WO2011141863A2 - Gaseous nitric oxide-sequestering products and processes of preparing same - Google Patents

Gaseous nitric oxide-sequestering products and processes of preparing same Download PDF

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Publication number
WO2011141863A2
WO2011141863A2 PCT/IB2011/052032 IB2011052032W WO2011141863A2 WO 2011141863 A2 WO2011141863 A2 WO 2011141863A2 IB 2011052032 W IB2011052032 W IB 2011052032W WO 2011141863 A2 WO2011141863 A2 WO 2011141863A2
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WIPO (PCT)
Prior art keywords
article
nitric oxide
chamber
enclosure
gno
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PCT/IB2011/052032
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English (en)
French (fr)
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WO2011141863A3 (en
Inventor
Yossef Av-Gay
Christopher C. Miller
Deborah Bohbot
Gilly Regev-Shoshani
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Enox Biopharma, Inc.
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Priority to US13/697,374 priority Critical patent/US20130078298A1/en
Priority to EP11780289.2A priority patent/EP2571541A4/de
Publication of WO2011141863A2 publication Critical patent/WO2011141863A2/en
Publication of WO2011141863A3 publication Critical patent/WO2011141863A3/en

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65D—CONTAINERS FOR STORAGE OR TRANSPORT OF ARTICLES OR MATERIALS, e.g. BAGS, BARRELS, BOTTLES, BOXES, CANS, CARTONS, CRATES, DRUMS, JARS, TANKS, HOPPERS, FORWARDING CONTAINERS; ACCESSORIES, CLOSURES, OR FITTINGS THEREFOR; PACKAGING ELEMENTS; PACKAGES
    • B65D25/00—Details of other kinds or types of rigid or semi-rigid containers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/46—Deodorants or malodour counteractants, e.g. to inhibit the formation of ammonia or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L28/00—Materials for colostomy devices
    • A61L28/0007—Materials for colostomy devices containing macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L28/00—Materials for colostomy devices
    • A61L28/0034—Use of materials characterised by their function or physical properties
    • A61L28/0038—Medicaments; Biocides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/04—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00—Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/14—Materials characterised by their function or physical properties, e.g. lubricating compositions
    • A61L29/16—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/16—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/02—Drugs for genital or sexual disorders; Contraceptives for disorders of the vagina
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B65—CONVEYING; PACKING; STORING; HANDLING THIN OR FILAMENTARY MATERIAL
    • B65B—MACHINES, APPARATUS OR DEVICES FOR, OR METHODS OF, PACKAGING ARTICLES OR MATERIALS; UNPACKING
    • B65B5/00—Packaging individual articles in containers or receptacles, e.g. bags, sacks, boxes, cartons, cans, jars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
    • A61L2300/114—Nitric oxide, i.e. NO

Definitions

  • the present invention in some embodiments thereof, relates to medical products and, more particularly, but not exclusively, to products having gaseous nitric oxide (NO) sequestered therein, to processes and systems for producing such products and to uses thereof.
  • NO gaseous nitric oxide
  • Infection is a constant risk to any healthy person, and poses even a higher risk to hospitalized patients. Infections which are a result of treatment in a hospital or a healthcare service unit are often termed nosocomial infections. The risk of infection is further increased when the natural infection barriers of skin or other epithelial surfaces are breached during a surgical procedure, and/or otherwise in cases where bacteria normally present on the skin or in the air is allowed to access the interior surfaces of the body.
  • Medical devices used in the care or treatment of people are sterilized before use. While medical devices that comprise metal, glass or other heat-stable materials can be treated with steam (autoclave) or hot air, the use of various polymers and plastics in medical devices may render heat-dependent sterilization unsuitable. Heat-sensitive medical devices are typically sterilized using gamma irradiation or sterilant gas technologies employing ethylene oxide, chlorine dioxide, hydrogen peroxide, and the like, which generally do not require heat application.
  • the medical device is only sterile until it is exposed to a non-sterile environment.
  • a non-sterile environment which, for example, may occur after a package containing the medical device is opened in a non-sterile environment, microbial contaminants in the air or in the subject's body (e.g., skin or intestinal flora) can contact the device. Under the generally warm and moist conditions found in a subject's body, the microbial contaminants can establish an infection. Contamination may even occur in a surgical environment.
  • Catheter-associated bacteriuria is the most common nosocomial infection worldwide. It accounts for up to 40 % of hospital-acquired infections in the US each year. CAB carries with it a 2.8-fold increased risk of death and results in bacteremia in approximately 3 % of patients, constituting a serious complication. Bacteriuria is typically caused by a single organism, with Escherichia coli (E. Coli) being the most frequently isolated species. Together, Escherichia coli and Pseudomonas aeruginosa account for over 39 % of cases. As a result of the widespread use of urinary catheterization, CAB results in considerable antimicrobial use. According to some studies, the daily rate of bacteriuria in catheterized patients ranges from 3 to 8 %; and the incidence of bacteriuria is directly related to the duration of catheterization.
