Lock solutions composed of a methanetrisdiazeniumdiolate salt and methods for making and using the same

Abstract

Described herein are methods for preventing microbial infections derived from an implanted catheters when not in use. In one aspect, the method involves filing the implanted catheter when not in use with a lock solution comprising a pharmaceutically acceptable salt of methanetrisdiazeniumdiolate and a pharmaceutically acceptable carrier. The lock solution releases nitric oxide and Azanone, which makes the lock solution a highly effective antimicrobial agent. The lock solution and use thereof can be effective in reducing or preventing particularly dangerous infections such as, for example, bloodstream infections.

Description

LOCK SOLUTIONS COMPOSED OF A METHANETRISDIAZENIUMDIOLATE SALT AND METHODS FOR MAKING AND USING THE SAMESTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under grant numberR01 HL134899 awarded by the NIH. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to co-pending U.S. Provisional Patent Application No. 63 / 625,408, filed on January 26, 2024, the contents of which are incorporated by reference herein in their entireties.BACKGROUND

[0003] Central venous catheters (CVCs) are used for long-term intravenous administration of fluids, medication, and nutritional solutions, although CVCs can also be used for more specialized treatments such as hemodialysis or hemodynamic monitoring1. In the Unites States alone, 5 million CVCs are inserted every year2. Catheter related bloodstream infections (CRBSIs) are one of the most common but most lethal complications of CVCs; annually, 250,000 CRBSIs occur in the US. Moreover, CVCs pose a greater risk of device related infection than any other type of medical device1. One source states that these infections predispose patients to increased likelihood of myocardial infarction, heart failure, and stroke due to microinflammation3.

[0004] Whenever the CVC is not actively being used, the catheter is filled with a lock solution. The lock solution is typically just saline sometimes doped with Heparin, but antibiotic lock solutions have become popular as they show promise to prevent and treat CRBSIs. While these antibiotic lock solutions do work to an extent, the complex nature of biofilms and bacteria in general, render these antibiotic lock solutions less effective than expected. For one, to disperse and kill bacteria in a biofilm the antibiotic concentration must be 1000 times the concentration used to treat a normal bacterial infection4. These large doses of antibiotics contribute to antibiotic resistance and may also pose a toxicity threat to the patient. It has been reported that up to 20% of the antibiotic lock solution leaks from the catheter distal end and enters the bloodstream5 6. This further decreases the efficacy of the antibiotic lock solution and can cause toxic levels of the antibiotic to accumulate in the bloodstream.SUMMARY

[0005] Described herein are methods for preventing microbial infections derived from an implanted catheters when not in use. In one aspect, the method involves filing the implanted catheter when not in use with a lock solution comprising a pharmaceutically acceptable salt of methanetrisdiazeniumdiolate and a pharmaceutically acceptable carrier. The lock solution releases nitric oxide and Azanone, which makes the lock solution a highly effective antimicrobial agent. The lock solution and use thereof can be effective in reducing or preventing particularly dangerous infections such as, for example, bloodstream infections.

[0006] Other compositions, apparatus, methods, features, and advantages will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional compositions, apparatus, methods, features and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Further aspects of the present disclosure will be more readily appreciated upon review of the detailed description of its various embodiments, described below, when taken in conjunction with the accompanying drawings.

[0008] Figure 1 shows the salt form of methanetrisdiazeniumdiolate.

[0009] Figures 2A-2D show (A) the UV-Vis curve of CTN using a concentration of 50 pg / mL; (B) the standard curve of CTN with the linear fit equation; (C) the UV-Vis curve of Ampicillin using 25 pg / mL; and (D) the standard curve of Ampicillin with the linear fit equation

[0010] Figures 3A-3B show the1HNMR of CTN B)13CNMR of CTN.

[0011] Figure 4 shows the FTIR spectrum of CTN.

[0012] Figure 5 shows the NO release via diffusion through catheter walls in physiological conditions.

[0013] Figures 6A-6C shows Viable CFU of bacteria attached to the outside surface area of the catheter A) S. aureus B) E. coli C) MRSA.

[0014] Figure 7 shows the cytocompatibility of CTN at different concentrations and Ampicillin at 10 mg / mL with three dilutions that represent different leakage percents into the bloodstream.

[0015] Figures 8A-8B show (A) the concentration of Ampicillin leached out from the walls of the catheter and (B) the percentage of the Ampicillin loaded into the catheter that leached out.

[0016] Figure 9 shows the storage Stability of CTN powder stored for a month in the three different temperature conditions.

[0017] The drawings illustrate only example embodiments and are therefore not to be considered limiting of the scope described herein, as other equally effective embodiments are within the scope and spirit of this disclosure. The elements and features shown in the drawings are not necessarily drawn to scale, emphasis instead being placed upon clearly illustrating the principles of the embodiments. Additionally, certain dimensions may be exaggerated to help visually convey certain principles. In the drawings, similar reference numerals between figures designate like or corresponding, but not necessarily the same, elements.DETAILED DESCRIPTION

[0018] Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.

[0019] Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0020] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0021] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps oroperational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0022] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0023] While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.

[0024] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0025] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.Definitions

[0026] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” “having,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms“consisting essentially of’ and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.

[0027] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutically acceptable carrier” includes, but is not limited to, mixtures or combinations of two or more such carriers, and the like.

[0028] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0029] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about x’ to about ‘y’”.

[0030] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1 % to about 5%, but also includeindividual values (e.g., about 1 %, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1 %; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0031] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0032] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.

[0033] Disclosed are the components to be used to prepare the compositions disclosed herein as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modificationsthat can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B- D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

[0034] It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.

[0035] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance and instances where it does not.

[0036] The term “prevent” or “preventing” as used herein is defined as eliminating or reducing the likelihood of the occurrence of one or more symptoms of a microbial infection when using the lock solutions described herein.

[0037] The term “pharmaceutically acceptable salts”, as used herein, means salts of the active principal agents which are prepared with acids or bases that are tolerated by a biological system or tolerated by a subject or tolerated by a biological system and tolerated by a subject when administered in a therapeutically effective amount. When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include, but are not limited to; sodium, potassium, calcium, ammonium, organic amino, magnesium salt, lithium salt, strontium salt or a similar salt. When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtainedby contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include, but are not limited to; those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like.

