Antiseptic compositions, methods and systems

Abstract

Antiseptic compositions comprising at least one salt of EDTA are disclosed. These compositions have broad spectrum antimicrobial and antifungal activity and they also have anticoagulant properties. The antiseptic compositions have also demonstrated activity in penetrating and breaking down microbial slime, or biofilms. They are safe for human and medical uses and may be used as prophylactic preparations to prevent infection, or to reduce the proliferation of and/or eliminate existing or established infections.

Claims

1. A method, comprising: contacting a medical device or a medical surface with a solution, wherein the solution comprises at least one ethylene diamine tetra acidic acid (EDTA) salt and a solvent, wherein the at least one EDTA salt comprises tetra-sodium EDTA, wherein the at least one EDTA salt further comprises tri-sodium EDTA, wherein the solution is at a pH greater than 8.5 and a concentration of at least 0.5% (w/v) EDTA.

2. The method of claim 1, wherein the solution is at a concentration of at most 30% (w/v) EDTA.

3. The method of claim 2, wherein the medical device is a veterinary instrument, a dental device, a periodontal device, a toothbrush, a contact lens, a catheter, a nasal tube, a throat tube or other medical tube.

4. The method of claim 1, wherein the solution further comprises between 0.5% and 40% (v/v) ethanol and a solvent.

5. The method of claim 1, wherein the solvent comprises ethanol.

6. The method of claim 1, wherein the contacting comprises transferring the solution to the medical device from a wipe, a bandage or a dressing.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A-1D show minimum inhibitory concentration (MIC) and minimum bactericidal (MBC) concentrations for various gram-positive and gram-negative bacterial organisms against EDTA salt solutions consisting essentially of: di-potassium EDTA; di-ammonium EDTA; di-sodium EDTA; tri-sodium EDTA and tetra-sodium EDTA, using the agpr dilution method. The bacterial organisms were isolated from catheter-related infections in human patients. Experimental techniques are described in Example 1.

(2) FIG. 2 shows MIC and MBC concentrations for various fungal organisms against different formulations of EDTA, using the agar dilution method. Experimental techniques are described in Example 1. The fungal organisms were collected from human patient samples.

(3) FIGS. 3A and 3B show MIC and MBC data for gram-positive and gram-negative bacterial organisms against EDTA salt solutions consisting essentially of: cupric di-sodium EDTA; magnesium di-sodium EDTA and ferric sodium EDTA. The bacterial organisms were isolated from catheter-related infections in human patients. Experimental techniques are described in Example 1.

(4) FIGS. 4A-4C show MIC and MBC data for various gram-positive and gram-negative bacterial organisms against combination EDTA salt solutions consisting essentially of cupric di-sodium and tetra-sodium EDTA; cupric di-sodium and di-potassium EDTA and and cupric di-sodium and di-ammonium EDTA. The bacterial organisms were isolated from catheter-related infections in human patients. Experimental techniques are described in Example 1.

(5) FIGS. 5A-5C show MIC and MBC data for various gram-positive and gram-negative bacterial organisms against combination EDTA salt solutions consisting essentially of: tetra-sodium and di-ammonium EDTA; tetra-sodium and di-potassium EDTA; and di-ammonium and di-potassium EDTA. The bacterial organisms were isolated from catheter-related infections in human patients. Experimental techniques are described in Example 1.

(6) FIG. 6 shows the minimum biofilm eradication concentration (MBEC) values for various organisms, expressed in mg/ml tetra-sodium EDTA (w/v) using the methodology described in Example 2.

(7) FIG. 7 shows experimental results produced by treating infected renal hemodialysis catheters with an antiseptic composition consisting essentially of tetra-sodium EDTA at a concentration of 40 mg/ml (w/v).

(8) FIG. 8 shows experimental results produced by treating infected renal hemodialysis catheters, as well as one arterial and one venous catheter, with an antiseptic composition consisting essentially of tetra-sodium EDTA at concentrations of 20-100 mg/ml (w/v).

DETAILED DESCRIPTION OF THE INVENTION

(9) EDTA is used at low concentrations in many compositions, in combination with other active components, as a stabilizer or preservative agent. Antiseptic compositions of the present invention comprise generally higher concentrations of EDTA and preferably comprise at least 1.0% EDTA salt(s), by weight per volume of solution (w/v), and may comprise up to 15% (w/v) EDTA salt(s). Antiseptic compositions comprising at least 2.0% (w/v) EDTA salt(s) and less than 10% (w/v) EDTA salt(s) are preferred for many applications. Antiseptic compositions comprising between 2.0% (w/v) EDTA salt(s) and 8.0% (w/v) EDTA salt(s) are preferred for many applications, and compositions comprising between 2.0% and 6.0% EDTA salt(s) are especially preferred for many applications. Exemplary compositions, described below, comprise 3.6-4.4% (w/v) EDTA salt(s) in aqueous solution.

(10) The desired EDTA salt(s) concentration for various applications may depend on EDTA salt(s) or combination of salts employed, the type of infection being treated and, to some degree, on the solvent used for antiseptic compositions. When aqueous solvents comprising ethanol are used, for example, the concentration of EDTA salt(s) required to provide the desired level of activity may be reduced compared to the EDTA salt(s) concentration used in compositions having water as the solvent. Antiseptic compositions comprising one or more EDTA salt(s) have demonstrated inhibitory and/or bactericidal efficacy at concentration ranges of 0.5% to 30% or more, as shown in the exemplary data provided below. Effective concentrations of desired EDTA salt(s) in antiseptic compositions of the present invention for inhibitory, bactericidal, fungicidal, biofilm eradication and other purposes, may be determined by routine experimentation, as described in the examples provided below.

(11) The British Pharmacopoeia (BP) specifies that a 5% solution of di-sodium EDTA has a pH of 4.0 to 5.5. The BP also specifies a pH range of 7.0 to 8.0 for solutions of tri-sodium EDTA. The pH values for other EDTA salts in aqueous solution are shown in Example 10, below. At physiological pH, the sodium salts of EDTA exist as a combination of di-sodium and tri-sodium EDTA, with the tri-sodium salt of EDTA being predominant. In the U.S., pharmaceutical di-sodium EDTA prepared for injection has generally been titrated with sodium hydroxide to a pH of 6.5 to 7.5. At this pH, the EDTA solution actually comprises primarily tri-sodium EDTA, with a lesser proportion of the di-sodium salt. Other compositions comprising sodium salts of EDTA that are used in medical or healthcare applications are generally adjusted to a pH that is substantially physiological.

(12) In certain embodiments, antiseptic compositions of the present invention comprise, or consist essentially of or consist of, a sodium EDTA salt (or combination of sodium EDTA salts) in solution at a pH higher than physiological, preferably at a pH of >8.0, or at a pH>8.5, or at a pH>9, or at a pH>9.5. In another embodiment, antiseptic compositions of the present invention comprise, or consist essentially of; or consist of, a sodium EDTA salt (or combination of sodium salts) in solution at a pH in the range between 8.5 and 12.5 and, in another embodiment, at a pH of between 9.5 and 11.5 and, in yet another embodiment, at a pH of between 10.5 and 11.5. When used herein, the term EDTA salt may refer to a single salt, such as a di-sodium or tri-sodium or tetra-sodium salt, or another EDTA salt form, or it may refer to a combination of such salts. The composition of EDTA salt(s) depends both on the EDTA salts used to formulate the composition, and on the pH of the composition. For antiseptic compositions of the present invention comprising sodium EDTA salt(s), and at the desired pH ranges (specified above), the sodium EDTA salts are predominantly present in both the tri-sodium and tetra-sodium salt forms.

