BROAD SPECTRUM ANTIMICROBIAL & ANTICOAGULANT COMPOSITION

Abstract

To reduce problems associated with clotting and thrombus formation, it is now common to lock intravascular access catheters between successive uses. Locking typically involves first flushing the catheter with saline to remove blood and other substances from the catheter lumen. After the catheter has been flushed, an anti-coagulant solution is injected to displace the saline and fill the lumen. Use of an effective antimicrobial substance along with anticoagulant during the same process is important to reduce the possibility of bacterial contamination and subsequent infection. The present invention deals with a safe, effective and economical catheter lock flush solution with excellent antimicrobial and anticoagulant activity. The unique ratio of the active components of the catheter lock solution used in the invention i.e. Sodium salt(s) of EDTA, Ethanol and D-mannitol with a suitable pH ensures broad-spectrum antimicrobial and optimum anticoagulant activity.

Claims

1. A catheter lock flush solution composition capable of providing synergistic broad-spectrum antimicrobial activity, comprising of sodium salt(s) of EDTA and Ethanol wherein the sodium salt(s) of EDTA is at a concentration ranging between 0.01-8% (w/v), and the Ethanol is at a concentration ranging between 0.1-50% (v/v) with a pH ranging between 7-11.

2. The catheter lock flush solution as per claim 1 wherein the concentration of sodium salt(s) of EDTA is 4% (w/v), the Ethanol is at a concentration of 12.5% (v/v) and the pH of the solution is 10.5+/?0.5.

3. The catheter lock flush solution as per claim 1 further comprises of D-mannitol at a concentration ranging between 0.1-10% (w/v) and preferably 0.73% (w/v).

4. A method for disinfecting a catheter comprising: introducing a disinfectant solution into an interior lumen of the catheter, wherein the disinfectant solution comprises of sodium salt(s) of EDTA, at a concentration of 4% (w/v), and the Ethanol, at a concentration of 25% (v/v) and D-mannitol 0.73% (w/v) with the pH being ranging between 7-11.

5. The method of disinfecting a catheter as per claim 4 wherein the pH of the solution is 7.5+/?0.5.

6. A method for disinfecting, maintaining catheter patency, and an International Normalized Ratio (INR) of at least 0.5 points higher than that of the INR of 10 U/ml heparin and the said method comprising of introducing a disinfectant solution into an interior lumen of the catheter, wherein the disinfectant solution comprises of sodium salt(s) of EDTA, at a concentration of 4% (w/v), and the Ethanol, at a concentration of 25% (v/v), D-mannitol 0.73% (w/v) with the pH being ranging between 7-11.

7. The method of disinfecting a catheter as per claim 6 wherein the pH of the solution is 7.5+/?0.5.

8. An anti-microbial solution for general purpose use comprising of sodium salt(s) of EDTA and Ethanol wherein the sodium salt(s) of EDTA is at a concentration of 4% (w/v), and the Ethanol is at a concentration of 12.5% (v/v) with an alkaline pH ranging between 8-11 and preferably 10.5+/?0.5.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0047] In the following discussion, the terms microbe or microbial will be used to refer to microscopic organisms or matter, including fungal and bacterial organisms, and possibly including viral organisms, capable of infecting humans. The term anti-microbial will thus be used herein to refer to a material or agent that kills or otherwise inhibits the growth of fungal and/or bacterial and possibly viral organisms.

[0048] The term disinfect will be used to refer to the reduction, inhibition, or elimination of infectious microbes from a defined system. The term disinfectant will be used herein to refer to a one or more anti-microbial substances used either alone or in combination with other materials such as carriers, solvents, or the like.

[0049] The term bactericidal activity is used to refer to an activity that at least essentially kills an entire population of bacteria, instead of simply just reducing or inhibiting their growth. The term fungicidal activity is used to refer to an activity that at least essentially kills an entire population of yeast, instead of simply just reducing or inhibiting their growth. Contamination of conduits, e.g., catheters, poses serious and substantial health risks and bactericidal disinfection is a significant priority.

[0050] The term infected system will be used herein to refer to a defined or discrete system or environment in which one or more infectious microbes are or are likely to be present. Examples of infected systems include a physical space such as a bathroom facility or operating room, a physical object such as food or surgical tool, a biological system such as the human body, or a combination of a physical object and a biological system such as a catheter or the like arranged at least partly within a human body. Tubes and other conduits for the delivery of fluids, in industrial and healthcare settings, may also define an infected system.

[0051] The term anticoagulation will be used to refer to the delay or abnormal blood clotting time when compared with a citrate control in a defined system. The term anticoagulant will be used herein refer to one or more anti-coagulant substances used either alone or in combination with other materials such as carriers, solvents, or the like.

[0052] In an aspect, the compositions of the present disclosure comprise concentrations of sodium salt(s) of EDTA, Ethanol and D-Mannitol at a pH ranging 4 to 12. The sodium salt(s) of EDTA, Ethanol and D-Mannitol may be used in compositions with distilled water or phosphate buffer saline as the solvent, or a combination thereof.

[0053] EDTA is used at low concentrations in many compositions, in combination with other active components, as a stabilizer or preservative agent. The compositions of the present disclosure comprise generally higher concentrations of EDTA.

[0054] 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. At physiological pH, the sodium salts of EDTA exist as a combination of di-sodium and tri-sodium EDTA, with the tri-sodium salt(s) 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.

[0055] 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 hyper-calcemia 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.

[0056] Ethanol has a molecular Formula of C.sub.2H.sub.5OH and a Molecular Weight of 46.07. Ethanol is also known as ethyl alcohol, grain alcohol, pure alcohol, hydroxyethane, drinking alcohol, ethyl hydrate and EtOH. Ethanol is readily available at a reasonable cost and is stable over time in solution.

[0057] Mannitol has a molecular formula C.sub.6H.sub.14O.sub.6 and a molecular weight of 182.17. Mannitol is also known as mannite or manna sugar. It is readily available at a reasonable cost and is stable over time in solution.

[0058] A sodium salt(s) of EDTA has been shown to have some anti-coagulant effect. Ethanol has been shown to have some anti-coagulant effect. Anticoagulant activity of Mannitol is unknown. The combination of sodium salt(s) of EDTA, Ethanol and Mannitol has an anti-coagulant effect.

[0059] Embodiments of the disclosed composition may comprise at least 0.01% of a sodium salt(s) of EDTA, by weight per volume solution (w/v) and up to 8% (w/v). Embodiments comprising at least 0.18% (w/v) of a sodium salt(s) of EDTA and up to 6% (w/v) are preferred for many applications, compositions comprising at least 1% (w/v) of a sodium salt(s) of EDTA and up to 5% (w/v) are also preferred for certain applications and compositions comprising about 4% (w/v) of a sodium salt(s) of EDTA are especially preferred.

[0060] Embodiments of the disclosed composition may comprise at least 0.1% Ethanol, by volume per volume solution (v/v) and up to 50% (v/v) Ethanol. Embodiments comprising at least 5% (v/v) Ethanol and up to 40% (v/v) Ethanol are preferred for many applications, compositions comprising at least 10% (v/v) Ethanol and up to 30% (v/v) Ethanol are also preferred for certain applications and compositions comprising about 25% (v/v) Ethanol are especially preferred.

[0061] Embodiments of the disclosed composition may comprise at least 0.01% of D-Mannitol, by weight per volume solution (w/v) and up to 10% (w/v). Embodiments comprising at least 0.1% (w/v) of D-Mannitol and up to 5% (w/v) are preferred for many applications, compositions comprising at least 0.5% (w/v) of D-Mannitol and up to 1% (w/v) are also preferred for certain applications and compositions comprising about 0.73% (w/v) of D-Mannitol are especially preferred.

[0062] The compositions comprising desired sodium salt(s) of EDTA, Ethanol and D-Mannitol concentrations for various applications may depend on the type of infection being treated and, to some degree, on the solvent used for disinfectant compositions. Effective concentrations of sodium salt(s) of EDTA, Ethanol and D-Mannitol in disinfectant compositions of the present disclosure for inhibitory, bactericidal, fungicidal, bio-film eradication and other purposes may be determined by routine experimentation.

[0063] In certain embodiments, disinfectant compositions of the present disclosure comprise, or consist essentially of, or consist of, sodium salt(s) of EDTA, Ethanol and D-Mannitol in solution at pH range 4 to 12, or at a pH range 6 to 10, or at a pH range 7 to 10, preferable at a pH range 7.5+/?0.5.