[0038] The term “pharmaceutically-acceptable carrier”, as used herein, means one or more of a pharmaceutically acceptable solutions or vehicles for introducing into an administered cater that is not in use. Examples of suitable pharmaceutically-acceptable carriers include, but are not limited to, water, saline, glucose solution, and phosphate buffer. In other aspects, pharmaceutically-acceptable carrier can include mannitol, normal serum albumin, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. The pharmaceutically-acceptable carrier can also include ppreservatives and other additives such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.

[0039] The terms “antimicrobial” and “antimicrobial characteristic” refer to the ability to kill and / or inhibit the growth of microorganisms. A substance having an antimicrobial characteristic may be harmful to microorganisms or microbes (e.g., bacteria, fungi, virus, protozoans, algae, and the like). A substance having an antimicrobial characteristic can kill the microorganism and / or prevent or substantially prevent the growth or reproduction of the microorganism.

[0040] The terms “bacteria” or “bacterium” include, but are not limited to, gram positive and gram negative bacteria. Bacteria can include, but are not limited to, Abiotrophia, Achromobacter, Acidaminococcus, Acidovorax, Acinetobacter, Actinobacillus, Actinobaculum, Actinomadura, Actinomyces, Aerococcus, Aeromonas, Afipia, Agrobacterium, Alcaligenes, Alloiococcus, Alteromonas, Amycolata, Amycolatopsis, Anaerobospirillum, Anabaena affinis and other cyanobacteria (including the Anabaena, Anabaenopsis, Aphanizomenon, Camesiphon, Cylindrospermopsis, Gloeobacter Hapalosiphon, Lyngbya, Microcystis, Nodularia, Nostoc, Phormidium, Planktothrix, Pseudoanabaena, Schizothrix, Spirulina, Trichodesmium, and Umezakia genera) Anaerorhabdus, Arachnia, Arcanobacterium,Arcobacter, Arthrobacter, Atopobium, Aureobacterium, Bacteroides, Balneatrix, Bartonella, Bergeyella, Bifidobacterium, Bilophila Branhamella, Borrelia, Bordetella, Brachyspira, Brevibacillus, Brevibacterium, Brevundimonas, Brucella, Burkholderia, Buttiauxella, Butyrivibrio, Calymmatobacterium, Campylobacter, Capnocytophaga, Cardiobacterium, Catonella, Cedecea, Cellulomonas, Centipeda, Chlamydia, Chlamydophila, Chromobacterium, Chyseobacterium, Chryseomonas, Citrobacter, Clostridium, Collinsella, Comamonas, Corynebacterium, Coxiella, Cryptobacterium, Delftia, Dermabacter, Dermatophilus, Desulfomonas, Desulfovibrio, Dialister, Dichelobacter, Dolosicoccus, Dolosigranulum, Edwardsiella, Eggerthella, Ehrlichia, Eikenella, Empedobacter, Enterobacter, Enterococcus, Erwinia, Erysipelothrix, Escherichia, Eubacterium, Ewingella, Exiguobacterium, Facklamia, Filifactor, Flavimonas, Flavobacterium, Francisella, Fusobacterium, Gardnerella, Gemella, Globicatella, Gordona, Haemophilus, Hafnia, Helicobacter, Helococcus, Holdemania Ignavigranum, Johnsonella, Kingella, Klebsiella, Kocuria, Koserella, Kurthia, Kytococcus, Lactobacillus, Lactococcus, Lautropia, Leclercia, Legionella, Leminorella, Leptospira, Leptotrichia, Leuconostoc, Listeria, Listonella, Megasphaera, Methylobacterium, Microbacterium, Micrococcus, Mitsuokella, Mobiluncus, Moellerella, Moraxella, Morganella, Mycobacterium, Mycoplasma, Myroides, Neisseria, Nocardia, Nocardiopsis, Ochrobactrum, Oeskovia, Oligella, Orientia, Paenibacillus, Pantoea, Parachlamydia, Pasteurella, Pediococcus, Peptococcus, Peptostreptococcus, Photobacterium, Photorhabdus, Phytoplasma, Plesiomonas, Porphyrimonas, Prevotella, Propionibacterium, Proteus, Providencia, Pseudomonas, Pseudonocardia, Pseudoramibacter, Psychrobacter, Rahnella, Ralstonia, Rhodococcus, Rickettsia Rochalimaea Roseomonas, Rothia, Ruminococcus, Salmonella, Selenomonas, Serpulina, Serratia, Shewenella, Shigella, Simkania, Slackia, Sphingobacterium, Sphingomonas, Spirillum, Spiroplasma, Staphylococcus, Stenotrophomonas, Stomatococcus, Streptobacillus, Streptococcus, Streptomyces, Succinivibrio, Sutterella, Suttonella, Tatumella, Tissierella, Trabulsiella, Treponema, Tropheryma, Tsakamurella, Turicella, Ureaplasma, Vagococcus, Veillonella, Vibrio, Weeksella, Wolinella, Xanthomonas, Xenorhabdus, Yersinia, and Yokenella. Other examples of bacterium include Mycobacterium tuberculosis, M. bovis, M. typhimurium, M. bovis strain BCG, BCG substrains, M. avium, M. intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans, M. avium subspecies paratuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus equi, Streptococcus pyogenes, Streptococcus agalactiae, Listeria monocytogenes, Listeria ivanovii, Bacillus anthracis, B. subtilis, Nocardia asteroides, and other Nocardia species, Streptococcus viridans group,Peptococcus species, Peptostreptococcus species, Actinomyces israelii and other Actinomyces species, and Propionibacterium acnes, Clostridium tetani, Clostridium botulinum, other Clostridium species, Pseudomonas aeruginosa, other Pseudomonas species, Campylobacter species, Vibrio cholera, Ehrlichia species, Actinobacillus pleuropneumoniae, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Legionella pneumophila, other Legionella species, Salmonella typhi, other Salmonella species, Shigella species Brucella abortus, other Brucella species, Chlamydi trachomatis, Chlamydia psittaci, Coxiella burnetii, Escherichia coli, Neiserria meningitidis, Neiserria gonorrhea, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Yersinia pestis, Yersinia enterolitica, other Yersinia species, Escherichia coli, E. hirae and other Escherichia species, as well as other Enterobacteria, Brucella abortus and other Brucella species, Burkholderia cepacia, Burkholderia pseudomallei, Francisella tularensis, Bacteroides fragilis, Fudobascterium nucleatum, Provetella species, and Cowdria ruminantium, or any strain or variant thereof. The gram-positive bacteria may include, but is not limited to, gram positive Cocci (e.g., Streptococcus, Staphylococcus, and Enterococcus). The gram-negative bacteria may include, but is not limited to, gram negative rods (e.g., Bacteroidaceae, Enterobacteriaceae, Vibrionaceae, Pasteurellae and Pseudomonadaceae).

