, 0.04 g CaCl 2 .2 H 2 O, 0.5 g MgSO 4 .7H 2 O, 0.1 mg CuSO 4 .5H 2 O, 0.1 mg ZnSO 4 .H 2 O, 0.1 mg FeSO 4 .7H 2 O, and 0.004 mg MnCl 2 .4H 2 O per liter, pH 7.2) at 22° C. in shake flasks at 220 rpm. Biofilms were grown as described below at 22° C. in minimal medium. Glutamate (130 mg/liter) was used as the sole carbon source.
  • Biofilms were grown as described previously (Sauer et.al., J. Bacteriol. 184:1140-1154 (2002),which is hereby incorporated by reference). Briefly, the interior surfaces of silicone tubing of a once-through continuous flow tube reactor system were used to cultivate biofilms at 22° C. Biofilms were harvested after 3 days (maturation-1 stage), 6 days (maturation-2 stage), and 9 days (dispersion stage) of growth under flowing conditions. Biofilm cells were harvested from the interior surface by pinching the tube along its entire length, resulting in extrusion of the cell material from the lumen. The resulting cell paste was collected on ice. Prior to sampling, the bulk liquid was purged from the tubing to prevent interference from detached, planktonic cells.
  • the population size of planktonic and biofilm cells was determined by the number of CFU by using serial dilution plate counts. To do so, biofilms were harvested from the interior surface after various periods of time of exposure to SOSs. Images of biofilms grown in once-through flow cells were viewed by transmitted light with an Olympus BX60 microscope (Olympus, Melville, N.Y.) and a — 100 magnification A100PL objective lens. Images were captured using a Magnafire cooled three-chip charge-coupled device camera (Optronics Inc., Galena, Calif.) and a 30-ms exposure. In addition, confocal scanning laser microscopy was performed with an LSM 510 Meta inverted microscope (Zeiss, Heidelberg, Germany). Images were obtained with a LD-Apochrome 40 — /0.6 lens and with the LSM 510 Meta software (Zeiss).
  • ORP water is effective against bacteria in bioflims.

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