[0064] In some embodiments, disinfectant compositions of the present disclosure consist of sodium salt(s) of EDTA, Ethanol and D-Mannitol as described above, and disinfectant solutions consist of sodium salt(s) of EDTA, Ethanol and D-Mannitol dissolved in a solvent, generally an aqueous solvent such as water or saline. Disinfectant compositions of the present disclosure consisting essentially of sodium salt(s) of EDTA, Ethanol and D-Mannitol are substantially free from other active substances having substantial antimicrobial and/or anti-fungal activity.

[0065] 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 the sodium salt(s) of EDTA and Ethanol composition in aqueous solution with a concentration of 0.12% (w/v) sodium salt(s) of EDTA that comprises tetra-sodium EDTA and 6.25% (v/v) Ethanol at a pH of 10.5+/?0.5.

[0066] Further, embodiments of the invention include compositions wherein the anti-microbial and/or antifungal activity is at least 60%, or 70% or 80% or 90% of the anti-microbial and/or antifungal activity of sodium salt(s) of EDTA that comprises tetra-sodium EDTA and Ethanol composition in aqueous solution with a concentration of 4% (w/v) sodium salt(s) of EDTA that comprises tetra-sodium EDTA and 12.5% (v/v) Ethanol at a pH of 10.5+/?0.5.

[0067] Further, embodiments of the invention include compositions wherein the anti-microbial and/or antifungal activity is at least 90% or higher of the anti-microbial and/or antifungal activity of sodium salt(s) of EDTA and Ethanol composition in aqueous solution with a concentration of 1-4% (w/v) sodium salt(s) of EDTA, 10-25% (v/v) Ethanol and Mannitol 0.5 to 1% (w/v) at a pH range of 7 to 11.

[0068] Also disclosed are methods of using the compositions. In an embodiment, a method for disinfecting a catheter is disclosed, the method comprising: introducing a composition into an interior lumen of the catheter, wherein the composition comprises of sodium EDTA, at a concentration ranging from 1 to 8% (w/v), the Ethanol, at a concentration ranging from 5 to 40% (v/v) and D-Mannitol, at a concentration ranging from 0.5 to 3% (w/v) with the pH ranging from mild acidic to alkaline about 6 to 11.

[0069] In another aspect, a method for disinfecting, maintaining catheter patency, and an International Normalized Ratio (INR) of at least 0.5 points higher than that of the INR of 10 U/ml heparin is provided, the said method comprising of introducing a composition into an interior lumen of the catheter, wherein the composition comprises of sodium salt(s) of EDTA, at a concentration ranging from 1 to 8% (w/v), the Ethanol, at a concentration ranging from 5 to 40% (v/v) and Mannitol, at a concentration ranging from 0.5 to 3% (w/v) with the pH ranging from mild acidic to alkaline about 6 to 11.

[0070] In some embodiments, disinfectant compositions of the present disclosure comprise sodium salt(s) of EDTA, Ethanol and D-Mannitol having specified concentrations, at specified pH ranges, and may contain additional materials. Other antimicrobial or bio-cidal components may be incorporated in disinfectant compositions of the present disclosure comprising sodium salt(s) of EDTA, Ethanol and D-Mannitol although the use of traditional antibiotics and bio-cidal agents is generally discouraged as a result of the potential dire consequences of the development of antibiotic and bio-cidal resistant organisms.

[0071] In some embodiments, disinfectant compositions of the present disclosure comprising sodium salt(s) of EDTA, Ethanol and D-Mannitol having specified concentration(s), at specified pH ranges, are substantially free from other active substances having substantial antimicrobial and/or anti-fungal activity. Other active and inactive components may also be incorporated in disinfectant compositions of the present disclosure comprising sodium salt(s) of EDTA, Ethanol and D-Mannitol, provided that they don't deleteriously affect the activity and/or stability of the sodium salt(s) of EDTA, Ethanol and D-Mannitol. Proteolytic agents may be incorporated in disinfectant compositions for some applications. Disinfectant compositions formulated for topical application have various creams, emollients, skin care compositions such as aloe-vera, and the like, for example. Disinfectant compositions of the present disclosure 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 sodium salt(s) of EDTA, Ethanol and D-Mannitol.

[0072] The compositions of the present disclosure may be used in a solution or dry form. In solution, the sodium salt(s) of EDTA, Ethanol and D-Mannitol are preferably dissolved in a solvent, which may comprise an aqueous solution, such as water or saline, or another biocompatible solution in which the sodium salt(s) of EDTA, Ethanol and D-Mannitol are soluble, or a combination thereof. In some embodiments, bio-compatible non-aqueous solvents may also be employed, provided that the sodium salt(s) of EDTA, Ethanol and D-Mannitol can be solubilized and remain in solution during storage and use.

[0073] Sodium salt(s) of EDTA, Ethanol and D-Mannitol solutions of the present disclosure are preferably provided in a sterile and non-pyrogenic form and may be packaged in any convenient fashion. In some embodiments, disinfectant sodium salt(s) of EDTA, Ethanol and D-Mannitol compositions of the present disclosure 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 sodium salt(s) of EDTA, Ethanol and D-Mannitol solutions may be provided. Multiple use vials, syringes or containers may also be provided. Systems of the present disclosure include such vials, syringes or containers containing the sodium salt(s) of EDTA, Ethanol and D-Mannitol solutions of the present disclosure.

[0074] The compositions of the present disclosure 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. Dry forms of the disinfectant compositions of the present disclosure may include hydrophilic polymers such as PVP, which tend to absorb water and provide lubricity, surfactants to enhance solubility and/or bulking and buffering agents to provide thermal as well as pH stability. Such substantially dry forms of the sodium salt(s) of EDTA, Ethanol and D-Mannitol compositions of the present disclosure 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 the sodium salt(s) of EDTA, Ethanol and D-Mannitol 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 the sodium salt(s) of EDTA, Ethanol and D-Mannitol compositions of the present disclosure are formulated such that in the presence of a solution, the substantially dry composition forms the sodium salt(s) of EDTA, Ethanol and D-Mannitol 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 sodium salt(s) of EDTA, Ethanol and D-Mannitol is accomplished upon extended exposure to solutions. In this embodiment, the substantially dry sodium salt(s) of EDTA, Ethanol and D-Mannitol solutions may provide disinfectant activity over an extended period of time and/or upon multiple exposures to solutions.

[0075] Compositions comprising Ethanol have a well-established safety profile in connection with medical usage and administration to humans. Ethanol is present, in combination with other components, in many solutions used in medical and human health applications. It has been established as safe, for human use, both in vitro and in vivo. Ethanol is readily available at a reasonable cost, and is stable over time in solution.

[0076] Mannitol is utilized in various medical applications, primarily as bulking agents. It is used either singly or in combination with other agents. It has been established as safe for human use, both in-vitro and in-vivo. It is readily available at a reasonable cost, and is stable over time in solution.

[0077] Formulation and production of disinfectant compositions of the present disclosure are generally straightforward. In one embodiment, desired disinfectant compositions of the present disclosure are formulated by dissolving the sodium salt(s) of EDTA, Ethanol and D-Mannitol in an aqueous solvent, such as distilled water or phosphate buffer saline, to the desired concentration and adjusting the solution pH to the desired level. In alternative embodiments, desired disinfectant compositions of the present disclosure are formulated by dissolving the sodium salt(s) of EDTA, Ethanol and D-Mannitol in a solvent to provide a concentrated, solubilized solution, and additional solvents or components may then be added, or the solubilized composition may be formulated in a form other than a solution, such as a topical preparation. The disinfectant solution may then be sterilized using conventional means, such as filtration and/or ultra-filtration, and other means. The solutions are preferably formulated using USP materials.