[0041] The terms “fungus” or “fungi” include, but are not limited to yeasts such as, for example, Candida albicans or other Candida spp. including C. glabrata, C. rugosa, C. parapsilosis, C. tropicalis, or C. dubliniensis Fungi can also include dermatophytes such as, for example, Trichophyton spp. and Microsporum spp. (e.g., T. rubrum, T. interdigitale, T. tonsurans, T. violaceum, T. concentricum, T. schoenleinii, T. soudanense, T. mentagrophytes, T. equinum, T. erinacei, T. verrucosum, M. audouinii, M. ferrugineum, M. canis, M. gypseum, M. nanum, and / or M. cookie).

[0042] As used herein, the term “subject” includes humans, mammals e.g., cats, dogs, horses, etc.), birds, and the like. Typical subjects to which embodiments of the present disclosure may be administered will be mammals, particularly primates, especially humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; and domesticated animals particularly pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like.Lock Solutions and Methods for Making and Using the Same

[0043] Described herein are methods for preventing microbial infections derived from an implanted catheters when not in use. Once a catheter has been implanted, when the catheter is not in use, it is typically filled with a lock solution. However, the catheter can be a source of infection. The lock solutions described herein address this problem.

[0044] The lock solution described herein is composed of a pharmaceutically acceptable salt of methanetrisdiazeniumdiolate and a pharmaceutically acceptable carrier. The structure of the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate is provided in Figure 1. Exemplary methods for producing the salt of methanetrisdiazeniumdiolate are provided in the Examples.

[0045] In one aspect, the salt of methanetrisdiazeniumdiolate releases nitric oxide (NO) for extended periods of times. Salts of methanetrisdiazeniumdiolate have numerous advantages over other NO donors that make them an ideal molecule for a lock solution. Methanetrisdiazeniumdiolate is not catalyzed by metals and catalysis via elevated temperature or light. The NO release from the molecule is still superior to most other NO donors.

[0046] A common struggle with nitric oxide-based therapy is providing a controlled release of the NO. An ideal NO donor releases NO in levels that provide the desired effect but do not cause unwanted side effects. A balance between increased NO flux and longer NO release is a constant struggle. The lock solutions described herein provide controlled and sustained release of NO. In one aspect, the lock solutions provide release of NO for at least one day, two days or three days. In another aspect, the lock solutions provide release of NO for one day to ten days.

[0047] In additional to releasing nitric oxide, the lock solutions described herein also can release Azanone (HNO). Azanone, also called nitroxyl, is a potent vasorelaxant, antiinflammatory agent, and anti-platelet agent21. It has been proven to have some antibacterial properties21 22. Two major cardiovascular mechanisms of HNO are its ability to increase myocardial contractility and to prevent further damage during reperfusion. Thus, the lock solutions described herein provide additional health benefits in addition to preventing microbial infections.

[0048] In one aspect, the lock solutions described herein can be produced by mixing the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate with the pharmaceutically acceptable carrier. For example, a powder of the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate can be mixed with the pharmaceutically acceptable carrier usingtechniques know in the art to produce a homogeneous solution. Once the lock solution is prepared, it can be injected into the implanted catheter using techniques known in the art. In certain aspects, additional antimicrobial agents can be added to the lock solution to further preview microbial infection as well as provide other health benefits.

[0049] In one aspect, the lock solutions described herein can be part of a kit. In one aspect, the lock solution comprising the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate and the pharmaceutically acceptable carrier are premade and present in a container such as, for example, a vial. In one aspect, the container is composed of a glass or other material that minimizes the exposure of the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate to light. In another aspect, the kit includes a container of the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate and a separate container of the pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate and the pharmaceutically acceptable carrier are mixed together prior to injecting into the catheter. The kit can include a syringe for injecting the lock solution into the catheter.

[0050] In one aspect, the pH of the lock solution can be varied to modify the release rate of nitric oxide. By adding an acid or base to the lock solution and adjusting the pH, NO release can be controlled. In one aspect, the pH of the lock solution is from 7.0 to 7.5.

[0051] The concentration of the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate can be varied depending upon the application. In one aspect, the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate is at a concentration of about 1 mg / mLto about 50 mg / mL ofthe lock solution. In another aspect, the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate is at a concentration of about 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL of the lock solution, where any concentration can be a lower and upper endpoint of a range (e.g., 5 mg / mL to 25 mg / mL).

[0052] The lock solution can be incorporated into any catheter that has been implanted into a subject where there is a risk of microbial infection. In one aspect, the device is urinary catheter or a peripheral intravenous catheter. In another aspect, the catheter is a central venous catheter. Not wishing to be bound by theory, NO produced by the lock solution can diffuse through the catheter wall and impart antimicrobial properties.

[0053] In one aspect, the lock solutions described herein can prevent catheter related bloodstream infections (CRBSIs) as NO is a small molecule that can diffuse through polymersreadily. In one aspect, the NO-releasing lock solutions can prevent intraluminal and extraluminal CRBSIs, the latter of which is not possible with antibiotic lock solutions.

[0054] The ability of the lock solutions to release both NO and Azanone provides biological effects that have not been possible with current NO donor lock solution technology. While most research has either focused on just NO release or just HNO release, the lock solutions described herein provide a synergistic effect by releasing both NO and Azanone. In additional to providing antimicrobial properties, the lock solutions described herein can provide synergistic antiplatelet adhesion properties, strong vasodilation, and protection of the heart tissue following myocardial ischemia. The lock solutions can also protect the patient from heart attack or stroke.Aspects

[0055] Aspect 1. A method for preventing a microbial infection in a subject by an implanted catheter, wherein when the implanted catheter is not in use, filing the implanted catheter with a lock solution comprising a pharmaceutically acceptable salt of methanetrisdiazeniumdiolate and a pharmaceutically acceptable carrier.