(13) In one embodiment, antiseptic compositions of the present invention comprise, or consist essentially of; a combination of at least the tri-sodium and tetra-sodium salts of EDTA. In another embodiment, antiseptic compositions of the present invention comprise, or consist essentially of, a combination of at least the tri-sodium and tetra-sodium salts of EDTA, in which at least 10% of the EDTA in the composition is present in the tetra-sodium salt form. In yet another embodiment, antiseptic compositions of the present invention comprise, or consist essentially of a combination of at least tri-sodium and tetra-sodium salts of EDTA, in which at least 50% and, in another embodiment, at least 60%, of the EDTA in the composition is present in the tri-sodium salt form. In another embodiment, antiseptic compositions of the present invention comprise, or consist essentially of a combination of di-sodium and tri-sodium and tetra-sodium EDTA, in which less than 10% of the EDTA in the composition is present in the di-sodium salt form.

(14) Antiseptic compositions comprising, or consisting essentially of, or consisting of EDTA salt(s) other than or in addition to sodium EDTA salts have different effective pH ranges. Effective pH ranges for desired EDTA salt(s) in antiseptic compositions of the present invention for inhibitory, bactericidal, fungicidal, biofilm eradication and other purposes, may be determined by routine experimentation.

(15) In some embodiments, antiseptic compositions of the present invention consist of the EDTA salt(s), as described above, and antiseptic solutions consist of EDTA salts dissolved in a solvent, generally an aqueous solvent such as water or saline. In other embodiments, antiseptic compositions of the present invention consist essentially of the EDTA salt(s), as described above, generally in an aqueous solvent such as water or saline. Antiseptic compositions of the present invention consisting essentially of an EDTA salt or a combination of EDTA salts are substantially free from other active substances having substantial antimicrobial and/or anti-fungal activity. Substantial antimicrobial and/or anti-fungal activity, in this context, means anti-microbial and/or antifungal activity that is at least 50% of the anti-microbial and/or antifungal activity of a sodium EDTA salt(s) composition in aqueous solution at a concentration of 4.0% (w/v) at a pH of 10.5.

(16) In some embodiments, antiseptic compositions of the present invention comprise EDTA salt(s) having specified concentration(s), at specified pH ranges, and may contain materials, including active components, in addition to the EDTA salts described above. Other antimicrobial or biocidal components may be incorporated in antiseptic compositions of the present invention comprising EDTA salt(s), although the use of traditional antibiotics and biocidal agents is generally discouraged as a consequence of the dire consequences of the development of antibiotic- and biocidal-resistant organisms. In some embodiments, antiseptic compositions of the present invention comprising EDTA salt(s) having specified concentration(s), at specified pH ranges, are substantially free from other active substances having substantial antimicrobial and/or anti-fungal activity.

(17) Other active and inactive components may also be incorporated in antiseptic compositions of the present invention comprising EDTA salt(s), provided that they don't deleteriously affect the activity and/or stability of the EDTA salt(s). Proteolytic agents may be incorporated in antiseptic compositions for some applications. Antiseptic compositions formulated for topical application have various creams, emoluments, skin care compositions such as aloe vera, and the like, for example. Antiseptic compositions of the present invention provided in a solution formulation may also comprise other active and inactive components, provided they don't interfere, deleteriously, with the activity and/or stability of the EDTA salt(s).

(18) The compositions of the present invention may be used in a solution or a dry form. In solution, the EDTA salt(s) are preferably dissolved in a solvent, which may comprise an aqueous solution, such as water or saline, or another biocompatible solution in which the EDTA salt(s) are soluble. Other solvents, including alcohol solutions, may also be used. In one embodiment, EDTA salt compositions of the present invention are formulated in a mixture of water and ethanol. Such solutions are highly efficacious and may be prepared by making a concentrated EDTA salt(s) stock solution in water and then introducing the desired concentration of ethanol. EDTA salt concentrations of from about 1.0 to 10%, w/v, are suitable, and ethanol concentrations of from more than about 0.5% and less than about 10%, v/v, provide effective antiseptic compositions. In some embodiments, EDTA salt concentrations of about 2.0% (w/v) in water with an ethanol concentration of about 1% (v/v) are highly effective against a broad spectrum of bacterial strains. When sodium EDTA salts are used, the pH ranges of these antiseptic compositions are as described above. Bio-compatible non-aqueous solvents may also be employed, provided the EDTA salt(s) can be solubilized and remain in solution during storage and use.

(19) EDTA solutions of the present invention are preferably provided in a sterile and non-pyrogenic form and may be packaged in any convenient fashion. In some embodiments, antiseptic EDTA compositions of the present invention may be provided in connection with or as part of a medical device, such as in a pre-filled syringe or another medical device. The compositions may be prepared under sterile, aseptic conditions, or they may be sterilized following preparation and/or packaging using any of a variety of suitable sterilization techniques. Single use vials, syringes or containers of EDTA solutions may be provided. Multiple use vials, syringes or containers may also be provided. Systems of the present invention include such vials, syringes or containers containing the EDTA solutions of the present invention.

(20) The compositions of the present invention may also be provided in a substantially dry form, such as a substantially dry coating on a surface of tubing, or a conduit, or a medical or industrial device such as a catheter or conduit, or a container, or the like. Such substantially dry forms of EDTA compositions of the present invention may be provided in a powder or lyophilized form that may be reconstituted to form a solution with the addition of a solvent. Substantially dry forms of EDTA compositions may alternatively be provided as a coating, or may be incorporated in a gel or another type of carrier, or encapsulated or otherwise packaged and provided on a surface as a coating or in a container. Such substantially dry forms of EDTA compositions of the present invention are formulated such that in the presence of a solution, the substantially dry composition forms an EDTA solution having the composition and properties described above. In certain embodiments, different encapsulation or storage techniques may be employed such that effective time release of the EDTA is accomplished upon extended exposure to solutions. In this embodiment, the substantially dry EDTA solutions may provide antiseptic activity over an extended period of time and/or upon multiple exposures to solutions.

(21) Compositions comprising EDTA have a well established safety profile in connection with medical usage and administration to humans. Doses of up to 3000 mg EDTA disodium are infused over 3 hours, on a daily basis, for the treatment of hypercalcemia in humans. This dose is well tolerated. EDTA salts are also present, in combination with other components, in many solutions used in medical and human health applications, and have been established as safe for human use, both in vitro and in vivo. EDTA salts are readily available at a reasonable cost, and are stable over time in solution.

(22) Formulation and production of antiseptic compositions of the present invention is generally straightforward. In one embodiment, desired antiseptic compositions of the present invention are formulated by dissolving one or more EDTA salt(s) in an aqueous solvent, such as purified water, to the desired concentration and adjusting the pH of the EDTA salt solution to the desired pH. In alternative embodiments, desired antiseptic compositions of the present invention are formulated by dissolving one or more EDTA salt(s) in a solvent in which the EDTA salt or combination of salts is soluble to provide a concentrated, solubilized EDTA salt solution, and additional solvents or components may then be added, or the solubilized EDTA salt composition may be formulated in a form other than a solution, such as a topical preparation. The antiseptic solution may then be sterilized using conventional means, such as autoclaving, UV irradiation, filtration and/or ultrafiltration, and other means. The preferred osmolarity range for EDTA solutions is from 240-500 mOsM/Kg, more preferably from 300-420 mOsm/Kg. The solutions are preferably formulated using USP materials.