[0078] Disinfectant compositions of the present disclosure comprising, or consisting essentially of, or consisting of, the sodium salt(s) of EDTA, Ethanol and D-Mannitol as described above are also useful for many other applications. Sodium salt(s) of EDTA, Ethanol and D-Mannitol solutions may be used as disinfectant solutions for soaking, or rinsing, or contacting medical, dental and veterinary surfaces and objects. Sodium salt(s) of EDTA, Ethanol and D-Mannitol solutions of the present disclosure may be used, for example, for storing and/or disinfectant contact lenses and other optical devices; for storing and/or disinfectant dental devices such as dentures, bridges, retainers, tooth brushes, and the like, and for storing and/or disinfectant medical and dental and veterinary devices and instruments. In these applications, the devices or surfaces may be contacted with sodium salt(s) of EDTA, Ethanol and Mannitol solutions of the present disclosure for a time sufficient to substantially eliminate microbial and/or fungicidal infections, or devices and surfaces may be soaked in sodium salt(s) of EDTA, Ethanol and D-Mannitol solutions for a desired time period. Sodium salt(s) of EDTA, Ethanol and D-Mannitol compositions of the present disclosure may additionally be used to disinfect water and other fluid supply lines. Disinfection of fluid supply lines may be accomplished by intermittently flushing the lines with sodium salt(s) of EDTA, Ethanol and D-Mannitol compositions of the present disclosure. Similarly, sodium salt(s) of EDTA, Ethanol and D-Mannitol compositions of the present disclosure may be used to eradicate bio-films and microbial (including some virus and protozoa) and fungal populations in water supply and storage devices.

[0079] Conduits may be treated with sodium salt(s) of EDTA, Ethanol and D-Mannitol solutions as a preventative disinfectant or as treatment following potential fungal or bacterial infection. The treatment of conduits can include locking, flushing, coating, or aerosol doses of the sodium salt(s) of EDTA, Ethanol and D-Mannitol solution. Examples of conduits that may be treated using the sodium salt(s) of EDTA, Ethanol and D-Mannitol solution include, but not limited to, water lines in dental or medical offices, lines carrying sterile fluids, catheters or ports that carry blood and/or other fluids into and out of the body, industrial water supply lines which develop large bio-film populations which effect the efficient flow of fluids as well as contaminating the fluids passing through the line, and airway support devices. Other examples include, but not limited to, consumption such as drink dispensers and food packaging. Conduits treated by the sodium salt(s) of EDTA, Ethanol and Mannitol solution are typically made of plastic, but the principles of the present disclosure may be applied to conduit device made of any material such as metal that delivers or carries fluid.

[0080] A sodium salt(s) of EDTA, Ethanol and Mannitol solution can be used in treatment of topical infections, including but not limited to skin, ear, anal, mouth, and vulvo/vaginal sites.

[0081] A sodium salt(s) of EDTA, Ethanol and Mannitol solution can be used in as an effective disinfectant for surfaces and equipment in industrial, medical, and household applications. A typical infected system would include the walls, floors, and commode in a lavatory. The delivery system will typically comprise a solvent and tools that allow flushing, locking, wiping, soaking, fogging, or coating of the surface defining the infected system.

[0082] A sodium salt(s) of EDTA, Ethanol and D-Mannitol solution can be used as an effective decontamination disinfectant for medical instruments and devices, dental (both consumer and professional) instruments and devices, and/or veterinary instruments and devices. A typical example would be a soak for disinfecting toothbrushes.

[0083] A sodium salt(s) of EDTA, Ethanol and D-Mannitol solution can be used as an effective disinfectant solution for optical contact lenses.

[0084] A sodium salt(s) of EDTA, Ethanol and D-Mannitol solution can be used as a treatment for catheters defining an infected system. The sodium salt(s) of EDTA, Ethanol and D-Mannitol solution may inhibit microbe colonization by treating the catheter with the solution at the prescribed concentration using a liquid lock prior to and in between infusions and/or by surface coating of catheter devices. A further application is the treatment of colonized or infected catheters by use of a liquid lock containing the sodium salt(s) of EDTA, Ethanol and D-Mannitol solution in the preferred concentration and pH.

[0085] Typically, the sodium salt(s) of EDTA, Ethanol and D-Mannitol solution, when used to treat catheters, are dissolved in water as a carrier, although other carriers may be used. Substances such as thrombolytics, sodium, alcohol, or reagents may also be added to the basic water/sodium salt(s) of EDTA, Ethanol and D-Mannitol solution.

Minimum Inhibitory Concentration (MIC)Experiments

[0086] Experiments were conducted to assess the minimum inhibitory concentration (MIC) both for sodium salt of EDTA that comprises tetra-sodium EDTA and Ethanol. This was conducted primarily, as the name suggests, to evaluate the minimum concentration where these reagents inhibit the growth of microorganisms. The method used was a variant of National, Committee for Clinical Laboratory Standards (NCCLS) macro-dilution procedure. In this experiment multiple concentrations of the solutions are prepared and exposed to the microorganisms for a period of 24 hours. Following incubation the growth is visually observed against the light. If the solutions exhibit turbidity/haziness it indicates growth. Clear solution indicates no growth. The minimum concentration at which no growth is observed is defined as the minimum inhibitory concentration (MIC).

[0087] The following solutions were prepared to evaluate the MIC values of sodium salt of EDTA that comprises tetra-sodium EDTA (TEDTA) and Ethanol. The reagents used were of Titriplex III GR [Ethylenedinitrilotetraacetic acid disodium salt (dihydrate)], Catalogue No 60841801001730, Lot No. QD3Q630747 Merck Specialties Private Limited; Ethanol Absolute, Catalogue No XK-13-011-00009, Lot No 20150125, Changshu Yangyuan Chemical, China.

TABLE-US-00001 TEDTA (CONC. WT %) ETHANOL (CONC. VOL %) 0.015 3.13 0.030 6.25 0.060 12.50 0.120 25.00 0.240 50.00

Synergy Experiments

[0088] Two sets of experiments were conducted to show an unexpected synergism of the disinfectant activity of a composition that includes both a sodium salt of EDTA that comprises tetra-sodium EDTA and Ethanol.

[0089] The first set of experiments conducted, were screening experiments using checkerboard titration strategy to assess if the combinations fall within a range having an Fractional Inhibitory Concentration (FIC) index value of ?1. The method used was a NCCLS macro-dilution procedure.

[0090] The second set of experiments conducted, were Rate Kill assay. A rate kill assay can confirm whether combinations are synergistic or not. In these assays the formulations are first exposed to organisms for a desired time (for the current formulations readings were taken at 1, 3 and 24 hours). Then a sample of the organisms and formulation mixture is serially diluted and plated to assess the log reduction. The organisms are allowed to grow and are checked for growth/log reduction after 24 hrs. The log reduction values obtained for individual components are compared with the combinations. Any combination(s) having >2 log reduction when compared with the most active compound used in the combination at any time point tested are labeled as synergistic (Comparison of methods for assessing synergic antibiotic interactions, M. L Mackay, K Miline, I. M. Gould, International journal of antimicrobial agents, 15 (2000) 125-129).

[0091] Based on the strategy stated above, experiments were conducted to investigate the effect of Ethanol on the antimicrobial activity of tetra-sodium EDTA. Reagents used were Titriplex III GR [Ethylenedinitrilotetraacetic acid disodium salt (dihydrate)], Catalogue No 60841801001730, Lot No. QD3Q630747 Merck Specialties Private Limited; Ethanol Absolute, Catalogue No XK-13-011-00009, Lot No 20150125, Changshu Yangyuan Chemical, China.

Checkerboard Titration Experiment(S. Aureus)

[0092] The Checkerboard Titration method was used to assess the interactions between TEDTA and Ethanol. The Checkerboard Titration method is a frequently used technique where, for example, each agent (TEDTA and Ethanol) was tested at multiple dilutions lower than the MIC. During this experiment, TEDTA and Ethanol were tested in the combinations to assess if the combinations have a Fractional Inhibitory Concentration (FIC) index of ?1. The following concentrations were tested:

TABLE-US-00002 CONCENTRATION CONCENTRATION TEDTA ETHANOL COMBINATION (WT %) (VOL %) 0.5 MIC + 0.5 MIC 0.120 12.5 0.25 MIC + 0.25 MIC 0.060 6.25 0.125 MIC + 0.125 MIC 0.030 3.125

[0093] Fractional Inhibitory Concentration (FIC) is defined as the MIC of the compound in combination divided by the MIC of the compound alone. If the FIC index is ?0.5, the combination is interpreted to be synergistic; <1 but >0.5as partially synergistic; =1 as additive; >1 but <4 as indifferent; and ?4 as antagonistic. In order to calculate the FIC index the following calculations are performed for compounds A and B:

FIC-A=(MIC of A in combination)/(MIC of A alone)

FIC-B=(MIC of B in combination)/(MIC of B alone)

FIC-combination=FIC-A+FIC-B

[0094] The MIC-TEDTA (MIC of TEDTA in combination with Ethanol), a minimum concentration of TEDTA, while in combination with Ethanol, which inhibited the growth of S. aureus in Mueller Hinton Broth (MHB) was found. In order to determine the MIC-Ethanol (MIC of Ethanol in combination with TEDTA), a minimum concentration of Ethanol, while in combination with TEDTA, which inhibited the growth of S. aureus in MHB was found. As per experiments conducted above, the MIC-TEDTA is 0.06% (w/v) for S. aureus and the MIC-ETH is 6.25% (w/v) for S. aureus. See table 4 for results.