[0056] Aspect 2. The method of Aspect 1 , wherein the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate is the sodium salt or the potassium salt.

[0057] Aspect 3. The method of Aspect 1 , wherein the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate is the potassium salt.

[0058] Aspect 4. The method of any one of Aspects 1-3, wherein the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate is at a concentration of about 1 mg / mL to about 50 mg / mL.

[0059] Aspect 5. The method of any one of Aspects 1-3, wherein the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate is at a concentration of about 5 mg / mL to about 25 mg / mL.

[0060] Aspect 6. The method of any one of Aspects 1-5, wherein the lock solution releases nitric oxide.

[0061] Aspect 7. The method of any one of Aspects 1-5, wherein the lock solution releases nitric oxide for at least three days.

[0062] Aspect 8. The method of any one of Aspects 1-7, wherein the lock solution releases Azanone.

[0063] Aspect 9. The method of any one of Aspects 1-8, wherein the lock solution has a pH of about 7.0 to about 7.5.

[0064] Aspect 10. The method of any one of Aspects 1-9, wherein the pharmaceutically acceptable carrier comprises water, saline, or a buffered solution.

[0065] Aspect 11. The method of any one of Aspects 1-10, wherein the microbial infection is caused by a microbe selected from the group consisting of bacteria, fungi, virus, protozoan, and algae.

[0066] Aspect 12. The method of any one of Aspects 1-10, wherein the microbial infection is caused by bacteria.

[0067] Aspect 13. The method of any one of Aspects 1-10, wherein the microbial infection is a bloodstream infection.

[0068] Aspect 14. The method of any one of Aspects 1-13, wherein the installed catheter is a central venous catheter, a peripheral intravenous catheter, or a urinary catheter.

[0069] Aspect 15. A kit comprising(a) a pharmaceutically acceptable salt of methanetrisdiazeniumdiolate;(b) a pharmaceutically acceptable carrier; and syringe.

[0070] Aspect 16. A kit comprising(a) lock solution comprising a pharmaceutically acceptable salt of methanetrisdiazeniumdiolate a pharmaceutically acceptable carrier; and(b) a syringe.EXAMPLES

[0071] Now having described the embodiments of the disclosure, in general, the examples describe some additional embodiments. While embodiments of the present disclosure are described in connection with the example and the corresponding text and figures, there is no intent to limit embodiments of the disclosure to these descriptions. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure.MATERIALS AND METHODS

[0072] Materials

[0073] Dimethylsulfoxide (DMSO), thiazolyl blue tetrazolium bromide (MTT), anhydrous magnesium sulfate, potassium hydroxide (KOH), sodium phosphate dibasic heptahydrate, and sodium phosphate monobasic hydrate were purchased from Millipore Sigma (Burlington, MA). Dulbecco’s Modification of Eagle’s Medium (DMEM), penicillin-streptomycin (P&S, 5,000U mL-1), trypsin-EDTA (0.05%), phosphate-buffered saline (PBS 1x) without calcium or magnesium salts, 3 A molecular sieves, acetone, anhydrous methanol, potassium chloride, sodium chloride was purchased from Thermo Fischer Scientific (Waltham, MA). Sodium Ampicillin was purchased from GoldBio (St. Louis, MO). Helixmark Dow SILASTIC silicone tubing (ID=1 .47 mm and OD=1.96 mm) and Fetal bovine serum (FBS) was purchased from VWR (Radnor, PA). Dowsil 3140 RTV silicone coating was purchased from Dow Inc. (Midland, Ml). A compressed Nitric oxide (NO) gas cylinder was purchased from Linde Gas and Equipment Inc. (Danbury, CT). An ultra-pure compressed Argon (Ar) gas cylinder was purchased from Airgas (Athens, GA). NIH / 3T3 mouse fibroblasts (ATCC® CRL-1658) were grown from stock cultures originally obtained from American Type Culture Collection (Manassas, VA). All chemicals were of reagent grade and used without further purification besides the following: The acetone was dried using magnesium sulfate and stored for short periods of time with activated 3 A sieves and under Ar. Phosphate buffered saline (PBS) was adjusted to a pH of 7.4 and deoxygenated by bubbling the liquid with Ar for 15-20 minutes.

[0074] Synthesis of Potassium Methanetrisdiazeniumdiolate

[0075] A custom reactor modeled after a hydrogenation reactor was used for this synthesis. Potassium methanetrisdiazeniumdiolate was synthesized using a revised protocol of a previously reported method18. As the reactor has two separate reactor vessels, two aliquots of the reaction mixture were made. The following provides the measurements for 1 aliquot of the reaction mixture. Briefly, 2.805 g (50 mmol) of KOH was dissolved by stirring into 75 mL of anhydrous MeOH in the reaction vessel. After full dissolution, 918 pL of acetone (12.5 mmol) was added to the vessel along with a stir bar. The reaction vessels were attached to the reactor and kept under continuous stirring. Next, the reactor was purged of any other gases using Ar. Then, the system was pressurized with NO gas pressurized to 80 psig and left to react for 24 hours. After 24 hours an off-white precipitate had formed, and the NO was slowly removed from the system. More Ar purges were done to make sure all the NO was removed from the system. The reaction vessels were disconnected, and the mixtures were vacuum filtered. The collected solid was washed with 150 mL of chilled anhydrous MeOH. The product was collected and dried in vacuo with desiccant for 2 days to remove residual solvent. After vacuum drying, the product was freeze-dried to remove any residual water. The product was kept in an Ar flushed vial stored in vacuo with a mixture of calcium sulfate and calcium chloride (Thermo Fischer Scientific) as desiccant, at -20°C. All calculations were made assuming the product was 100% pure.

[0076] Ultraviolet-Visible light Spectroscopy

[0077] The Amax of CTN and the Ampicillin was confirmed using an Agilent Cary 60 UV-Vis spectrometer. Respectively, 50 ug / mL and 25 ug / mL of CTN and Ampicillin were dissolved in PBS. The spectrum was acquired with 1 mL of the solution in a semi-quartz cuvette and was baseline corrected with plain PBS. Standard curves for each compound were also obtained from multiple dilution runs on the UV-vis spectrometer (each point used for the data fit is the mean of 3 replicates). For CTN Amax=264 nm and for Sodium Ampicillin Amax=203 nm.