(23) Antiseptic compositions consisting of, or consisting essentially of, or comprising tri- or tetra-sodium salt(s), or a mixture of tri- and tetra-sodium salts, are preferred for many applications and may be prepared using sodium salts of EDTA other than tri- and tetra-sodium salts, such as di-sodium EDTA, which is readily available. Di-sodium EDTA solutions have a lower pH in solution than the desired pH range of compositions of the present invention but, upon pH adjustment to the desired range using a pH adjustment material, such as sodium hydroxide, sodium acetate, and other well-known pH adjustment agents, EDTA solutions prepared using di-sodium salts are converted to the preferred combination di- and/or tri- and/or tetra-sodium salt EDTA compositions of the present invention. Thus, different forms and combinations of EDTA salts may be used in the preparation of EDTA compositions of the present invention, provided that the pH of the composition is adjusted to the desired pH range prior to use. In one embodiment, antiseptic compositions consisting of a mixture of primarily tri- and tetra-sodium EDTA is provided by dissolving di-sodium EDTA in an aqueous solution, 3%-5% on a weight/volume basis, and adding sodium hydroxide in a volume and/or concentration sufficient to provide the desired pH of >8.5 and <12.0.

(24) Antiseptic compositions of the present invention comprising, or consisting essentially of or consisting of EDTA salt(s) as described above are also useful for many other applications. EDTA solutions may be used as antiseptic solutions for soaking, or rinsing, or contacting medical, dental and veterinary surfaces and objects. EDTA solutions of the present invention may be used, for example, for storing and/or sanitizing contact lenses and other optical devices; for storing and/or sanitizing dental devices such as dentures, bridges, retainers, tooth brushes, and the like, and for storing and/or sanitizing medical and dental and veterinary devices and instruments. In these applications, the devices or surfaces may be contacted with EDTA solutions of the present invention for a time sufficient to substantially eliminate microbial and/or fungicidal infections, or devices and surfaces may be soaked in EDTA solutions for a desired time period. EDTA compositions of the present invention may additionally be used to sanitize water and other fluid supply lines. Sanitizing of fluid supply lines may be accomplished by intermittently flushing the lines with EDTA compositions of the present invention. Similarly, EDTA compositions of the present invention may be used to eradicate biofilms and microbial (including some virus and protozoa) and fungal populations in water supply and storage devices.

(25) Numerous experimental tests and procedures have been carried out using EDTA containing compositions of the present invention to establish their properties and their efficacy as antiseptic compositions. Several experimental procedures are described in detail below. These procedures and the experimental results are being provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

EXAMPLE 1

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Data for Organisms Against Different Formulations of EDTA, Using the Agar Dilution Method

(26) The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) for various gram-positive and gram-negative bacterial and yeast organisms were established for several different formations of EDTA. The MICs and MBCs for various organisms were also tested in combinations of EDTA salt(s). The agar dilution method (protocol described below) was used.

(27) The gram-positive and gram-negative bacterial organisms were isolated from human patients having catheter-related infections to ensure that the bacterial stains were actively pathogenic and were of the type common in human catheter-related bacterial infections. The yeast organisms were collected from patients having serious septicemic infections. The organisms were catalogued and maintained in the laboratory of Peter Kite at the University of Leeds.

(28) Various EDTA salt solutions and combination EDTA salt solutions were prepared by dissolving relevant reagent grade EDTA salts in distilled water to the desired EDTA salt concentration (w/v). Concentrated stock EDTA salt solutions were prepared for each EDTA salt or EDTA salt combination for determining the MIC and MBC for various organisms. Tetra- and tri-sodium EDTA solutions were prepared using the tetra- and tri-sodium salts of EDTA rather than using di-sodium EDTA and adjusting the pH of the solution to achieve the desired pH ranges. EDTA salt solutions were sterilized prior to use and stored at 4 C.

(29) Agar Dilution Method Protocol

(30) Making the Agar

(31) Place 2 liters of nutrient agar into a steam bath and leave for about 1 hour (until molten). Allow the agar to cool to 50 C. Collect 20 sterile (125 mL) glass bottles and allocate 100 mL of the nutrient agar to each one. To this add 0.5, 1.0, 1.5, 2.0, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 and 100 mg/mL of Tetra-sodium EDTA (or other EDTA salt or EDTA salt combination being tested), using a stock solution at 200 mg/mL. Pour 20 mL agar into a sterile petri dish and allow to set. Pour 3 further plates. Label the plates with the concentration of EDTA they contain. Do this for each concentration. These plates can then be stored, until they are needed, in a 4 C. fridge.
Inoculating the Plates Grow overnight cultures of 23 Gram-positive organisms and 19 Gram-negative organisms in nutrient broth. Dilute each culture to 10.sup.6 cfu/mL, using Phosphite buffered saline (PBS). Use an automatic plate inoculator to inoculate each plate with 21 organisms. Incubate the plates overnight at 37 C. Next day score + or for growth. Use sterile filter paper to transfer the growth from the initial plates to fresh Cled agar plates to determine the MBC's. Incubate the replica plates overnight at 37 C. Next day score + or for growth. The MIC and MBC were described as, the lowest concentration at which there was no growth.

(32) Results are shown in FIGS. 1A-5C. FIGS. 1A-ID show MIC and MBC data (presented as mg/ml EDTA solution, w/v) for many gram-positive and gram-negative organisms against EDTA salt solutions consisting essentially of: di-potassium EDTA; di-ammonium EDTA; di-sodium EDTA; tri-sodium EDTA and tetra-sodium EDTA. FIG. 2 shows MIC and MBC data (presented as mg/ml EDTA solution w/v) for yeasts against EDTA salt solutions consisting essentially of: tetra-sodium EDTA; di-potassium EDTA; and di-ammonium EDTA.

(33) FIGS. 3A and 3B show MIC and MBC data (presented as mg/ml EDTA solution, w/v) for gram-positive and gram-negative organisms against EDTA salt solutions consisting essentially of: cupric di-sodium EDTA; magnesium di-sodium EDTA and ferric sodium EDTA.

(34) FIGS. 4A-4C show MIC and MBC data (presented as mg/ml EDTA solution, w/v) for gram-positive and gram-negative organisms against combination EDTA salt solutions consisting essentially of: cupric di-sodium and tetra-sodium EDTA; cupric di-sodium and dipotassium EDTA; and cupric di-sodium and di-ammonium EDTA.

(35) FIGS. 5A-5C show MIC and MBC data (presented as mg/ml EDTA solution, w/v) for gram-positive and gram-negative organisms against combination EDTA salt solutions consisting essentially of: tetra-sodium and di-ammonium EDTA; tetra-sodium and dipotassium EDTA; and di-ammonium and di-potassium EDTA.

(36) Several of the EDTA salts and EDTA salt combinations were effective in inhibiting and/or eliminating a broad spectrum of bacterial strains at reasonable concentrations. Prior medical testing and use has established good biocompatibility profiles for the use of sodium EDTA salts in humans and animals, while the biocompatibility of other EDTA salts has not yet been established. Tetra- and tri-sodium EDTA salts appeared to be the most efficacious against a broad spectrum of pathogenic bacteria, they have been or could easily be established to be biocompatible for human and veterinary use, and they are cost effective and stable. Tetra-sodium EDTA salt is additionally active as an anticoagulant and is highly soluble in aqueous solvents. Based on these factors and the experiments outlined above, tetra- and tri-sodium EDTA salts were chosen as the most promising candidates for antiseptic compositions of the present invention.

EXAMPLE 2

Minimum Biofilm Eradication (MBEC) Data for Organisms Against Tetra-Sodium EDTA, Using the Modified Calgary Method

(37) Biofilm formation is an important factor in bacterial contamination. An effective antiseptic composition preferably has the ability to reduce the proliferation of biofilm, or prevent or inhibit the formation of biofilms. We therefore tested our candidate tetra-sodium EDTA antiseptic solution to determine whether it could prevent or inhibit the formation of biofilms. The minimum biofilm eradication concentration (MBEC) for various organisms against tetra-sodium EDTA was established using a modified Calgary device method. The Calgary method is described in the CANADIAN JOURNAL OF VETERINARY RESEARCH, 66:8692 (2002) and in U.S. Pat. No. 6,599,714. The method and results are described below.