  • CAUTI Catheter-associated urinary tract infection
  • CAB is difficult to treat with current antimicrobial strategies because of the antibiotic resistance biofilm that forms from free-floating bacteria that adhere to the surface of catheters and colonize them. These biofilm bacteria are highly differentiated and extremely resistant to antibiotics. Biofilms are especially relevant in catheterization, as indwelling urinary catheters provide a surface for the attachment of microbial host cell binding receptors that are recognized by bacterial adhesins. This in turn facilitates microbial adhesion and subsequent colonization with uropathogens. Catheterization also disrupts the uroepithelial mucosa, exposing new binding sites for bacterial adhesins. Once biofilms are established they shed cells that seed other sections of the catheter and bladder. They also protect pathogenic bacteria from antimicrobials and the host immune response.
  • Silver is a very effective antibacterial substance and silver alloy coated-catheters have been utilized in recent years in an effort to reduce infection rates.
  • silver oxide catheters were not associated with a statistically significant reduction in CAB, and other meta-analyses have similarly concluded that silver oxide coated catheters are ineffective.
  • IDSA Infectious Diseases Society of America
  • Antibiotic-coated catheters may reduce the risk of CAB by preventing or delaying onset in hospitalized patients [Darouiche et al., J infect diseases 176: 1 109- 1 112, 1997; Stickler, 2008, supra], and have demonstrated antimicrobial effects against bacteriuria pathogens in several in vitro studies [Johnson et al. Antimicrob agents and chemother 43: 2990-2995, 1999; Darouiche et al., 1997, supra], and some in vivo studies also report positive effects [Jacobsen et al. Clinic Microbiol Reviews 21 : 26-59, 2008].
  • Ciprofloxacin, gentamicin, norflaxin, nitrofurazone, and combinations of compounds such as chlorhexidine and protamine sulfate have been successfully incorporated into catheter coatings [Jacobson et al., 2008, supra].
  • Nitrofurans have proven to be effective against a wide spectrum of gram-positive and gram negative bacteria, including a variety of strains of the common urinary pathogens. In vitro studies indicate that nitrofurazone catheters might have a stronger antibacterial effect than silver hydrogel catheters [Bjarnsholt et al.2007, supra]. However, nitrofurans have been demonstrated to be ineffective against most strains of Pseudomonas aeruginosa, and they do not inhibit viruses or fungi. Nitrofurazone catheters are also very expensive.
  • BV Bacterial vaginitis
  • Vulvovaginal candidiasis is the second most common cause of vaginitis (after bacterial vaginitis), diagnosed in up to 40 % of women with vaginal complaints [Achkar and Fries, 2010, Clinical Microbiology Reviews, April 2010, Vol. 23, No. 2: pp. 253-273; Andersen et al, 2004, JAMA 291 : pp. 1368-1379; Bauters et al, 2002, Am. J. Obstet. Gynecol. 187: pp. 569-574). Approximately 75 % of women experience at least one occurrence of VVC during their childbearing years, and half of these women have at least one recurrence [Sobel, The Lancet. Volume 369, Issue 9577, 9 June 2007- 15 June 2007: pp. 1961-1971].
  • VVC affects primarily healthy women, with risk factors for infection including frequent sexual intercourse, receptive oral sex, high-estrogen oral contraceptives, condoms, and spermicides, hormone replacement therapy, uncontrolled diabetes mellitus, conditions with high reproductive hormone levels, genetic predispositions pregnancy, and antibiotic usage [Achkar and Fries, 2010, supra].
  • C. albicans is the most common species identified in VVC cases, with an estimated incidence of 76 to 89 %.
  • Candida albicans is an opportunistic fungus capable of colonizing the vaginal mucosa, which is present in the vaginal tracts of up to 70 % of non-pregnant women [Bauters et al., 2002, supra; Beigi et al, 2004, Obstet Gynecol. Nov; 104 (5 Pt l):pp. 926-30].
  • individuals with vaginal colonization of Candida are asymptomatic.
  • Candida Upon disturbance in the balance between colonization and the host, Candida can cause vulvovaginal candidiasis (VVC), resulting in clinical signs and symptoms of inflammation [Achkar and Fries, 2010, supra]. The transition from asymptomatic colonization to symptomatic candidiasis may also result from factors that enhance fungus virulence (Cassone et al., 2007).
  • VVC is commonly managed with intravaginal azole regimens of varying duration, there is a growing trend toward shorter therapies, including single-dose treatments.