[0095] Thus, the FIC-TEDTA is 0.06/0.24, which equals 0.25. The FIC-Ethanol is 6.25/25, which equals 0.25. Thus, the FIC-combination is 0.25+0.25, which equals 0.50. Accordingly, the combination of TEDTA and Ethanol unexpectedly has synergistic results. That is, embodiments of the combination of TEDTA and Ethanol provides results that are, unexpectedly, greater than the total effects of each agent operating by itself. This synergistic effect of embodiments of the combination of TEDTA and Ethanol may provide enhanced activity against bio-films. Without being bound to theory, it is suspected that TEDTA will degrade the bio-film, thus making the sessile microorganisms planktonic, allowing Ethanol to act against the more susceptible planktonic microorganisms. See table 4 for results.

Checkerboard Titration ExperimentC. albicans

[0096] The following concentrations were tested:

TABLE-US-00003 CONCENTRATION CONCENTRATION TEDTA ETHANOL COMBINATION (WT %) (VOL %) 0.5 MIC + 0.5 MIC 0.120 1.5625 0.25 MIC + 0.25 MIC 0.060 0.78125 0.125 MIC + 0.125 MIC 0.030 0.39062

[0097] The MIC-TEDTA (MIC of TEDTA in combination with Ethanol), a minimum concentration of TEDTA, while in combination with Ethanol, that inhibited the growth of C. albicans in MHB was found. In order to determine the MIC-Ethanol (MIC of Ethanol in combination with TEDTA), a minimum concentration of Ethanol, while in combination with TEDTA, that inhibited the growth of C. albicans in MHB was found. As per experiments conducted above, the MIC-TEDTA is 0.06% (w/v) for C. albicans and the MIC-Ethanol is 0.781% (v/v) for C. albicans. See table 5 for results.

[0098] Thus, the FIC-TEDTA is 0.06/0.24, which equals 0.25. The FIC-Ethanol is 0.781/3.125, which equals 0.25. Thus, the FIC-combination is 0.25+0.25, which equals 0.5. Accordingly, the combination of TEDTA and Ethanol unexpectedly has synergistic results. That is, embodiments of the combination of TEDTA and Ethanol provides results that are, unexpectedly, greater than the total effects of each agent operating by itself. This synergistic effect of embodiments of the combination of TEDTA and Ethanol may provide enhanced activity against bio-films. Without being bound to theory, it is suspected that TEDTA will degrade the bio-film, thus making the sessile microorganisms planktonic, allowing Ethanol to act against the more susceptible planktonic microorganisms.

Checkerboard Titration ExperimentP. aeruginosa

[0099] The following concentrations were tested:

TABLE-US-00004 CONCENTRATION CONCENTRATION TEDTA ETHANOL COMBINATION (WT %) (VOL %) 0.48 MIC + 0.5 MIC 0.120 3.125 0.24 MIC + 0.25 MIC 0.060 1.5625 0.12 MIC + 0.125 MIC 0.030 0.78125

[0100] The MIC-TEDTA (MIC of TEDTA in combination with Ethanol), a minimum concentration of TEDTA, while in combination with Ethanol, that inhibited the growth of P. aeruginosa in MHB was found. In order to determine the MIC-Ethanol (MIC of Ethanol in combination with TEDTA), a minimum concentration of Ethanol, while in combination with TEDTA, that inhibited the growth of P. aeruginosa in MHB was found. As per experiments conducted above, the MIC-TEDTA is 0.12% (w/v) for P. aeruginosa and the MIC-Ethanol is 3.125% (v/v) for P. aeruginosa. See table 6 for results.

[0101] Thus, the FIC-TEDTA is 0.12/0.25, which equals 0.48. The FIC-Ethanol is 3.125/6.25, which equals 0.5. Thus, the FIC-combination is 0.48+0.50, which equals 0.98. Accordingly, the combination of TEDTA and Ethanol unexpectedly has partially synergistic results. That is, embodiments of the combination of TEDTA and Ethanol provides results that are, unexpectedly, greater than the total effects of each agent operating by itself. This partially synergistic effect of embodiments of the combination of TEDTA and Ethanol may provide enhanced activity against bio-films. Without being bound to theory, it is suspected that TEDTA will degrade the bio-film, thus making the sessile microorganisms planktonic, allowing Ethanol to act against the more susceptible planktonic microorganisms.

Rate Kill Study

[0102] This study was conducted in two parts to assess [0103] a) the synergy and [0104] b) the effect of pH and D-Mannitol on the antimicrobial efficacy of the EDTA and ethanol based formulations.

Part A: Synergy Assessment

[0105] Rate Kill AssayS. aureus

[0106] The following solutions were prepared:

TABLE-US-00005 COMPOSITION WT % OR VOL % TEDTA 0.06 wt % TEDTA 0.12 wt % TEDTA 4.00 wt % Ethanol 6.25 vol % Ethanol 12.50 vol % Ethanol 25.00 vol % TEDTA + Ethanol 0.06 wt % + 6.25 vol % TEDTA + Ethanol 0.12 wt % + 12.50 vol % TEDTA + Ethanol 4.00 wt % + 12.50 vol % TEDTA + Ethanol 4.00 wt % + 25.00 vol %

[0107] Each solution was then combined with S. aureus and the log reduction of the S. aureus was measured at 1, 3 and 24 hours. The difference in log reduction was 4.33 at 1 hour and 4.25 at 3 hours when EDTA+Ethanol combinations were compared with the most active component in the combination alone. See table 7 for results. Accordingly, the data shows that TEDTA and Ethanol solutions are synergistic. That is, embodiments of the combination of TEDTA and Ethanol provides results that are, unexpectedly, greater than the total effects of each agent operating by itself. This synergistic effect of embodiments of the combination of TEDTA and Ethanol is expected to provide enhanced activity against bio-films. Without being bound to theory, it is suspected that TEDTA salt(s) will degrade the bio-film, thus making the sessile microorganisms planktonic, allowing Ethanol to act against the more susceptible planktonic microorganisms.

Rate Kill AssayC. albicans

[0108] The following solutions were prepared:

TABLE-US-00006 COMPOSITION WT % OR VOL % TEDTA 0.06 wt % TEDTA 0.12 wt % TEDTA 4.00 wt % Ethanol 0.78 vol % Ethanol 1.56 vol % Ethanol 12.50 vol % Ethanol 25.00 vol % TEDTA + Ethanol 0.06 wt % + 0.78 vol % TEDTA + Ethanol 0.12 wt % + 1.56 vol % TEDTA + Ethanol 4.00 wt % + 12.50 vol % TEDTA + Ethanol 4.00 wt % + 25.00 vol %

[0109] Each solution was then combined with C. albicans and the log reduction of the C. albicans was measured at 1, 3 and 24 hours. The difference in log reduction was 2.90 at 1 hour for the 4 wt % TEDTA+12.5 vol % Ethanol combination when compared with the most active ingredient i.e. 12.5 vol % Ethanol in the combination. See table 8 for results. Accordingly, the data shows that TEDTA and Ethanol solutions are synergistic. That is, embodiments of the combination of TEDTA and Ethanol provides results that are, unexpectedly, greater than the total effects of each agent operating by itself. This synergistic effect of embodiments of the combination of TEDTA and Ethanol is expected to provide enhanced activity against bio-films. Without being bound to theory, it is suspected that TEDTA salt(s) will degrade the bio-film, thus making the sessile microorganisms planktonic, allowing Ethanol to act against the more susceptible planktonic microorganisms.