(38) Tetra-sodium EDTA salt solutions were prepared by dissolving reagent grade tetra-sodium EDTA salt in distilled water to the desired EDTA salt concentration (w/v).

(39) Concentrated stock tetra-sodium EDTA salt solutions were prepared for determining the MBEC for various organisms in a sessile or biofilm form. Tetra-sodium EDTA solutions were sterilized prior to use and stored at 4 C.

(40) Method

(41) Forming Biofilm:

(42) Use 100 mL of Muller Hinton overnight broth of required organism. Pipette 200 uL into all the wells in a 96 well microtitre tray. Place on lid with 96 pins. Incubate on an orbital shaker for 24 hours at 37 C. at a speed of 200 rpm.
Susceptibility Test: Use biofilm formed above. Place lid (with pins) into a new 96 well microtitre tray containing 250 uL of required concentrations of test agent. Incubate for 1 to 24 hours at 37 C. (Not on shaker). At time intervals of 1, 3, 6, and 24 hours, remove 4 pins for each concentration from the lid by inserting a screwdriver and snapping pin off into the well. Place 3 pins for each concentration into a 5 ml wash of PBS and invert once. Place the three pins into 3 mL of PBS and sonicate for 15 minutes. Plate out 2 uL onto 3CLED plates and spread using a sterile plastic spreader. Incubate at 37 C. overnight. Read colony counts next day. Place the remaining loose pin (for each concentration) into 600 uL of 4% formal saline for SEM.

(43) The MBEC values for various organisms, expressed in mg/ml tetra-sodium EDTA (w/v) determined using this method, are shown in FIG. 6. The results demonstrate that 40 mg/ml tetra-sodium EDTA (4% w/v) was an effective biofilm eradication concentration for all microbial populations tested.

(44) Exemplary data generated by MBEC experiments for various microorganisms are provided below. Tetra-sodium EDTA was used for all experiments, which were performed in triplicate.

(45) TABLE-US-00001 Conc. Colony Colony Colony Colony EDTA count/mL count/mL count/mL count/mL mg/mL after 1 hour after 3 hours after 6 hours after 24 hours 0 40152 53285 64234 6133 48175 62044 56934 4960 43796 61314 76642 5120 5 0 520 80 0 0 540 80 0 0 620 133 730 10 0 0 0 0 0 0 0 0 0 0 0 0 15 0 0 0 0 0 0 0 0 0 0 0 0 20 0 0 0 0 0 0 0 0 0 0 0 0 For 250 E. coli, the MBEC = 10 mg/mL tetra-sodium EDTA.

Orgamism: J26 Pseudomonas aeruginosa

(46) TABLE-US-00002 Conc. Colony Colony Colony Colony EDTA count/mL count/mL count/mL count/mL mg/mL after 1 hour after 3 hours after 6 hours after 24 hours 0 86861 4400 92701 66667 89781 3060 79562 35036 94891 3080 83212 41606 5 0 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 15 0 0 0 0 0 0 0 0 0 0 0 0 20 0 0 0 0 0 0 0 0 0 0 0 0 For J26 Pseudomonas aeruginosa, the MBEC = <5 mg/mL tetra-sodium EDTA.

Organism: 292 Enterobacter cloacae

(47) TABLE-US-00003 Conc. Colony Colony Colony Colony EDTA count/mL count/mL count/mL count/mL mg/mL after 1 hour after 3 hours after 6 hours after 24 hours 0 1.00E+06 103704 94444 91241 1.00E+06 118519 131481 116667 1.00E+06 107407 100000 131481 5 69343 35036 36496 0 67153 15974 32197 0 67153 19697 39416 0 10 38686 12035 80 0 42336 17803 219 0 40909 18561 0 0 15 8000 8133 379 0 8533 8133 219 0 7467 8267 133 0 20 13786 2840 0 0 12473 2820 0 0 14661 2600 0 0 For 292 Enterobacter cloacae, the MBEC = <5 mg/mL tetra-sodium EDTA.

Organism: H Enterococcus sp.

(48) TABLE-US-00004 Conc. Colony Colony Colony Colony EDTA count/mL count/mL count/mL count/mL mg/mL after 1 hour after 3 hours after 6 hours after 24 hours 0 5600 3520 4000 6133 8133 3980 3440 4720 6800 3920 3760 4640 5 1380 780 80 0 1160 580 100 0 1140 500 120 0 10 40 0 0 0 100 0 0 0 20 0 0 0 15 40 0 20 0 0 0 0 0 80 0 20 0 20 1480 730 160 0 1560 379 160 0 2000 320 140 0 For H. Enterococcus sp., the MBEC = <5 mg/mL tetra-sodium EDTA.

Organism: J22 Enterobacter cloacae

(49) TABLE-US-00005 Conc. Colony Colony Colony Colony EDTA count/mL count/mL count/mL count/mL mg/mL after 1 hour after 3 hours after 6 hours after 24 hours 0 124074 107407 105556 101852 116667 91241 105556 120370 112963 98540 92701 100000 5 6400 267 2040 0 5200 133 2160 0 8933 379 1820 0 10 3540 1920 267 80 3040 2900 160 1532 3760 2340 219 800 15 2620 1560 740 0 2100 1740 720 0 2720 1580 920 0 20 2040 80 960 0 2360 1460 840 0 1620 133 560 0 For J22 Enterobacter cloacae, the MBEC = 15 mg/mL tetra-sodium EDTA.

Organism: R81 Proteus vulgaris

(50) TABLE-US-00006 Conc. Colony Colony Colony Colony EDTA count/mL count/mL count/mL count/mL mg/mL after 1 hour after 3 hours after 6 hours after 24 hours 0 62044 81752 112963 59259 55474 73723 103704 68519 54015 78832 107407 59124 5 3160 160 3460 0 4000 400 3120 0 4000 160 3140 0 10 1520 730 400 0 1920 533 1460 0 1900 438 160 0 15 2960 379 1100 0 2580 80 780 0 2560 400 1220 0 20 4560 400 1520 0 4480 320 1280 0 2820 240 720 0 For R81 Proteus vulgaris, the MBEC = <5 mg/mL tetra-sodium EDTA.

EXAMPLE 3

In Vitro Catheter Lock Treatment Procedure on Patient Positive Catheters

(51) A catheter lock treatment procedure using the candidate 40 mg/ml (4% w/v) tetra sodium EDTA solution was developed and used for sample patient hemodialysis catheters that tested positive for various microbial infections. Catheters that were determined to have microbial infections were subjected to the catheter lock treatment using tetra-sodium EDTA and colony counts were taken at various time points. In a first experiment, all catheters were treated with a 4% w/v tetra-sodium EDTA solution while in a second experiment, catheters were treated with tetra-sodium EDTA solutions at various concentrations. Tetra-sodium EDTA solutions were prepared and stored as described above with reference to Examples 1 and 2. The procedure and results are described below.