[0078] Fourier Transform Infrared Spectroscopy (FTIR)

[0079] FTIR analysis of CTN was obtained with the KBr (Thermofischer scientific) method using a spectrum two Perkin Elmer FT-IR spectrometer. 64 scans were completed with a resolution of 1 cm1. Prominent peaks: 3402 cm1, 2992 cm1, 1633 cm1, 1403 cm1, 1395 cnr1, 1359 cm1, 1340 cm1, 1296 cm1, 1276 cm1, 1255 cm1,1218 cm1,1196 cm1, and 1159 cm1

[0080] Nuclear Magnetic Resonance Spectroscopy (NMR)

[0081] NMR spectra were obtained using an Ascend 400 MHz magnet with a Bruker Advance 111 HD Nanobay console. The solvent system used was 90% H20 and 10% D2O. For the1H-NMR spectrum, 256 scans were done and for the13C-NMR spectrum 1024 scans were done.

[0082] Lock Solution and Catheter Preparation

[0083] CTN powder and Sodium Ampicillin powder were dissolved in PBS at a concentration of 20 mg / mL and 10 mg / mL, respectively. Certain lengths of the silicone tubing were cut, and both ends were sealed overnight using the RTV silicone rubber.

[0084] Nitric Oxide Detection

[0085] Nitric oxide release from the CTN lock solution was measured using chemiluminescence with a Sievers 280i Nitric Oxide analyzer (Boulder, CO). Prior to use, the NOA calibration was completed with a Zysense NO tank at 0 and 45 ppm using N2(Airgas) as a balance. NO released during the experiment was swept into the analyzer using N2gas at a rate of 200 mL / min. To test the NO release via diffusion from the catheter, 3 cm long sealed catheters were filled with the CTN solution, and the catheters were submerged in PBS in the reaction chamber, the flux values were normalized to the outer surface area of the catheter. The data reported is the mean ± SD (n > 3).

[0086] Bacterial Adhesion Prevention

[0087] Single isolated colonies of each bacteria strain (Staphylococcus aureus, Escherichia coli, or Methicillin resistant Staphylococcus aureus) were inoculated in 20 mL of Luri Bertani (LB) broth and grown over night at 37 °C shaking at 150 rpm. The cultures werecentrifuged at 3500 rpm for 7 min and washed with sterile PBS. Bacteria inoculums were confirmed to be in the logarithmic growth stage using a UV-vis spectrophotometer to detect optical density (OD) at 600 nm. The final OD of each bacteria culture was adjusted to 0.1 before exposure. Sealed catheters were injected with either PBS, ampicillin, or CTN lock solution and submerged in 4 mL of 0.1 OD bacteria solution. All samples were then incubated at 37 °C, 150 rpm for 24 h protected from ambient light. To quantify the efficacy of each lock solution, catheter samples were taken out of the bacteria solution after 24 h, washed in fresh pbs, and cut open to complete remove all of the injected lock solution. Open catheter samples were then submerged in 1 mL of PBS, followed by 1 min of homogenizing and 1 min of vortexing to remove all adhered bacteria. Diluted bacteria suspensions were plated on LB agar plates using a spiral plater (Eddy Jet 2W, IUL Instruments) with the Log mode 50 L setting. Plates were incubated overnight at 37 °C, viable colony-forming units were then quantified using a colony counter (Sphere Flash, IUL Instruments) and normalized to the catheter sample’s surface area. Percent reduction in the viability of bacteria was determined with respect to the control samples containing a PBS lock solution. The final data is reported as the mean ± SD (n > 3).

[0088] Cell Culture

[0089] 3T3 fibroblast cultures were developed from cryopreserved stocks and subcultured in complete DMEM supplemented with 10% FBS (10% v / v) and P&S (1 % v / v). Cells were grown at 37 °C under a CO2-humidified atmosphere and grown up to ~70% monolayer coverage. Upon reaching this threshold, media was aspirated, monolayers were washed with PBS (1x), and treated with trypsin-EDTA to detach cells. Detached cells were collected via centrifugation (200 ref, 5 min) and resuspended in media. Suspended cells were counted using an EVETM automated cell counter (NanoEnTek) and seeded onto polystyrene 96-well plates at a density of 5,000 cells well-1 . Plates were incubated for 24 h prior to treatment with lock solutions.

[0090] Direct Contact Lock Solution Leakage Cytotoxicity Screening

[0091] Cytocompatibility screen of the CTN and Ampicillin lock solutions was assessed following our prior protocols for lock solution screening8. Following initial seeding and 24 incubation, seeded well plates were washed and treated with complete DMEM supplemented with various concentrations of CTN and ampicillin lock solutions at dilutions of 0.06 0.09 0.2 %v / v to mimic the potential for lock solution leakage from catheters in the physiological environment (Figure 7). These dilution ratios were based on prior clinical evidence of lock solution leakage from central venous catheters into the bloodstream, corresponding tovariable dilution rates with respect to total blood volume. Each lock solution concentration and dilution ratio were plated across quintuplicate wells, with cells incubated for an additional 24 h. Afterwards, exposure media was decanted and replaced with MTT reagent (0.5 mg mL-1) in PBS (1x). Cells were incubated for an additional 3 h, with the remaining MTT dye aspirated and resulting formazan precipitate dissolved in DMSO. Well plates were then read for absorbance with a BioTek Cytation 5 plate reader (Agilent) at 570 nm with a separate reference measurement at 690 nm. The average difference in absorbance between the two measurements (A570-690) for each treatment was used to calculate the relative cellular viability of treated cells compared to untreated cells according to Equation 1. Results are reported as the mean cellular viability of 3T3 cells ± standard deviation (SD, N > 3 independent biological repeats).% Cell Viability

[0092] Leaching of Lock Solutions

[0093] Leaching of the lock solutions through the silicone catheters was assessed using UV-Vis spectroscopy. Briefly, catheters containing the Ampicillin solution, CTN solution, or PBS were incubated in PBS for 24 hours at 37°C using a surface area to volume ratio of 6 cm2to 1 mL (CTN was done in a 1 cm2to 1 mL ratio). After 24 hours, the incubated PBS leachate solutions from the CTN and ampicillin catheters were run on the UV-Vis spectrometer with the baseline correction of the PBS filled catheter leachates to account for oligomer leaching. The final data shows the 4 replicates as independent points on top on the mean ± SD.