Rate Kill AssayP. aeruginosa

[0110] The following solutions were prepared:

TABLE-US-00007 COMPOSITION WT % OR VOL % TEDTA 0.06 wt % TEDTA 0.12 wt % TEDTA 4.00 wt % Ethanol 1.56 vol % Ethanol 3.13 vol % Ethanol 12.50 vol % Ethanol 25.00 vol % TEDTA + Ethanol 0.06 wt % + 1.563 vol % TEDTA + Ethanol 0.12 wt % + 3.125 vol % TEDTA + Ethanol 4.00 wt % + 12.50 vol % TEDTA + Ethanol 4.000 wt % + 25.00 vol %

[0111] Each solution was then combined with P. aeruginosa and the log reduction of the P. aeruginosa was measured at 1, 3 and 24 hours. The difference in log reduction was 4.07 at 1 hour and 2.78 at 3 hours for the 4 wt % TEDTA+12.5 vol % Ethanol combination when compared with the most active ingredient i.e. 12.5 vol % Ethanol in the combination. See table 9 for results. Accordingly, the data shows that TEDTA and Ethanol solutions are synergistic. That is, embodiments of the combination of TEDTA and Ethanol provides results that are, unexpectedly, greater than the total effects of each agent operating by itself. This synergistic effect of embodiments of the combination of TEDTA and Ethanol is expected to provide enhanced activity against bio-films. Without being bound to theory, it is suspected that TEDTA salt(s) will degrade the bio-film, thus making the sessile microorganisms planktonic, allowing Ethanol to act against the more susceptible planktonic microorganisms.

[0112] The synergistic effect (rate kill assay) and partial/fully synergistic effect (checkerboard titration) provides significant, practical advantages for uses of embodiments of the combination of TEDTA and Ethanol. As noted, bio-films are a significant problem in a variety of fields. The bio-film protective substance, often referred to as extra-cellular polymeric substance (EPS), polysaccharide covering or glycocalyx, provides a protection to bio-films that are difficult to inhibit or eradicate. A solution that can inhibit or eradicate a bio-film is an important alternative. The proper use of antibiotics to eradicate a bio-film is costly, time consuming and may result in the development of antibiotic resistant bacterial strains, which cannot be effectively treated. Thus, embodiments of the present invention should prevent the overuse of broad-spectrum antibiotics and continued unnecessary catheter removal and replacement procedures.

Part B: Effect pH & D-Mannitol Addition Assessment

Rate Kill Assay

[0113] The following solutions were prepared:

TABLE-US-00008 COMPOSITION WT % OR VOL % pH EDTA + Ethanol + 4.00 wt % + 25.00 vol % + 7.5 D-Mannitol 0.73 wt % EDTA + Ethanol + 1.50 wt % + 12.50 vol % + 7.5 D-Mannitol 0.73 wt % EDTA + Ethanol 1.50 wt % + 12.50 vol % 7.5 D-Mannitol 0.73 wt % 7.5 EDTA + Ethanol + 4.00 wt % + 25.00 vol % + 11.0 D-Mannitol 0.73 wt % EDTA + Ethanol + 1.50 wt % + 12.50 vol % + 11.0 D-Mannitol 0.73 wt % EDTA + Ethanol 1.50 wt % + 12.50 vol % 11.0 D-Mannitol 0.73 wt % 11.0

[0114] Each solution was then combined with S. aureus and the log reduction of the S. aureus was measured at 1, 3 and 24 hours. The data shows enhancement in antimicrobial efficacy (>2 logs at 1 & 3 hrs time point) with pH increase from 7.5 to 11 for EDTA+Ethanol+D-Mannitol. Further, D-Mannitol exhibits >4 log reduction, which is unexpectedly high both at pH 7.5 & 11. See table 10 for results.

[0115] Each solution was then combined with C. albicans and the log reduction of the C. albicans was measured at 1, 3 and 24 hours. The data exhibits that with increase in pH, the antimicrobial efficacy remains similar for EDTA+Ethanol+D-Mannitol. This particular finding would be extremely beneficial since it would allow the formulation to be used at physiological pH without compromising the activity. Further, D-Mannitol exhibits >5 log reduction, which is unexpectedly high both at pH 7.5 & 11. See table 11 for results.

[0116] Each solution was then combined with P. aeruginosa and the log reduction of the P. aeruginosa was measured at 1, 3 and 24 hours. The data exhibits that with increase in pH, the antimicrobial efficacy remains similar for EDTA+Ethanol+D-Mannitol. This particular finding would be extremely beneficial since it would allow the formulation to be used at physiological pH without compromising the activity. Further, D-Mannitol exhibits >5 log reduction, which is unexpectedly high, both at pH 7.5 & 11. See table 12 for results.

[0117] Further, the embodiments of the combination of EDTA, Ethanol and D-Mannitol are expected to provide enhanced activity against bio-films. Without being bound to theory, it is suspected that D-Mannitol will enhance antimicrobial sensitivity of bio-films, while EDTA salt(s) will degrade the bio-film, thus making the sessile microorganisms planktonic, allowing Ethanol to act against the more susceptible planktonic microorganisms.

Prothrombin Time (PT) Experiments:

[0118] Experiments were conducted to assess the prothrombin time (PT) both for sodium salt of EDTA that comprises tetra-sodium EDTA and Ethanol. In addition, combination solutions that comprise of tetra-sodium EDTA and Ethanol were also tested. This was conducted primarily, to evaluate to what extent the solutions delays the clotting time when compared with the citrated control.

[0119] The blood was drawn from healthy males in the age group of 18 to 40 years with no history of any kind of anticoagulant therapy etc. Blood samples were collected in plain tubes containing the solutions in a ratio of 1:10, usually 1.8 or 2.7 ml blood is mixed with 0.2 or 0.3 ml solution. Plasma was obtained from each venous sample by centrifugation. Following centrifugation the red cells were removed by carefully pipetting the layer off to avoid the platelet layer. PT was then measured by STA-Neoplastine CI Plus (Diagnostica Stago, Asnieres, France).

[0120] This study was conducted in two parts to assess the [0121] a) anticoagulant activity of EDTA, Ethanol and in Combination. [0122] b) effects of pH and D-Mannitol on anticoagulant activity of EDTA and Ethanol based formulations.

Part A

[0123] Following solutions were prepared for PT assessment:

TABLE-US-00009 COMPOSITION WT % OR VOL % TEDTA 4.0 wt % Ethanol 12.5 vol % Ethanol 25.0 vol % TEDTA + Ethanol 4.0 wt % + 12.5 vol % TEDTA + Ethanol 4.0 wt % + 25.0 vol %

[0124] 10 U/ml of Heparin solution sample were provided by the medical practitioners. The manufacturer was Troikka Pharmaceuticals.

[0125] The above solutions were assessed for PT. See table 13 for results. From the table it is evident that the formulations containing TEDTA and Ethanol have higher PT when compared with heparin alone. This clearly indicates the anticoagulant capability of the formulations.

Part B

[0126]

TABLE-US-00010 COMPOSITION WT % OR VOL % pH EDTA + Ethanol + 4.00 wt % + 25.00 vol % + 7.5 D-Mannitol 0.73 wt % EDTA + Ethanol + 1.50 wt % + 12.50 vol % + 7.5 D-Mannitol 0.73 wt % EDTA + Ethanol 1.50 wt % + 12.50 vol % 7.5 D-Mannitol 0.73 wt % 7.5 EDTA + Ethanol + 4. 00 wt % + 25.00 vol % + 11.0 D-Mannitol 0.73 wt % EDTA + Ethanol + 1.50 wt % + 12.50 vol % + 11.0 D-Mannitol 0.73 wt % EDTA + Ethanol 1.50 wt % + 12.50 vol % 11.0 D-Mannitol 0.73 wt % 11.0

[0127] The above solutions were assessed for PT. See table 14 for results. From the table it is evident that a) the lower pH formulations have slightly better anticoagulant activity, b) addition of D-Mannitol helps in maintaining the PT for EDTA and Ethanol based formulations. In addition, it apparent that the formulations containing EDTA, Ethanol and D-Mannitol have higher PT when compared with heparin alone (see table 13). This clearly indicates the anticoagulant capability of the formulations.

[0128] From the foregoing, it should be clear that the present disclosure may be embodied in forms other than those discussed above; the scope of the present disclosure should be determined by the following claims and not the detailed discussion presented above.