(52) Method

(53) Renal hemodialysis catheters removed on suspicion of infection were screened, by flushing 1 mL of sterile Phosphate buffed saline down each lumen. Quantitative culture was performed using 1 and 10 uL aliquots spread onto blood agar plates and incubated. The catheters were initially stored at 4 C. until after screening and the external lumen sterilized with an alcohol wipe. Prior to lock treatment testing, the screened positive catheters were locked with nutrient broth using a 5 mL syringe and incubated overnight at 37 C. to ensure biofilm viability and to ensure total colonization of all the endoluminal surfaces with the infecting organism. After overnight incubation each catheter lumen was flushed with 5 mL of sterile saline and 21 cm pieces were cut from the distal end, each placed in 1 mL of IM sterile calcium chloride, (for neutralization of agent) one for Scanning electron microscopy (SEM) and the other for culture, in sterile universal containers. For the culture procedure the universal was placed in a sonication bath for 15 mins at room temperature and then vortexed for 20 secs. Quantitative culture was performed using aliquots of 1 ul and 10 ul plated on blood agar plates and spread by means of sterile plastic L shaped rods, incubated at 37 C. overnight, and colonies counted next day. The catheter was flushed and locked with the appropriate concentration of tetra-sodium EDTA lock fluid and incubated at 37 C. for 18 hrs. At 3, 6 and 18 hrs incubation 21 cm pieces of the distal end of the catheter were cut off and neutralised in 1 mL of 1 M sterile calcium chloride solution. The quantitative count procedure was followed, at each time interval, as previously described and one piece retained for SEM.

(54) Seventeen (17) infected renal hemodialysis catheters were treated with an antiseptic composition consisting of tetra-sodium EDTA at a concentration of 40 mg/ml (4% w/v). The results are shown in FIG. 7. Ten (10) additional infected renal hemodialysis catheters, as well as one arterial and one venous catheter were treated with an antiseptic composition consisting of tetra-sodium EDTA at concentrations of 20-100 mg/ml (2-10% w/v). The results are shown in FIG. 8.

(55) The results demonstrate that 40 mg/ml (4% w/v) tetra-sodium EDTA is efficacious to kill or to dramatically reduce the population of most organisms after a 24 hour treatment. This concentration of tetra-sodium EDTA is safe for use in connection with humans and other animals and is considered to be efficacious and a desired concentration for antiseptic compositions and methods of the present invention.

EXAMPLE 4

The Effect of Tetra-Sodium EDTA on Acanthamoeba and the Effect of Tetra-Sodium EDTA Treated Klebsiella on Acanthamoeba

(56) Several species of Acanthamoeba are capable of infecting humans and Acanthamoebic infections often result as a consequence of improper storage of contact lenses and other medical devices that come into contact with the human body. Acanthamoebae feed on bacterial populations and are resistant to many treatments. We tested the effect of tetra-sodium EDTA, prepared as described above on Acanthamoeba populations. Tetra-sodium EDTA compositions were also prepared using Pages saline and physiological saline as solvents. We also tested the effect of tetra-sodium EDTA-treated Klebsiella on Acanthamoeba experimentally using the following methodology.

(57) The Effect of Tetra-Sodium EDTA on Acanthamoeba

(58) Method

(59) Incubate a fresh blood agar plate with Klebsiella edwardsii at 37 C. 18 hours prior to testing. Using a stock solution of Tetra-sodium EDTA (100 mg/ml.), make a concentration of 22 and 44 mg/mL in Page's saline. Place 9 mL of each concentration into a sterile glass test tube. Place 9 mL of sterile Page's saline in to another sterile glass test tube to act as a control. Make a suspension of Klebsiella edwardsii in 6 mL sterile Page's saline. Adjust to McFarland standard 5. Add 1 mL of the suspension to each serial dilution and the control. Due to the dilution factor of the Klebsiella suspension each concentration will now be at 20 and 40 mg/mL. The control still contains no Tetra-sodium EDTA. Repeat all the concentrations in physiological saline. Vortex to mix. Each tube should now contain a suspension of Klebsiella at McFarland 0.5. Scrape the surface of the whole of the Acanthamoeba plate and suspend in 1.5 mL of Page's saline. Vortex. Add 200 uL of the Acanthamoeba suspension to each serial dilution and the control. Place the test tubes into a 30 C. incubator for 24 hours After incubation centrifuge each universal for 10 minutes at 3000 rpm Pour off the supernatant and resuspend the pellet Place duplicate 10 uL of each dilution and the control onto a non-nutrient agar plate with a lawn of Klebsiella. Cut a groove down the center of each plate to prevent migration and place 10 uL of the dilution being tested on each side. Mark each inoculation site with a black marker pen. Incubate plates for 72 hours at 30 C. Check for growth of Acanthamoeba by direct visualization of the plates using a 10 magnification eyepiece light microscope, starting at each inoculation site.

Growth after 24 Hours Incubation with Tetra-Sodium EDTA

(60) TABLE-US-00007 Concentration of EDTA mg/ml. Growth of in (solution) Acanthamoeba O (Pages saline) +++ 0 (Pages saline) +++ 20 (Pages saline) ++ 20 (pages saline) ++ 40 (Pages saline) 40 (Pages saline) 0 (physiological saline) +++ 0 (physiological saline) +++ 20 (physiological saline) ++ 20 (physiological saline) ++ 40 (physiological saline) 40 (physiological saline) ++

Growth after 24 Hours Iacubatioa with Tetra-Sodium EDTA (Repeat)

(61) TABLE-US-00008 Concentration of EDTA Growth of (mg/mL) Acanthamoeba 0 (Pages saline) +++ 0 (Pages saline) +++ 20 (Pages saline) ++ 20 (Pages saline) ++ (trophozoites present) 40 (Pages saline) 40 (Pages saline) 0 (physiological saline) +++ 0 (physiological saline) +++ 20 (physiological saline) 20 (physiological saline) 40 (physiological saline) +++ 40 (physiological saline) ++ (trophozoites present)

Growth after 48 Hours Incubation with Tetra-Sodium EDTA

(62) TABLE-US-00009 Concentration of EDTA Growth of (mg/mL) Acanthamoeba 0 (Pages saline) +++ (trophozoites present) 0 (Pages saline) +++ (trophozoites present) 20 (Pages saline) 20 (Pages saline) 40 (Pages saline) 40 (Pages saline) 0 (physiological saline) +++ 0 (physiological saline) +++ 20 (physiological saline) 20 (physiological saline) 40 (physiological saline) 40 (physiological saline)

(63) The results demonstrate that 20-40 mg/ml (2-4% w/v) tetra-sodium EDTA in Pages and physiological saline is effective to reduce, or substantially eliminate, Acanthamoeba populations after 48 hours of exposure. Tetra-sodium EDTA compositions prepared using water as the solvent were also effective (data not shown). These results indicate that the antiseptic compositions of the present invention are suitable for application as soaking solutions for various medical instruments and devices, including contact lenses and dental/orthodontic/periodontic devices. Antiseptic compositions of the present invention are also effective to substantially eliminate Acanthamoeba populations in other applications, including in fresh and sea water storage and distribution systems, in heating, venting and air conditioner units, humidifiers, dialysis units, and the like.

(64) Acanthamoeba feed on bacterial populations. We therefore tested whether a bacterial population that was treated with antiseptic EDTA compositions of the present invention would have any effect on Acanthamoeba feeding on the treated bacterial population.