[0094] Storage Stability of Potassium methanetrisdiazeniumdiolate

[0095] For a month, the dry storage stability of the CTN powder was assessed weekly in 3 different temperature conditions: room temperature(~23°C), 4°C, and -20°C. CTN was weighed out in 12 small vials and the masses were recorded. The vials were stored at different temperature conditions (4 vials in each condition). On day 7, a vial from the three different temperature conditions was taken, and based on the initial mass of that sample, the powder in the vial was diluted to 50 g / mL with PBS. Subsequently, this solution was run on the UV- Vis spectrometer with a baseline correction of PBS. The final mass was calculated using the absorbance value at the Amaxand the standard curve. This was repeated for days 14, 21 , and 28. The data shown represents the mean ± SD (n > 3).

[0096] Statistical Analyses

[0097] All statistical comparisons were calculated using GraphPad Prism 10 (GraphPad Software, San Diego, GA). Comparisons between the different lock solutions were made using ordinary one-way analysis of variance (ANOVA) with Tukey’s method for correction of multiple comparisons. Values of p < 0.05 were deemed significant.RESULTS AND DISCUSSION

[0098] Synthesis and Chemical Characterization

[0099] Although the potassium salt of CTN has been made once before, slight changes in the procedure have led to slightly different analytical characteristics than previously reported. These differences seem to confirm the theorized mechanism of the Traube reaction and help identify the minor products formed during the reaction as well as the products remaining after degradation of the product.

[0100] The UV-Vis curve in this work for CTN (Figure 2A) exhibits the same Amaxas previous work18, and is consistent with a n- ir* transition. The standard curve shows an excellent linear fit in micromolar concentrations (Figure 2B). For ampicillin the Amax(Figure 2C) was the same as previous literature10. The standard curve did provide a good fit (Figure 2D), but in concentrations higher than 25 pg / mL a bathochromic shift was seen. The reason for this shift remains unknown, but it made quantifying ampicillin quite difficult. Furthermore, after long periods of time in aqueous solutions the Amax for ampicillin appeared at 208 nm, which signified a degradation product of ampicillin.

[0101] The three previous fabrications of different salts of CTN1820report extremely clean FTIR and NMR spectra and claim no other byproduct formation. This is in contradiction to the earlier findings of T raube as well as MacDonald and Masson15 16. This current work supports these original findings.

[0102] By using Potassium instead of Sodium in this reaction a more stable product is obtained. During this study, our group confirmed that the potassium salt product has a higher detonation point18and is also less likely to form a hydrate product than the sodium-based salts. The reaction of NO with alkoxides has been proven to result in aldehydes that react further either through the T raube reaction itself or the Cannizzaro reaction16 17. Thus, using a hydroxide salt for the reaction instead of an alkoxide salt minimizes the side reactions with NO. Despite this, potassium hydroxide in methanol creates an equilibrium reaction to form potassium methoxide salt and water, so methoxide formation is unavoidable. However, because potassium hydroxide is deliquescent, even reagent grade potassium hydroxide will contain some water. When dissolved in methanol this shifts the equilibrium to favor theconversion of the water and methoxide back to hydroxide and methanol. On the1HNMR spectra (Figure 3A), the small peak at 8.35 ppm is attributed to the hydrogen of Potassium formate. This is expected as any remaining methoxide reacts with NO to form formaldehyde, which undergoes the Cannizzaro reaction to form methanol and formic acid. In neutral pH, formic acid deprotonates and remains as the formate ion.

[0103] MacDonald and Masson claim that one of the minor products ofthe Traube reaction is acetic acid16. Like formic acid, in neutral pHs, acetic acid prefers to stabilize as the acetate ion. The1HNMR (Figure 3A) shows a peak at 1 .82 ppm that is attributed to the methyl group of acetate. The remaining peaks on the proton spectrum identify the other byproducts of the reaction. The large peak at 7.52 ppm is the single hydrogen of potassium methanetrisdiazeniumdiolate. The peak at 6.32 ppm is showing the compound (N2O2K)2CHCOOK according to Arulsamy and Bohle18. The two peaks at 5.89 and 5.82 ppm likely both correspond to the potassium salt of Traube’s anion. We would expect to see just one single peak, but it’s possible that the two hydrogens are appearing to be diastereotopic due to some difference in the two diazeniumdiolate groups; some possibilities include protonation of one or more of the negatively charged oxygen atoms, change in one of the diazeniumdiolate groups from the usual cis conformation to trans, or deuterium hydrogen exchange. The small peak at 2.43 ppm is the two methyl groups of acetone and the peak at 3.26 ppm is the methyl group of methanol. This suggests that the product still had residual solvent trapped in the crystalline structure. Despite the solvent impurities, potassium carboxylate salts, and the presence of the other carbon bound diazeniumdiolates with a degree of substitution of two, the peak attributed to the desired product is much larger than any ofthe other peaks. The pure potassium methanetrisdiazeniumdiolate was so concentrated in the NMR sample that its13C satellites can be seen without any enhancement, which is exceedingly rare.

[0104] To further show the high purity of potassium methanetrisdiazeniumdiolate in this product, no other peaks besides the large peak corresponding to the desired product at 96.69 ppm, are seen on the13C NMR (Figure 3B). The FTIR (Figure 4) further confirms the identification of all the compounds in the produced CTN.

[0105] The broad and medium strength band centered at 3402 cm1is that of an alcohol, which we can safely assume is methanol. The strong peak at 2992 cm1is from the methine carbon of pure potassium methanetrisdiazeniumdiolate. The medium band at 1633 cm1is from a carbon oxygen double bond; due to the band having a lower frequency than usually seen for carbonyl compounds, the band is attributed to a carboxyl compound, in this case,potassium acetate. The numerous strong bands in the region highlighted in pink are all from the presence of N2O2_groups and match the literature for the tri-substituted potassium salt18.

[0106] Having studied the rapid detonation of CTN with high temperatures and superacids, we strongly assume that the aqueous degradation of CTN results in all gaseous products-methane, nitric oxide, and Azanone- and the carboxylate salts-formate and acetate. Further studies are in progress to confirm this theory.