  • women with chronic or persistent yeast infections (RVVC) are less likely to respond to shorter treatment regimens, and symptomatic relapse occurs in more than half of RVCC cases within a short time of treatment cessation.
  • Bacterial vaginosis is recognized as being the most common cause of vaginal discharge. BV typically results from anaerobic bacterial infections from species such as Gardnerella vaginalis, Mycoplasma hominis, and Ureaplasma urealyticum, although aerobic bacteria have also been observed as causative agents.
  • Nitric oxide is a small, naturally produced, hydrophobic, free-radical gas that has a major role in innate immunity. NO exhibits broad reactivity and rapid diffusive properties through biological liquids and lipid membranes, with a short half- life in a physiological milieu [Subczynski and Wisniewska. 2000. Acta Biochim.Pol. 47:613-625]. Overproduction of NO induced by the enzymatic activity of inducible nitric oxide synthase (iNOS) in various cell types has been shown to play a vital role in several inflammatory and immunoregulatory processes. NO has been shown to play important roles in vasodilatation, neurotransmission, angiogenesis, modulation of wound healing, and nonspecific responses to infection ).
  • iNOS inducible nitric oxide synthase
  • NO has been shown to be bacteriostatic and bactericidal.
  • Miller et al. [Nitric Oxide 20: 16-23, 2009] demonstrated that multiple 30 minute treatments of 160 ppm nitric oxide resulted in over a 5 log 10 colony forming unit per milliliter (CFU/ml) decrease in the bacterial load of Staphylococcus aureus, E. coli and Pseudomonas aeruginosa.
  • Nitric oxide is also known to function as an antimicrobial agent.
  • Gaseous nitric oxide at a concentration of about 200 ppm has been demonstrated to clear pneumonia caused by pathogens such as Pseudomonas aeruginosa or S. aureus [McMullin et al., 2005, Respir. Care 5: 1451-1456].
  • Topical applications of gaseous nitric oxide at about 200 ppm has been demonstrated to inhibit or prevent growth of a variety of microbial pathogens including P. aeruginosa, S. aureus, E. coli, Streptococcus spp. and Candida albicans [Ghaffari et al., 2005, Nitric Oxide, 14:21-29].
  • Nitric oxide has also been demonstrated to inhibit replication of a variety of viral pathogens including influenza virus, retroviruses, rhabdoviruses e.g. vesicular stomatitis virus, flavivirus e.g. Japanese Encephalitis virus [Rimmelzwaan et al., 1999, J. Virol 73(10): 8880-8883 and references cited therein].
  • influenza virus retroviruses
  • rhabdoviruses e.g. vesicular stomatitis virus
  • flavivirus e.g. Japanese Encephalitis virus [Rimmelzwaan et al., 1999, J. Virol 73(10): 8880-8883 and references cited therein].
  • gaseous nitric oxide may be administered to humans having breathing problems since it was found to have beneficial effects due to its bronchodilatory and vasodilatory activity.
  • nitric oxide is rather complicated for use as a gas.
  • colorless gaseous NO may react with oxygen under certain conditions, yielding nitrogen dioxide (N0 2 ), a reddish-brownish gas with much higher toxicity than NO.
  • Nitric oxide-producing agents have been used to keep various medical and other devices substantially free of bacteria by their capacity to release nitric oxide under certain conditions.
  • WO 1995/024908, WO 2006/037105, WO 2007/1028657, WO 2002/1056864, WO 2006/1084910 and WO 2006/1078786 teach various industrial materials and polymers comprising nitric oxide precursors, which are capable of generating some amount of NO in situ when the material comes in contact with physiological media, or upon other chemical or physical stimuli.
  • U.S. Patent Application Nos. 2010/0268149 and 201 1/0076313, and WO 2009/036571 teach nitric oxide gas-releasing conduit configured for surgical implantation through a patient's tympanic membrane.
  • Regev-Shoshani et al. disclose studies conducted for investigating the potential of NO to prevent biofilm formation.
  • Catheters impregnated with gaseous nitric oxide (gNO) demonstrated slow-release of nitric oxide over a 14-day period, were rendered antiseptic, and were able to prevent bacterial colonization and biofilm formation on their luminal and exterior surfaces.
  • NO impregnated catheters inhibited the growth of Escherichia coli within the surrounding media and eradicated bacterial concentrations of up to 10 4 CFU/ml.
  • a process of preparing an article having gaseous nitric oxide sequestered therewithin comprising: placing an article within a chamber; generating a reduced pressure in the chamber; and filling the chamber with a gaseous nitric oxide- containing environment, thereby preparing the article having gaseous nitric oxide sequestered therewithin.
  • generating the reduced pressure comprises reducing the pressure by from - 1 psi to -50 psi.