The Effect of Tetra-Sodium EDTA Treated Klebsiella on Acaothamoeba

(65) Method

(66) Incubate a fresh blood agar plate with Klebsiella edwardsii at 37 C. 18 hours prior to testing. Using a stock solution of Tetra-sodium EDTA (100 mg/mL), make a concentration of 22 and 44 mg/mL in Page's saline. Place 9 mL of each concentration into a sterile glass test tube. Place 9 mL of sterile Page's saline into another sterile glass test tube to act as a control. Make a suspension of Klebsiella edwardsii in 6 mL sterile Page's saline. Adjust to McFarland standard 5. Add 1 mL of the suspension to each serial dilution and the control. Due to the dilution factor of the Klebsiella suspension each concentration will now be at 20 and 40 mglmL. The control still contains no Tetra-sodium EDTA. Repeat all the concentrations in physiological saline. Vortex to mix. Each tube should now contain a suspension of Klebsiella at McFarland 0.5. Incubate tubes at 37 C. overnight. Next day, centrifuge tubes at 300 rpm for 10 minutes. Tip off supernatant; add 10 mL fresh saline or Page's saline, resuspend and re-centrifuge. Tip off supernatant and resuspend in 1 mL of either saline or Page's saline. Scrape the surface of the whole of the Acanthamoeba plate and suspend in 1.5 mL of Page's saline. Vortex. Add 200 uL of the Acanthamoeba suspension to 3 tubes containing 9 mL saline and 3 tubes containing 3 mL Page's saline. Label each tube as if they were the EDTA concentrations used in the incubation with the Klebsiella. Add the 1 mL of resuspended Klebsiella to the appropriate tube containing Acanthamoeba. Place the test tubes in to a 30 C. incubator for 24 hours. Set up another set of tubes to incubate Klebsiella with the EDTA at 37 C., overnight as before. After incubation centrifuge each tube containing the Acanthamoeba for 10 minutes at 3000 rpm. Pour off the supernatant and resuspend the pellet. Place duplicate 10 uL of each dilution and the control onto a non-nutrient agar plate with a lawn of Klebsiella (not incubated with EDTA). Cut a groove down the center of each plate to prevent migration and place 10 uL of the dilution being tested on each side. Mark each inoculation site with a black marker pen. Incubate plates at 30 C. Check for growth of Acanthamoeba by direct visualization of the plates using a 10-magnification eyepiece light microscope, starting at each inoculation site. Place the remainder of the Acanthamoeba suspension into a fresh set of tubes containing either fresh saline or fresh Page's saline. Wash and resuspend the Klebsiella, that has been incubated overnight with the EDTA, as 30 before and add to each appropriate tube containing the Acanthamoeba. Incubate the tubes at 30 C. overnight. After incubation centrifuge each universal for 10 minutes at 3000 rpm. Pour off the supernatant and resuspend the pellet. Place duplicate 10 uL of each dilution and the control onto a non-nutrient agar plate with a lawn of Klebsiella (not incubated with EDTA). Cut a groove down the center of each plate to prevent migration and place 10 uL of the dilution being tested on each side. Mark each inoculation site with a black marker pen. Incubate plates at 30 C. Check for growth of Acanthamoeba by direct visualization of the plates using a 10-magnification eyepiece light microscope, starting at each inoculation site.

Growth of Acanthamoeba after 24 Hours Incubation with Klebsiella (Previously Incubated with EDTA)

(67) TABLE-US-00010 Concentration of EDTA Growth of (mg/mL) Acanthamoeba 0 (Pages saline) +++ 0 (Pages saline) 20 (Pages saline) ++ 20 (Pages saline) ++ 40 (Pages saline) ++ 40 (Pages saline) O(physiological saline) +++ O(physiological saline) +++ 20 (physiological saline) ++ 20 (physiological saline) ++ 40 (physiological saline) 40 (physiological saline)

Growth of Acanthamoeba after 48 Hours Incubation with Klebsiella (Previously Treated with EDTA)

(68) TABLE-US-00011 Concentration of EDTA Growth of (mg/ml.) Acanthamoeba O(Pages saline) +++ O(Pages saline) +++ 20 (Pages saline) + 20 (Pages saline) 40 (Pages saline) 40 (Pages saline) O(physiological saline) +++ O(physiological saline) +++ 20 (physiological saline) 20 (physiological saline) 40 (physiological saline) 40 (physiological saline)

(69) These results demonstrate that growth of Acanthamoeba can be arrested and Acanthamoeba populations can be substantially eliminated by treating bacterial populations on which they feed with antiseptic EDTA compositions of the present invention. Antiseptic EDTA compositions having a tetra-sodium EDTA concentration of from 20-40 mg/ml, (24% w/v) were effective. This substantiates the usefulness of antiseptic compositions of the present invention for applications such as soaking solutions for various medical instruments and devices, including contact lenses and dental/orthodontic/periodontic devices, as well as for other applications such as fresh and sea water storage and distribution systems, in heating, venting and air conditioner units, humidifiers, dialysis units, and the like.

EXAMPLE 5

(70) Experiments were conducted to determine whether tetra-sodium EDTA compositions prevent the attachment of and adherence to silicon tubing of microorganisms. If attachment of and adherence to silicon tubing of microorganisms can be prevented, the formation of biofilms can be reduced. The experimental protocol used and the results obtained are provided below.

(71) Method

(72) Fill 1 cm sections of silicon tubing with molten wax to seal each endolumen, harden at 4 C. Place 4 sections into 30 mL sterile Phosphate buffered saline (PBS) as a control. Place 8 sections into 30 mL 4% tetra-sodium EDTA. After 30 minutes, place the 4 sections from the PBS and 4 of the sections from the 4% tetra-sodium EDTA into clean containers on a hot block, and allow to dry. Transfer the remaining 4 sections into 30 mL sterile PBS to rinse, then allow to air dry as before. Once dried place all 12 sections into 10 cfu/mL mixed organisms (overnight cultures of Klebsiella pneumoniae and CNS grown in nutrient broth at 37 C.), incubate at 37 C. After 30 minutes remove 2 sections of each type and rinse in 230 mL sterile PBS. Air dry as before. Using separate washes and drying vessels for each type prevent contamination. Place each section into 1 mL PBS in a centrifuge tube, sonicated in a sonicating water bath for 15 minutes. Plate out each tube, in duplicate, on the automatic plate inoculator, 50 uL on a log dilution. Incubate the plates at 37 C. overnight. Read colony counts on automatic plate reader Protocol. Repeat after 6 hours. The results for control and EDTA-treated catheter sections are shown below.

(73) TABLE-US-00012 Type of Number of Colony catheter Time of catheter Colony counting counting section incubation section NEAT (cfu/mL) 1/10(cfu/mL) Control 30 min 1 240 0 220 0 Control 30 min 2 280 1 140 0 Control 6 hours 1 1480 0 1120 0 Control 6 hours 2 5200 7800 5467 5800 Air-dried 30 min 1 240 1333 EDTA 400 800 Air-dried 30 min 2 267 0 EDTA 720 0 Air-dried 6 hours 1 1280 16800 EDTA 1120 8800 Air-dried 6 hours 2 2240 21333 EDTA 2340 16000 Rinsed 30 min 1 267 0 EDTA 379 0 Rinsed 30 min 2 1040 0 EDTA 0 0 Rinsed 6 hours 1 1980 9600 EDTA 1740 12800 Rinsed 6 hours 2 3600 19000 EDTA 3660 8600

(74) The results for the neat EDTA solution were found to be more reproducible, and these were therefore analysed further. As sections were placed in 1 mL counts per mL are equal to counts per section.

(75) TABLE-US-00013 Type of catheter Mean colony count after Mean colony count after 6 section 30 minutes (cfu/section) hours (cfu/section) Control 880 3317 Air-dried EDTA 407 1745 Rinsed EDTA 421 2745

(76) TABLE-US-00014 Mean % reduction Mean % reduction in cfu/section from the cfu/section from the Type of catheter section control after 30 minutes control after 6 hours Air-dried EDTA 53.8% 47.4% Rinsed EDTA 52.2% 17.3% Repeated over 24 hours with Klebsiella + CNS:

(77) TABLE-US-00015 Mean colony Mean colony count Mean colony count count after 24 Type of catheter after 30 minutes after 6 hours hours section (cfu/section) (cfu/section) (cfu/section) Control 377 9205 105806 Air-dried EDTA 273 3720 70370 Rinsed EDTA 474 9499 77051

(78) TABLE-US-00016 Mean % Mean % Mean % reduction in cfu/ reduction in cfu/ reduction in Type of section from section from cfu/section from catheter the control after the control after the control after section 30 minutes 6 hours 24 hours Air-dried EDTA 27.4% 59.6% 33.5% Rinsed EDTA +25.7% +31.9% 27.2% +Denotes increase in mean cfu/section from control
Results for Pseudomonas aeruginosa:

(79) TABLE-US-00017 Mean Mean Mean colony colony count colony count count after 24 Type of catheter after 30 minutes after 6 hours hours section (cfu/section) (cfu/section) (cfu/section) Control 6400 341994 1290000 Air-dried EDTA 4108 30000 474494 Rinsed EDTA 5200 153758 1150000

(80) TABLE-US-00018 Mean % Mean % Mean % reduction in reduction in reduction in Type of cfu/section from cfu/section from cfu/section from catheter the control after the control after the control after section 30 minutes 6 hours 24 hours Air-dried EDTA 35.8 91.2 63.2 Rinsed EDTA 18.8 55.0 10.9

(81) These results demonstrate at least a short team reduction in bacterial populations on both air-dried and rinsed catheter sections.