[0107] Nitric Oxide Release

[0108] A common struggle with nitric oxide-based therapy is providing a controlled release of the NO. An ideal NO donor releases NO in levels that provide the desired effect but do not cause unwanted side effects. A balance between increased NO flux and longer NO release is a constant struggle. CTN provides both high flux of NO and extended NO release.

[0109] NO based lock solutions have the potential to change the management of CRBSIs as NO is a small molecule that can diffuse through polymers readily. Therefore, NO lock solutions should theoretically treat and prevent intraluminal and extraluminal CRBSIs. The latter of which is not possible with antibiotic lock solutions. However, research of NO based lock solutions has mainly focused on intraluminal CRBSIs. A major cause of this gap in research is because the researched NO-based lock solutions could not provide a high enough extraluminal NO release to provide antimicrobial benefits.

[0110] CTN lock solution exhibited therapeutic levels of NO by diffusion across the catheter walls for 72 hours (Figure 5). The average extraluminal NO release was 2.83 ±1.1 flux units, 1.13 ± 0.15 flux units, and 0.89 ± 0.28 flux units for days 1 ,2, and 3 respectively. CTN also showed an ideal NO release profile-that is one that does not exhibit an initial burst release but instead follows a logarithmic release.

[0111] As shown by Arnold et al., CTN NO release is very pH dependent19. This allows the release of NO to be manipulated simply by adding an acid or a base to the lock solution.

[0112] Antibacterial Prevention

[0113] NO has shown its effectiveness as a potent antimicrobial, but also provides different antimicrobial benefits based on the concentration of NO23. For example, a lower concentration of NO will kill planktonic bacteria. An increased concentration will disperse a biofilm, but an even higher concentration of NO is required to kill the now planktonic bacteria from the dispersed biofilm. However, unlike antibiotics, the required concentration of NO to kill a biofilm is not an unsafe amount. Also, bacteria have not shown resistance to NO7, so it is unlikely that NO concentrations will need to be increased to provide the same effect.

[0114] In this study we tested the CTN lock solution against a commonly used antibiotic lock solution, 10 mg / mL Ampicillin6. While these studies show the ability for the lock solution to prevent extraluminal CRBSIs, studies are currently underway to show the prevention of intraluminal CRBSIs. As seen in Figures 6A-6C, CTN showed significantly better prevention of infection by Staph aureus, E. coli, and MRSA.

[0115] Compared to control catheters, CTN showed a 99.98%, 99.98%, and 99% reduction in viable bacteria adhered to the catheter surface for S. Aureus, E. coli, and MRSA, respectively.Table 1. Reduction of viable bacteria for CTN and Ampicillin expressed in log reductions.

[0116] Cytotoxicity

[0117] The relative viability of the 3T3 fibroblast cells was used to determine the maximum concentration of CTN so that in the case of three different leakage percents into the blood would still be cytocompatible. Per ISO standards a relative viability of 70% was the cut off for the cytotoxicity limit, therefore the concentration 20 mg / mL CTN was used forthe lock solution.

[0118] Leaching

[0119] Ampicillin’s ability to prevent extraluminal bacterial adhesion was a counterintuitive result at first. During all studies with locked catheters, special care was taken when sealing and injecting lock solutions into catheters to make sure no holes were present in the catheter. After all studies the catheters were tested for leaking by applying significant force on the catheterto check that the catheters still were completely sealed. By the laws of mass transport, it seemed very likely that the salts of the lock solutions could diffuse through the catheter walls into the surrounding environment. Leaching studies with locked catheters were done to prove this theory. Figures 8A-8B show the results of the leaching of ampicillin through the catheter. No detectable leaching of CTN was found during this study.

[0120] The leaching of ampicillin through the catheter explains the extraluminal bacterial prevention. To our knowledge, leaching of lock solutions from catheters in clinical settings has never been studied. However, leaking from the open distal end of the CVC has been reported524. It’s likely that both leaching and leaking of lock solutions occur in the body, but it’s not possible to distinguish between the two phenomena in the dynamic conditions of blood in the body. In fact, the rapid flow of blood throughout the body explains why even if antibiotic lock solutions leak into the blood stream, antibiotic lock therapy does not prevent extraluminal infection. The constant flow of the circulatory system and the dilution of the leaked antibiotic in the bloodstream prevent the antibiotics from having any effects on the extraluminal portion of the catheter.

[0121] The lack of detectable leaching of CTN through the catheter cannot be explained yet. A reasonable theory is that because CTN reacts with the water it is dissolved in, the diffusion rate is much slower compared to ampicillin; also, CTN’s reaction products are gaseous and do diffuse through the catheter. More studies are being conducted to test the leaching of CTN from the catheters.

[0122] Storage Stability

[0123] Figure 9 shows that temperature does not strongly affect the degradation of CTN. While storage at -20°C did show slightly enhanced stability compared to the other conditions, the difference is not significant. Also in all three temperature conditions, CTN did not show any degradation over the period of 1 month.

[0124] This makes CTN even more attractive as to use as an NO donor, as its storage conditions do not show significant degradation of the product. So, vials of CTN could be stored in hospital settings very easily without worrying about degradation of the compound.