TABLE-US-00011 TABLE 1 MINIMUM INHIBITORY CONCENTRATION (MIC) VIA MACRO-DILUTION METHOD - RESULTS MICROORGANISM - S. aureus TEDTA ETHANOL (CONC (CONC WT %) GROWTH GROWTH GROWTH VOL %) GROWTH GROWTH GROWTH 0.015 +VE +VE +VE 3.13 +VE +VE +VE 0.030 +VE +VE +VE 6.25 +VE +VE +VE 0.060 +VE +VE +VE 12.50 +VE +VE +VE 0.120 +VE +VE +VE 25.00 ?VE ?VE ?VE 0.240 ?VE ?VE ?VE 50.00 ?VE ?VE ?VE POSITIVE +VE +VE +VE CONTROL NEGATIVE ?VE ?VE ?VE CONTROL TEDTAMIC 0.24 WT % ETHANOLMIC 25 VOL %

TABLE-US-00012 TABLE 2 MINIMUM INHIBITORY CONCENTRATION (MIC) VIA MACRO-DILUTION METHOD - RESULTS MICROORGANISM - C. albicans TEDTA ETHANOL (CONC (CONC WT %) GROWTH GROWTH GROWTH VOL %) GROWTH GROWTH GROWTH 0.015 +VE +VE +VE 3.125 ?VE ?VE ?VE 0.030 +VE +VE +VE 6.25 ?VE ?VE ?VE 0.060 +VE +VE +VE 12.50 ?VE ?VE ?VE 0.120 +VE +VE +VE 25.00 ?VE ?VE ?VE 0.240 ?VE ?VE ?VE 50.00 ?VE ?VE ?VE POSITIVE +VE +VE +VE CONTROL NEGATIVE ?VE ?VE ?VE CONTROL TEDTAMIC 0.24 WT % ETHANOLMIC 3.125 VOL %

TABLE-US-00013 TABLE 3 MINIMUM INHIBITORY CONCENTRATION (MIC) VIA MACRO-DILUTION METHOD - RESULTS MICROORGANISM - P. aeruginosa TEDTA ETHANOL (CONC (CONC WT %) GROWTH GROWTH GROWTH VOL %) GROWTH GROWTH GROWTH 0.125 +VE +VE +VE 3.13 +VE +VE +VE 0.250 ?VE ?VE ?VE 6.25 ?VE ?VE ?VE 0.500 ?VE ?VE ?VE 12.50 ?VE ?VE ?VE 1.000 ?VE ?VE ?VE 25.00 ?VE ?VE ?VE 1.500 ?VE ?VE ?VE 50.00 ?VE ?VE ?VE POSITIVE +VE +VE +VE CONTROL NEGATIVE ?VE ?VE ?VE CONTROL TEDTAMIC 0.25 WT % ETHANOLMIC 6.25 VOL %

TABLE-US-00014 TABLE 4 SYNERGY EVALUATION STUDY - RESULTS STRATEGY - CHECKERBOARD TITRATION; METHOD - MACRO DILUTION; MICROORGANISM - S. aureus TEDTA ETHANOL TEDTA ETHANOL (CONC (CONC (CONC (CONC WT %) VOL %) WT %) VOL %) IND. MIC GROWTH IND. MIC GROWTH COMB FIC GROWTH 0.24 1.00 ?VE 25.000 1.00 ?VE 0.12 0.50 +VE 12.500 0.50 +VE 0.12 12.500 1.00 ?VE 0.06 0.25 +VE 6.250 0.25 +VE 0.06 6.250 0.50 ?VE 0.03 0.13 N/D 3.125 0.13 N/D 0.03 3.125 0.25 +VE FIC = 0.50 - HENCE TEDTA + ETHANOL BASED COMBINATION SOLUTIONS HAVE SYNERGISTIC ACTION AGAINST S. aureus ?VENO GROWTH +VEGROWTH N/DNOT DONE INDINDIVIDUAL REAGENTS COMBCOMBINATION SOLUTION FICFRACTIONAL INHIBITORY CONCENTRATION FIC = 1 (ADDITIVE); FIC = <1 BUT >0.5 PARTIAL SYNERGY; FIC <0.5 SYNERGY

TABLE-US-00015 TABLE 5 SYNERGY EVALUATION STUDY - RESULTS STRATEGY - CHECKERBOARD TITRATION; METHOD - MACRO DILUTION; MICROORGANISM - C. albicans TEDTA ETHANOL TEDTA ETHANOL (CONC (CONC (CONC (CONC WT %) VOL %) WT %) VOL %) IND MIC GROWTH IND MIC GROWTH COMB FIC GROWTH 0.24 1.00 ?VE 3.125 1.00 ?VE 0.12 0.50 ?VE 1.563 0.50 +VE 0.12 1.563 1.00 ?VE 0.06 0.25 ?VE 0.781 0.25 +VE 0.06 0.781 0.50 ?VE 0.03 0.13 N/D 0.391 0.13 N/D 0.03 0.391 0.25 +VE FIC = 0.50 - HENCE TEDTA + ETHANOL BASED COMBINATION SOLUTIONS HAVE SYNERGISTIC ACTION AGAINST C. albicans ?VENO GROWTH +VEGROWTH N/DNOT DONE INDINDIVIDUAL REAGENTS COMBCOMBINATION SOLUTION FICFRACTIONAL INHIBITORY CONCENTRATION FIC = 1 (ADDITIVE); FIC = <1 BUT >0.5 PARTIAL SYNERGY; FIC <0.5 SYNERGY

TABLE-US-00016 TABLE 6 SYNERGY EVALUATION STUDY - RESULTS STRATEGY - CHECKERBOARD TITRATION; METHOD - MACRO DILUTION; MICROORGANISM - P. aeruginosa TEDTA ETHANOL TEDTA ETHANOL (CONC (CONC (CONC (CONC WT %) VOL %) WT %) VOL %) IND. MIC GROWTH IND. MIC GROWTH COMB FIC GROWTH 0.24 0.96 ?VE 6.250 1.00 ?VE 0.12 0.48 +VE 3.125 0.50 +VE 0.12 3.125 0.98 ?VE 0.06 0.24 +VE 1.563 0.25 +VE 0.06 1.563 0.49 +VE 0.03 0.781 0.245 +VE FIC = 0.98 - HENCE TEDTA + ETHANOL BASED COMBINATION SOLUTIONS HAVE PARTIALLY SYNERGISTIC ACTION AGAINST P. aeruginosa ?VENO GROWTH +VEGROWTH N/DNOT DONE INDINDIVIDUAL REAGENTS COMBCOMBINATION SOLUTION FICFRACTIONAL INHIBITORY CONCENTRATION FIC = 1 (ADDITIVE); FIC = <1 BUT >0.5 PARTIAL SYNERGY; FIC <0.5 SYNERGY

TABLE-US-00017 TABLE 7 SYNERGY EVALUATION STUDY - RESULTS STRATEGY - RATE KILL; METHOD - PLATE COUNT; MICROORGANISM - S. aureus TEDTA EtOH TEDTA ETHANOL (CONC (CONC (CONC (CONC DIFF IN SOLN WT %) LOG SOLN VOL %) LOG SOLN WT %) VOL %) LOG LOG TIME CODE (IND) RED CODE (IND) RED CODE (COMB) RED COMBIND RED 1 HR 1 0.06 3.60 4 6.25 3.58 7 0.06 6.25 3.60 2 0.12 3.53 5 12.50 3.67 8 0.12 12.50 3.67 3 4.0 3.74 6 25.00 8.00 9 4.0 12.50 8.00 9-5 4.33 10 4.0 25.00 8.00 3 HR 1 0.06 3.55 4 6.25 3.62 7 0.06 6.25 3.70 2 0.12 3.51 5 12.50 3.65 8 0.12 12.50 4.03 3 4.0 4.02 6 25.00 7.90 9 4.0 12.50 7.90 9-5 4.25 10 4.0 25.00 7.90 24 HR 1 0.06 3.06 4 6.25 4.15 7 0.06 6.25 4.22 2 0.12 3.00 5 12.50 7.30 8 0.12 12.50 7.30 3 4.0 7.30 6 25.00 7.30 9 4.0 12.50 7.30 10 4.0 25.00 7.30 COMBINATION IS SYNERGISTIC IF THE DIFFERENCE IN LOG REDUCTION IS GREATER THAN 2 LOGS WHEN COMBINATION IS COMPARED WITH THE MOST ACTIVE COMPONENT. FROM THE ABOVE DATA IT IS EVIDENT THAT THE TEDTA + ETHANOL COMBINATION HAS SYNERGISTIC ACTION AGAINST S. aureus.