EXAMPLE 6

Altered MBC Values when Tetra-Sodium EDTA b Combined with Ethanol

(82) Solutions having a range of tetra-sodium EDTA concentrations (0, 0.1, 0.5, 1, 2, 3, 4 and 8 mg/ml, w/v) were formulated with water and ethanol (to achieve final ethanol concentrations of 0, 0.1, 0.5, 1, 5, 10, 20 and 40%, in water) to test the efficacy of EDTA solutions alone, with alcohol solutions alone, and with EDTA/alcohol solutions. Concentrated stock solutions of tetra-sodium EDTA were prepared in distilled water and ethanol was added to the concentrated aqueous stock solutions to provide the appropriate ethanol concentration.

(83) Method

(84) Culture an organism in nutrient broth overnight at 37 C. Stock solutions of alcohol and tetra-sodium EDTA are used to fill in a grid pattern in 96 well plates (one per culture), using EDTA solutions having 0, 0.1, 0.5, 1, 2, 3, 4 and 8 mg/ml tetra-sodium concentration, w/v, in isopropyl alcohol solvents containing 0, 0.1, 0.5, 1, 5, 10, 20 and 40% alcohol, v/v, in water. Each well contains 150 uL of each diluent and 50 uL of organism at 110 cfu/mL. At time periods of 5 minutes, 6 hours and 24 hours each well is cultured by placing a 96 pin lid over the plate (and into each well) then transferring the lid to a 96 well plates, containing 300 uL fresh nutrient broth in each well. Incubate overnight at 37 C. Incubate each inoculum plate at 37 C. during incubation period. Record the turbidity of each well after 24 hours.

The Results for Several Organisms are Shown Below

(85) TABLE-US-00019 MBC tetra- MBC tetra-sodium sodium MBC EDTA (mg/mL) + Organism EDTA (mg/inL) alcohol (%) alcohol (%) E. coli 3 10 0.5 and 0.5 Proteus sp, 3 10 2 and 1 CNS (I) 8 10 2 and 1 Klebsiella sp. 8 10 1 and 1 Staphylococcus 0.1 0.1 0.1 and 0.1 aureus Pseudomonas sp. 2 10 2 and 1 CNS (IT) 8 10 0.5 and 0.5

(86) Tetra-sodium EDTA solutions in water were more effective in killing the microorganisms tested than were the ethanol (alone) solutions. Combination tetra-sodium EDTA in alcohol solutions killed the microorganisms tested at the lowest concentrations. The 2 mg/ml tetra-EDTA in 1% alcohol solution provided excellent results and had a bactericidal effect on all organisms tested. This antiseptic solution is effective at lower concentrations of tetra-sodium EDTA and ethanol than tetra-sodium EDTA solutions in water and than ethanol alone, and it is cost effective, safe and convenient to make and use. In addition to solution formulations, antiseptic compositions of the present invention comprehend EDTA in a mixed aqueous solvent and ethanol for topical use.

EXAMPLE 7

Solubility of Tetra-Sodium EDTA in Ethanol and Effect on pH

(87) The solubility of tetra-sodium EDTA in ethanol was tested, and the pH of various tetra-sodium solutions in alcohol solvents was measured

(88) Method

(89) Tetra-sodium EDTA was weighed out in duplicates though the range 10-100 mg in 1.5 mL size Eppendorf tubes. 1 mL of 74% Ethanol was added to each tube and vortexed for 30 s. To the duplicate set of weighed Tetra-sodium EDTA, 0.5 ml sterile distilled water was added and vortex mixed, followed by 0.5 ml of 74% Ethanol. Each of the tubes of Tetra-sodium EDTA was tested for pH where solubility was observed.

(90) The experimental results demonstrated that tetra-sodium EDTA was completely insoluble in a 74% Ethanol solution. The results furthermore demonstrated that, when tetra-sodium was dissolved in distilled water in concentrations in the range of 10-100 mg/ml, w/v, the tetra-sodium EDTA remained in solution when ethanol was added. A preferred technique thus involves solubilizing EDTA salt(s) in an aqueous solution first, and then adding ethanol or another solvent in which the EDTA salt(s) are less soluble, or insoluble. Prepared in this fashion, EDTA salt solutions are expected to be stable over time. The measured pH values for various solutions were as follows:

(91) TABLE-US-00020 74% Ethanol, alone pH 7.8 Water pH 7.1 +10 mg tetra-EDTA pH 9.0 +20 mg tetra-EDTA pH 10.8 +40 mg tetra-EDTA pH 11 +80 mg tetra-EDTA pH 11.15 +100 mg tetra-EDTA pH 11.25

EXAMPLE 8

Effect of Autoclaving at 121 C. on Tetra-Sodium EDTA Solutions

(92) We tested the effect of autoclaving on tetra-sodium EDTA solutions to determine whether autoclaving could be used to sterilize tetra-sodium EDTA solutions prior to use. The methodology used and results are described below.

(93) Method

(94) Make duplicates of 0, 20, 80 and 100 mg/mL of Tetra-sodium EDTA in sterile water and sterile, molten nutrient agar at 50 C. Leave one set at room temperature (not heated) and autoclave one set (heated). Next day place all agar bottles in a steamer to melt for 40 minutes.
Measuring the Zones of Diffusion Using a cork borer, punch out two holes in 16 fresh blood agar plates. Make a 0.5 McFarland suspension of CNS and spread using a sterile swab over the plates to create a lawn. Pipette 50 uL of each of the Tetra-sodium solutions into duplicate punched out holes and incubate at 37 C. overnight. Next day measure the zones of diffusion and record the results.

(95) The results, measured in zone sizes (mm), are presented below. The zone sizes of the controls were plotted against concentration, to allow determination of actual EDTA concentrations in the test samples, which are also presented below. These results demonstrate that autoclaving of tetra-sodium EDTA compositions, whether in sterile water or in agar, does not materially affect the antimicrobial activity of the tetra-sodium EDTA compositions.

(96) Zone Sizes in Mm

(97) TABLE-US-00021 Concentration EDTA in EDTA in EDTA in of EDTA Sterile Water autoclaved EDTA autoclaved mg/mL (control) Sterile Water in Agar Agar 0 0 0 0 0 0 0 0 0 20 13.2 11.6 13.5 12.7 13.2 11.6 13.5 12.7 80 16.1 15.2 17.2 15.3 15.3 16.1 15.2 17.2 100 17.1 17.0 17.1 16.4 17.1 17.0 17.1 16.4

Actual Concentration of EDTA

(98) TABLE-US-00022 Concentration of EDTA in EDTA in EDTA in initial EDTA Sterile Water autoclaved EDTA autoclaved mg/mL (control) Sterile Water in Agar Agar 0 0 0 0 0 20 20 16 26 19 80 80 60 101 62 100 100 98 100 83

EXAMPLE 9

Effect of Autoclaving at 121 C. on Different Formulations of EDTA

(99) We tested the effect of autoclaving on different formulations of EDTA solutions to determine whether autoclaving could be used to sterilize various EDTA solutions prior to use. The methodology used and results are described below.