[0125] I should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations, and are set forth only for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiments of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure.REFERENCES(1) Gahlot, R.; Nigam, C.; Kumar, V.; Yadav, G.; Anupurba, S. Catheter-related bloodstream infections. International journal of critical illness and injury science 2014, 4 (2), 162.(2) Kornbau, C.; Lee, K. C.; Hughes, G. D.; Firstenberg, M. S. Central line complications. International journal of critical illness and injury science 2015, 5 (3), 170.(3) Barraclough, K. A.; Hawley, C. M.; Playford, E. G.; Johnson, D. W. Prevention of access- related infection in dialysis. Expert review of anti-infective therapy 2009, 7 (10), 1 185-1200.(4) Kumar, A.; Chordia, N. Bacterial Resistance Against Antibiotics. Springer International Publishing, 2017; pp 171-192.(5) Polaschegg, H.-D. Loss of catheter locking solution caused by fluid density. ASAIO journal 2005, 51 (3), 230-235.(6) Robinson, J. L.; Tawfik, G.; Saxinger, L.; Stang, L.; Etches, W.; Lee, B. Stability of heparin and physical compatibility of heparin / antibiotic solutions in concentrations appropriate for antibiotic lock therapy. Journal of Antimicrobial Chemotherapy 2005, 56 (5), 951-953.(7) Privett, B. J.; Broadnax, A. D.; Bauman, S. J.; Riccio, D. A.; Schoenfisch, M. H. Examination of bacterial resistance to exogenous nitric oxide. Nitric oxide 2012, 26 (3), 169-173.(8) Ashcraft, M.; Douglass, M.; Garren, M.; Mondal, A.; Bright, L. E.; Wu, Y.; Handa, H. Nitric Oxide-Releasing Lock Solution for the Prevention of Catheter-Related Infection and Thrombosis. ACS applied bio materials 2022, 5 (4), 1519-1527.(9) Estes Bright, L. M.; Garren, M. R. S.; Douglass, M.; Handa, H. Synthesis and Characterization of Nitric Oxide-Releasing Ampicillin as a Potential Strategy for Combatting Bacterial Biofilm Formation. ACS Applied Materials & Interfaces 2023, 15 (12), 15185-15194.(10) Chug, M. K.; Griffin, L.; Garren, M.; Tharp, E.; Nguyen, G. H.; Handa, H.; Brisbois, E. J. Antimicrobial efficacy of a nitric oxide-releasing ampicillin conjugate catheter lock solution on clinically-isolated antibiotic-resistant bacteria. Biomaterials Science 2023, 11 (19), 6561-6572.(1 1) Kumar, R.; Massoumi, H.; Chug, M. K.; Brisbois, E. J. S-Nitroso-N-acetyl-l-cysteine Ethyl Ester (SNACET) Catheter Lock Solution to Reduce Catheter-Associated Infections. ACS applied materials & interfaces 2021 , 13 (22), 25813-25824.(12) Batchelor, M. M.; Reoma, S. L.; Fleser, P. S.; Nuthakki, V. K.; Callahan, R. E.; Shanley, C. J.; Politis, J. K.; Elmore, J.; Merz, S. I.; Meyerhoff, M. E. More lipophilic dialkyldiamine- based diazeniumdiolates: synthesis, characterization, and application in preparing thromboresistant nitric oxide release polymeric coatings. Journal of medicinal chemistry 2003, 46 (24), 5153-5161.(13) Maloney, S. E.; Grayton, Q. E.; Wai, C.; Uriyanghai, U.; Sidhu, J.; Roy-Chaudhury, P.; Schoenfisch, M. H. Nitric Oxide-Releasing Hemodialysis Catheter Lock Solutions. / ACS Applied Materials & Interfaces 2023.(14) Coneski, P. N.; Schoenfisch, M. H. Competitive formation of N-diazeniumdiolates and N- nitrosamines via anaerobic reactions of polyamines with nitric oxide. Organic letters 2009, 11 (23), 5462-5465.(15) Traube, W. Ueber Isonitramine. Berichte der deutschen chemischen Gesellschaft 1894, 27 (2), 1507-1510.(16) MacDonald, G.; Masson, O. LXXVI. — A product of the action of nitric oxide on sodium ethylate. Journal of the Chemical Society, Transactions 1894, 65, 944-949.(17) Derosa, F.; Keefer, L. K.; Hrabie, J. A. Nitric Oxide Reacts with Methoxide. The Journal of Organic Chemistry 2008, 73 (3), 1139-1142. DOI: 10.1021 / jo7020423.(18) Arulsamy, N.; Bohle, D. S. Multiplicity control in the polygeminal diazeniumdiolation of active hydrogen bearing carbons: chemistry of a new type of trianionic molecular propeller. Journal of the American Chemical Society 2001, 123 (44), 10860-10869.(19) Arnold, E. V.; Citro, M. L; Keefer, L. K.; Hrabie, J. A. A nitric oxide-releasing polydiazeniumdiolate derived from acetonitrile. Organic Letters 2002, 4 (8), 1323-1325.(20) Jeong, H.; Park, S.; Park, K.; Kim, M.; Hong, J. Sustained nitric oxide-providing small molecule and precise release behavior study for glaucoma treatment. Molecular Pharmaceutics 2020, 17 (2), 656-665.(21) Muniz Carvalho, E.; Silva Sousa, E. H.; Bernardes-Genisson, V.; Gonzaga De Franca Lopes, L. When NO. 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Claims

CLAIMS1 . A method for preventing a microbial infection in a subject by an implanted catheter, wherein when the implanted catheter is not in use, filing the implanted catheter with a lock solution comprising a pharmaceutically acceptable salt of methanetrisdiazeniumdiolate and a pharmaceutically acceptable carrier.

2. The method of claim 1 , wherein the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate is the sodium salt or the potassium salt.

3. The method of claim 1 , wherein the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate is the potassium salt.

4. The method of claim 1 , wherein the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate is at a concentration of about 1 mg / mL to about 50 mg / mL.

5. The method of claim 1 , wherein the pharmaceutically acceptable salt of methanetrisdiazeniumdiolate is at a concentration of about 5 mg / mL to about 25 mg / mL.

6. The method of claim 1 , wherein the lock solution releases nitric oxide.

7. The method of claim 1 , wherein the lock solution releases nitric oxide for at least three days.

8. The method of claim 1 , wherein the lock solution releases Azanone.

9. The method of claim 1 , wherein the lock solution has a pH of about 7.0 to about 7.5.

10. The method of claim 1 , wherein the pharmaceutically acceptable carrier comprises water, saline, or a buffered solution.

11. The method of any one of claims 1-10, wherein the microbial infection is caused by a microbe selected from the group consisting of bacteria, fungi, virus, protozoan, and algae.

12. The method of any one of claims 1-10, wherein the microbial infection is caused by bacteria.

13. The method of any one of claims 1-10, wherein the microbial infection is a bloodstream infection.

14. The method of any one of claims 1-10, wherein the installed catheter is a central venous catheter, a peripheral intravenous catheter, or a urinary catheter.

15. A kit comprising(a) lock solution comprising a pharmaceutically acceptable salt of methanetrisdiazeniumdiolate a pharmaceutically acceptable carrier; and(b) a syringe.

16. A kit comprising(a) a pharmaceutically acceptable salt of methanetrisdiazeniumdiolate;(b) a pharmaceutically acceptable carrier; and(c) syringe.

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