TABLE-US-00018 TABLE 8 SYNERGY EVALUATION STUDY - RESULTS STRATEGY - RATE KILL; METHOD - PLATE COUNT; MICROORGANISM - C. albicans TEDTA EtOH TEDTA EtOH (CONC (CONC (CONC (CONC DIFF IN SOLN WT %) LOG SOLN VOL %) LOG SOLN WT %) VOL %) LOG LOG TIME CODE (IND) RED CODE (IND) RED CODE (COMB) RED COMBIND RED 1 HR 1 0.06 3.44 4 0.78 3.43 8 0.06 0.78 3.48 2 0.12 3.46 5 1.56 3.45 9 0.12 1.56 3.50 3 4.0 3.92 6 12.50 5.00 10 4.0 12.50 7.90 10-6 2.90 7 25.00 7.90 11 4.0 25.00 7.90 3 HR 1 0.06 3.48 4 0.78 3.47 8 0.06 0.78 3.60 2 0.12 3.51 5 1.56 3.49 9 0.12 1.56 3.64 3 4.0 7.90 6 12.50 7.90 10 4.0 12.50 7.90 7 25.00 7.90 11 4.0 25.00 7.90 24 HR 1 0.06 3.32 4 0.78 3.22 8 0.06 0.78 7.60 2 0.12 3.38 5 1.56 3.28 9 0.12 1.56 7.60 3 4.0 7.60 6 12.50 7.60 10 4.0 12.50 7.60 7 25.00 7.60 11 4.00 25.00 7.60 COMBINATION IS SYNERGISTIC IF THE DIFFERENCE IN LOG REDUCTION IS GREATER THAN 2 LOGS WHEN COMBINATION IS COMPARED WITH THE MOST ACTIVE COMPONENT. FROM THE ABOVE DATA IT IS EVIDENT THAT THE TEDTA + ETHANOL COMBINATION HAS SYNERGISTIC ACTION AGAINST C. albicans.

TABLE-US-00019 TABLE 9 SYNERGY EVALUATION STUDY - RESULTS STRATEGY - RATE KILL; METHOD - PLATE COUNT; MICRO ORGANISM - P. aeruginosa TEDTA EtOH TEDTA EtOH (CONC (CONC (CONC (CONC DIFF IN SOLN WT %) LOG SOLN VOL %) LOG SOLN WT %) VOL %) LOG LOG TIME CODE (IND) RED CODE (IND) RED CODE (COMB) RED COMBIND RED 1 HR 1 0.06 3.75 4 1.56 3.76 8 0.06 1.563 5.12 2 0.12 3.83 5 3.13 3.76 9 0.12 3.125 5.20 3 4.0 4.23 6 12.50 4.13 10 4.0 12.50 8.20 10-6 4.07 7 25.00 8.20 11 4.0 25.00 8.20 3 HR 1 0.06 3.89 4 1.56 3.65 8 0.06 1.563 6.08 2 0.12 4.32 5 3.13 3.73 9 0.12 3.125 8.08 3 4.0 5.12 6 12.50 5.30 10 4.0 12.50 8.08 10-6 2.78 7 25.00 8.08 11 4.0 25.00 8.08 24 HR 1 0.06 4.32 4 1.56 3.64 8 0.06 1.563 7.90 2 0.12 7.90 5 3.13 7.90 9 0.12 3.125 7.90 3 4.0 7.90 6 12.50 7.90 10 4.0 12.50 7.90 7 25.00 7.90 11 4.0 25.00 7.90 COMBINATION IS SYNERGISTIC IF THE DIFFERENCE IN LOG REDUCTION IS GREATER THAN 2 LOGS WHEN COMBINATION IS COMPARED WITH THE MOST ACTIVE COMPONENT. FROM THE ABOVE DATA IT IS EVIDENT THAT THE TEDTA + ETHANOL COMBINATION HAS SYNERGISTIC ACTION AGAINST P. aeruginosa.

TABLE-US-00020 TABLE 10 pH AND D-MANNITOL ADDITION EFFECT EVALUATION STUDY - RESULTS STRATEGY - RATE KILL; METHOD - PLATE COUNT; MICRO ORGANISM - S. aureus EDTA EtOH MAN AVG. SOLN (CONC (CONC (CONC LOG LOG LOG TIME CODE WT %) VOL %) WT %) pH RED RED RED 1 HR 1 4.00 25.00 0.73 7.5 5.03 5.05 5.04 2 1.50 12.50 0.73 7.5 4.70 4.70 4.70 3 1.50 12.50 0.00 7.5 4.49 4.49 4.49 4 0.00 0.00 0.73 7.5 4.53 4.53 4.53 5 4.00 25.00 0.73 11.0 7.58 7.58 7.58 6 1.50 12.50 0.73 11.0 4.77 4.77 4.77 7 1.50 12.50 0.00 11.0 4.59 4.58 4.59 8 0.00 0.00 0.73 11.0 4.38 4.38 4.38 3 HR 1 4.00 25.00 0.73 7.5 5.50 5.46 5.48 2 1.50 12.50 0.73 7.5 4.95 4.95 4.95 3 1.50 12.50 0.00 7.5 4.64 4.64 4.64 4 0.00 0.00 0.73 7.5 4.67 4.66 4.66 5 4.00 25.00 0.73 11.0 7.58 7.58 7.58 6 1.50 12.50 0.73 11.0 6.97 7.27 7.12 7 1.50 12.50 0.00 11.0 6.62 7.10 6.86 8 0.00 0.00 0.73 11.0 4.47 4.47 4.47 24 HR 1 4.00 25.00 0.73 7.5 5.65 5.68 5.67 2 1.50 12.50 0.73 7.5 4.98 4.98 4.98 3 1.50 12.50 0.00 7.5 4.70 4.70 4.70 4 0.00 0.00 0.73 7.5 4.75 4.74 4.75 5 4.00 25.00 0.73 11.0 7.53 7.53 7.53 6 1.50 12.50 0.73 11.0 7.53 7.53 7.53 7 1.50 12.50 0.00 11.0 7.53 7.53 7.53 8 0.00 0.00 0.73 11.0 4.59 4.59 4.59 WITH PH INCREASE LOG REDUCTION IS GREATER THAN 2 LOGS AT 1 & 3 HRS FOR 4% EDTA + 25% ETHANOL + 0.73% MANNITOL; D-MANNITOL EXHIBITS > 4 LOG REDUCTION AGAINST S. aureus

TABLE-US-00021 TABLE 11 pH AND D-MANNITOL ADDITION EFFECT EVALUATION STUDY - RESULTS STRATEGY - RATE KILL; METHOD - PLATE COUNT; MICRO ORGANISM - C. albicans EDTA EtOH MAN AVG. SOLN (CONC (CONC (CONC LOG LOG LOG TIME CODE WT %) VOL %) WT %) pH RED RED RED 1 HR 1 4.00 25.00 0.73 7.5 7.55 7.55 7.55 2 1.50 12.50 0.73 7.5 5.71 5.69 5.70 3 1.50 12.50 0.00 7.5 5.69 5.75 5.72 4 0.00 0.00 0.73 7.5 5.37 5.39 5.38 5 4.00 25.00 0.73 11.0 7.55 7.55 7.55 6 1.50 12.50 0.73 11.0 5.88 5.84 5.86 7 1.50 12.50 0.00 11.0 6.07 6.14 6.10 8 0.00 0.00 0.73 11.0 5.27 5.27 5.27 3 HR 1 4.00 25.00 0.73 7.5 7.55 7.55 7.55 2 1.50 12.50 0.73 7.5 6.21 6.21 6.21 3 1.50 12.50 0.00 7.5 6.29 6.25 6.27 4 0.00 0.00 0.73 7.5 5.55 5.57 5.56 5 4.00 25.00 0.73 11.0 7.55 7.55 7.55 6 1.50 12.50 0.73 11.0 6.51 6.51 6.51 7 1.50 12.50 0.00 11.0 6.85 7.07 6.96 8 0.00 0.00 0.73 11.0 5.42 5.40 5.41 24 HR 1 4.00 25.00 0.73 7.5 7.53 7.53 7.53 2 1.50 12.50 0.73 7.5 7.53 7.53 7.53 3 1.50 12.50 0.00 7.5 7.53 7.53 7.53 4 0.00 0.00 0.73 7.5 5.76 5.74 5.75 5 4.00 25.00 0.73 11.0 7.53 7.53 7.53 6 1.50 12.50 0.73 11.0 7.53 7.53 7.53 7 1.50 12.50 0.00 11.0 7.53 7.53 7.53 8 0.00 0.00 0.73 11.0 5.55 5.58 5.56 WITH PH INCREASE ANTIMICROBIAL EFFICACY REMAINS SIMILAR FOR 4% EDTA + 25% ETHANOL + 0.73% D-MANNITOL D-MANNITOL EXHIBITS > 5 LOG REDUCTION AGAINST C. albicans