(100) Method

(101) Make Up the Agar

(102) Place 50 mL of Nutrient agar solution into 7100 mL sterile glass bottles. Add no EDTA powder to the first bottle (labelled 0). Add 2000 mg of EDTA powder to the second bottle (labelled 40 mg/mL auto). Add 4000 mg of EDTA powder to the third bottle (labelled 80 mg/mg auto). Add 5000 mg of EDTA powder to the fourth bottle (labelled 100 mg/mL auto. Add no EDTA to bottles five, six and seven (but label them 40, 80 and 100 mg/mL NON 25 autoclaved), leave at room temperature. Do this for each EDTA formulation to test, and autoclave all bottles, marker auto, at 121 C. for 20 minutes. Next day Place all bottle in a steam bath to melt the agar for pouring. Once melted allow to cool to 50 C. before adding the appropriate amount of EDTA to the bottles labelled NON autoclaved. All bottles are now ready to be tested.
Measuring the Zones of Diffusion Using a cork borer, punch out 2 holes in 7 fresh blood agar plates. Make a 0.5 McFarland suspension of CNS and spread using a sterile swab over the plates to create a lawn. Pipette 150 ul of each bottle into 2 separate punched out holes' and incubate at 37 C. overnight. Do this for each EDTA formulation. Next day measure the zones of diffusion and record results. Duplicate holes were used and 2 measurements per zone were made.

(103) Cupric and ferric EDTA solutions did not produce any zones. The effect of heat upon these solutions therefore cannot be measured using this method. The zone sizes measured for di-ammonium EDTA, di-potassium EDTA and magnesium EDTA solutions are provided below. The zone sizes of the controls (no heat) were plotted against concentration to allow determination of actual EDTA concentrations in the test (heated) samples, and results are provided below.

Zones Sizes (mm)

(104) TABLE-US-00023 Concentration Diammonium Diammonium Dipotassium Dipotassium Magnesium Magnesium of EDTA EDTA EDTA EDTA EDTA EDTA EDTA (mg/mL) No heat Heated No heat Heated No heat Heated 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 40 18.3 17.9 16.2 15.5 6.8 10.6 18.3 17.9 16.2 15.5 6.8 10.6 18.3 17.9 16.2 15.5 6.8 10.6 18.3 17.9 16.2 15.5 6.8 10.6 80 19.7 19.7 18.9 18.3 10.0 10.8 19.7 19.7 18.9 18.3 10.0 10.8 19.7 19.7 18.9 18.3 10.0 10.8 19.7 19.7 18.9 18.3 10.0 10.8 100 20.0 20.6 18.2 20.0 8.3 11.8 20.0 20.6 18.2 20.0 8.3 11.8 20.0 20.6 18.2 20.0 8.3 11.8 20.0 20.6 18.2 20.0 8.3 11.8
Actual Values of Autoclaved EDTA

(105) TABLE-US-00024 Di-potassium Magnesium Concentration of Di-ammonium EDTA EDTA EDTA mg/mL EDTA heated heated heated 0 0 0 0 40 39 38 >140 80 80 71 >140 100 150 >140 >140

(106) The results demonstrate that autoclaving did not diminish the efficacy of most EDTA salt compositions and autoclaving of antiseptic compositions of the present invention may therefore be carried out following preparation to provide sterile antiseptic compositions.

EXAMPLE 10

pH Values of EDTA Salts, Calcium Chloride and Sodium Citrate

(107) The pH values of various EDTA salt, calcium chloride and sodium citrate solutions, using distilled water as the solvent, and at specified concentrations, were measured. Results are shown below.

(108) TABLE-US-00025 Free acid EDTA 10% pH 4.7 Di-ammonium EDTA 10% pH 4.38 Calcium Sodium EDTA 10% pH 6.68 Di-potassium EDTA 10% pH 4.5 Copper EDTA 10% pH 6.15 Tetra-sodium EDTA 10% pH 11.6 2% pH 11 Calcium chloride neutralised TS EDTA pH 7.3 Calcium chloride, 1 molar pH 3.8 Sodium citrate 50%, 25% pH 8.5

EXAMPLE 11

Confirmation of the Anti-Coagulant Properties of EDTA Solutions

(109) We verified the anti-coagulant properties of EDTA solutions using the following methodology Method 100 ul aliquots of a range of concentrations (0.5-100 mg/mL) of tetra-sodium or di-sodium EDTA solutions, adjusted to a pH of 11.0-11.6, were placed in plastic capped tubes. 900 uL of fresh blood from healthy volunteers was added to each aliquot of EDTA solution and mixed gently by inversion of the blood tubes at regular intervals. The results revealed that control tubes containing blood without EDTA solution had clotting times of 10-23 minutes. Tubes containing di-sodium EDTA solutions all had clotting times in excess of 5 days. Tetra-sodium EDTA tubes >1 mg/mL had clotting times in excess of 5 days. Tetra-sodium EDTA tubes having a concentration of 0.5 mg/mL clotted in 28 minutes. Tetra-sodium EDTA is therefore effective as an anticoagulant at concentrations in excess of 1 mg/ml (1% w/v).

EXAMPLE 12

Osmolarity of Tetra-Sodium Salt Suspensions

(110) The osmolarity and red cell lysis of tetra-sodium EDTA solutions in water and physiological saline having various concentrations was tested using standard laboratory techniques. Red cell lysis was tested by adding 50 ul EDTA blood in 2 ml each concentration of each solution for 2 hours. The Plasma Osmolarity range was 275-295 mlosmol.

(111) TABLE-US-00026 Osmolarity Red Cell Lysis 2% Tet Sod EDTA in Distilled Water 142 m/osmol ++ 4% Tet Sod EDTA in Distilled Water 277 + 2% Tet Sod EDTA in Physiological Saline 219 +/ 4% Tet Sod EDTA in Physiological Saline 588

EXAMPLE 13

Efficacy of Three EDTA Salts on the Dissolution of Artificial Urine Crystals (AUC)

(112) One problem with urinary catheters is that urine crystals tend to accumulate on the surface of the catheter. The deposit of urine crystals may promote microbial colonization and/or the formation of biofilms, as well as reducing flow through the catheter. It would be desirable to use a sanitizing composition in connection with urinary catheters that reduces the formation of urine crystals. The efficacy of three EDTA salt solutions on the dissolution of artificial urine crystals was tested using the methodology described below.

(113) Materials:

(114) Artificial urine in 25 ml plastic universal container with urease, incubated at 45 C. for 7 days. Di-ammonium, Di-potassium and tetra-sodium EDTA solutions at 100 mg/ml.
Method: Centrifuge artificial urine crystals at 4000 rpm for 2 mins. Decant supernatant and wash crystals in water followed by centrifugation. Resuspend crystals to 1 ml in water and aliquot 200 ul into four universal containers. Add 4 ml 100 mg/ml solution of each EDTA salt and water as a control to each universal at room temp. After 1, 2 and 3 hours visually observe dissolution of crystals compared to the control.

(115) The results are shown below. All of the EDTA salt solutions reduced the urine crystal deposit compared to an aqueous solution. EDTA salt solutions are therefore suitable for use with urinary catheters.

(116) TABLE-US-00027 Solution Crystal Deposit Water + AUC +++++ Tetra-sodium EDTA + AUC ++ Di-ammonium EDTA + AUC + Di-potassium EDTA + AUC +/