TABLE-US-00022 TABLE 12 pH AND D-MANNITOL ADDITION EFFECT EVALUATION STUDY - RESULTS STRATEGY - RATE KILL; METHOD - PLATE COUNT; MICRO ORGANISM - P. aeruginosa EDTA EtOH MAN AVG. SOLN (CONC (CONC (CONC LOG LOG LOG TIME CODE WT %) VOL %) WT %) pH RED RED RED 1 HR 1 4.00 25.00 0.73 7.5 7.60 7.60 7.60 2 1.50 12.50 0.73 7.5 7.60 7.60 7.60 3 1.50 12.50 0.00 7.5 7.60 7.60 7.60 4 0.00 0.00 0.73 7.5 5.41 5.39 5.40 5 4.00 25.00 0.73 11.0 7.60 7.60 7.60 6 1.50 12.50 0.73 11.0 6.56 6.56 6.56 7 1.50 12.50 0.00 11.0 6.42 6.49 6.45 8 0.00 0.00 0.73 11.0 5.42 5.42 5.42 3 HR 1 4.00 25.00 0.73 7.5 7.59 7.59 7.59 2 1.50 12.50 0.73 7.5 7.59 7.59 7.59 3 1.50 12.50 0.00 7.5 7.59 7.59 7.59 4 0.00 0.00 0.73 7.5 5.73 5.75 5.74 5 4.00 25.00 0.73 11.0 7.59 7.59 7.59 6 1.50 12.50 0.73 11.0 7.59 7.59 7.59 7 1.50 12.50 0.00 11.0 7.59 7.59 7.59 8 0.00 0.00 0.73 11.0 5.74 5.80 5.77 24 HR 1 4.00 25.00 0.73 7.5 7.55 7.55 7.55 2 1.50 12.50 0.73 7.5 7.55 7.55 7.55 3 1.50 12.50 0.00 7.5 7.55 7.55 7.55 4 0.00 0.00 0.73 7.5 6.51 6.44 6.47 5 4.00 25.00 0.73 11.0 7.55 7.55 7.55 6 1.50 12.50 0.73 11.0 7.55 7.55 7.55 7 1.50 12.50 0.00 11.0 7.55 7.55 7.55 8 0.00 0.00 0.73 11.0 6.55 6.25 6.40 WITH PH INCREASE ANTIMICROBIAL EFFICACY REMAINS SIMILAR FOR 4% EDTA + 25% ETHANOL + 0.73% D-MANNITOL D-MANNITOL EXHIBITS > 5 LOG REDUCTION AGAINST P. aeruginosa.

TABLE-US-00023 TABLE 13 PART A - PROTHROMBIN TIME (PT) STUDY - RESULTS OBJECTIVE - TO ASSESS INTERACTION AMONG EDTA & ETHANOL FOR ANTICOAGULANT ACTIVITY AVERAGE STD SAMPLES INR-A INR-B INR-C INR-D INR IN INR CITRATED CONTROL 1.000 1.000 1.000 1.000 1.000 0.000 HEPARIN SODIUM 10 IU/ML 1.219 1.260 1.503 1.354 1.334 0.126 4 WT % TEDTA 1.891 1.937 2.229 2.087 2.036 0.153 4 WT % TEDTA + 25 VOL % ETHANOL 2.052 2.041 2.409 2.470 2.243 0.228 4 WT % TEDTA + 12.5 VOL % ETHANOL 2.099 2.099 2.146 2.134 2.120 0.024 25 VOL % ETHANOL 61.930 61.930 61.930 NOT DONE 61.930 0.000 12.5 VOL % ETHANOL 61.930 61.930 61.930 NOT DONE 61.930 0.000 THE ABOVE STUDIES WERE DONE ON TWO DIFFERENT DAYS AND USING DIFFERENT BLOOD FROM MALE POPULATION WITHIN THE AGE RANGE OF 18-40 YEARS. INR-A & INR-B - DURING FIRST ROUND WHILE INR-C & INR-D DURING SECOND ROUND. PTPROTHROMBIN TIME CITRATED CONTROL - 13.8 SECS ASSAY CONTROL - 13.8 SECS INTERNATION STANDARDIZATION INDEX (ISI) - 1.34 INR = (SAMPLE PT/ASSAY CONTROL PT){circumflex over ()}ISI FROM THE ABOVE RESULTS IT IS EVIDENT THAT 4% TEDTA + 25% ETHANOL & 4% TEDTA + 12.5% ETHANOL HAVE HIGHER INR WHEN COMPARED WITH 10 IU/ML HEPARIN. 4% TEDTA + 25% ETHANOL HAVE HIGHER INR WHEN COMPARED WITH 10 IU/ML AS WELL AS 4% TEDTA+12.5% ETHANOL.

TABLE-US-00024 TABLE 14 PART B - PROTHROMBIN TIME (PT) STUDY - RESULTS OBJECTIVE - TO ASSESS EFFECTS OF pH AND D-MANNITOL ADDITION TO EDTA & ETHANOL BASED FORMULATIONS SAMPLES WT % OR VOL % pH INR CITRATED CONTROL N/A N/A 1.00 EDTA + Ethanol + 4.00 wt % + 25.00 vol % + 7.5 2.54 D-Mannitol 0.73 wt % EDTA + Ethanol + 1.50 wt % + 12.50 vol % + 7.5 1.72 D-Mannitol 0.73 wt % EDTA + Ethanol 1.50 wt % + 12.50 vol % 7.5 1.74 D-Mannitol 0.73 wt % 7.5 N/A EDTA + Ethanol + 4.00 wt % + 25.00 vol % + 11.0 2.21 D-Mannitol 0.73 wt % EDTA + Ethanol + 1.50 wt % + 12.50 vol % + 11.0 1.71 D-Mannitol 0.73 wt % EDTA + Ethanol 1.50 wt % + 12.50 vol % 11.0 1.64 D-Mannitol 0.73 wt % 11.0 N/A THE ABOVE STUDY WAS DONE USING BLOOD FROM MALE POPULATION WITHIN THE AGE RANGE OF 18-40 YEARS. PTPROTHROMBIN TIME CITRATED CONTROL - 11.9 SECS ASSAY CONTROL - 11.9 SECS INTERNATION STANDARDIZATION INDEX (ISI) - 1.24 INR = (SAMPLE PT/ASSAY CONTROL PT){circumflex over ()}ISI FROM THE ABOVE RESULTS IT IS EVIDENT THAT A) pH HAS SLIGHT EFFECT - I.E. LOWER THE PH HIGHER IS THE PT TIME. B) ADDITION OF MANNITOL HELPS IN MAINTAINING THE ANTICOGULANT ACTIVITY OF EDTA AND ETHANOL BASED FORMULATIONS.

[0129] A lock flush composition comprising of sodium salt(s) of EDTA, Ethanol and D-Mannitol in solution, wherein the sodium salt(s) of EDTA is at a concentration of at least 1% (w/v) and up to 8% (w/v), the Ethanol is at a concentration of at least 5% (v/v) and up to 40% (v/v) and D-Mannitol is at a concentration of at least 0.5% (w/v) and up to 3% (w/v), the lock flush composition has a pH of at least 6.0 and up to 11.0, the lock flush composition is packaged in a sterile, non-pyrogenic form, and the lock flush composition is biocompatible for use in in-dwelling access catheters, urinary catheters, nasal tubes and throat tubes.