  • reducing the pressure is such that a humidity in the article is reduced by at least 50 %.
  • reducing the pressure is such that an amount of oxygen in the article is reduced by at least 50 %
  • filling the chamber with the gaseous nitric oxide-containing environment is effected for a time period that ranges from 30 minutes to 24 hours.
  • the article includes at least a portion of a surface configured to sequester nitric oxide.
  • the article includes at least a portion of a surface configured to sequester at least 1 ppm nitric oxide per cm 3 .
  • the article includes at least a portion of a surface configured to sequester at least 200 ppm nitric oxide per cm 3 .
  • At least a portion of the article comprises a plurality of voids for sequestering nitric oxide.
  • the article is a medical device.
  • the medical device is selected from the group consisting of a catheter, an endotracheal tube, a tampon, a tubing, a prosthetic, a medical implant, an artificial joint, an artificial valve, a needle, an intravenous access device, a cannula, a biliary stent, a nephrostomy tube, a vascular graft, an infusion pump, an adhesive patch, a suture, a fabric, a mesh, a polymeric surgical tool or instrument, an intubation device, prosthetics, artificial joints, artificial valves, adhesive patches, sutures, fabrics, a cardiovascular stent, a cardiac surgery device, an orthopedic surgery device, an orthodontic or periodontic device, a dental surgery device, a veterinary surgery device, a bone scaffold, a hemodialysis tubing or equipment, a blood exchanging device, an implantable prostheses, a bandage, a heart valve, an ophthalmic device and a breast implant
  • the medical device is an implantable device.
  • the medical device is selected from the group consisting of an indwelling catheter and a tracheal tube.
  • the catheter is selected from the group consisting of urinary catheter, central venous catheter, biliary vascular catheter, pulmonary artery catheter, peripheral venous catheter, arterial line, central venous catheter, peritoneal catheter, epidural catheter and central nervous system catheter.
  • the medical device is a tampon.
  • the medical device comprises a polymeric material.
  • the polymeric material is a hydrophobic polymeric material.
  • the polymeric material is selected from the group consisting of silicone, polyacetal, polyurethane, polyester, polytetrafluoroethylene, polyethylene, polymethylmethacrylate, polyhydroxyethyl methacrylate, polyvinyl alcohol, polypropylene, polymethylpentene, polyetherketone, polyphenylene oxide, polyvinyl chloride, polycarbonate, polysulfone, acrylonitrile- butadiene-styrene, polyetherimide, polyvinylidene fluoride, polysiloxane, fluorinated polysiloxane, polyanhydride, ethylene vinyl acetate, methacrylic acid, ethylene oxide, propylene oxide, polystyrene, ethylene-propylene rubber, fluoroelastomer, silastic elastomer, polyethylene tetrephthalate, colloidion, carbothane, nylon, poly(L-lactide), poly(DL-lactide),
  • the polymeric material is a hydrophilic or amphiphilic polymeric material.
  • the polymeric material is selected from the group consisting of rayon, cotton and cellulosic polymer (as in the case of, for example, tampons).
  • the article is selected from the group consisting of a packaged article and a bare (unpackaged) article.
  • the packaged article comprises a gas-permeable package.
  • placing the article in the chamber comprises positioning the article within an enclosure.
  • the enclosure is a non-gas permeable enclosure.
  • the process further comprises, subsequent to the filling, sealing the enclosure.
  • the process further comprises disposing a desiccant within the enclosure.
  • the process further comprises disposing a nitric oxide indicator within the enclosure.
  • an article having a gaseous nitric oxide sequestered therewithin prepared by the process described hereinabove.
  • the article further comprises an enclosure.
  • an article having sequestered therewithin at least 1 ppm nitric oxide per cm 3 and comprising less than 1 ppm per cm 3 nitrogen-containing and/or oxygen containing reactive species.
  • the article has sequestered therein from 1 ppm to 200 ppm per cm 3 nitric oxide.
  • the sequestered nitric oxide is releasable in an aqueous solution during a time period that ranges from 1 hour to 1 month.
  • any of the articles described herein further comprises an enclosure.
  • the enclosure comprises a gaseous nitric oxide-containing environment.
  • the environment is an ambient environment.
  • the enclosure is a non-gas permeable enclosure.
  • any of the articles described herein further comprises a desiccant disposed within the enclosure. According to some embodiments of the invention, any of the articles described herein further comprises a nitric oxide indicator disposed within the enclosure.
  • any of the articles described herein comprises at least 1 ppm per cm 3 nitric oxide sequestered therewithin.
  • any of the articles described herein comprises at least 200 ppm per cm 3 nitric oxide sequestered there within.
  • the sequestered nitric oxide is releasable is an aqueous solution during a time period that ranges from 1 hour to 1 month.
  • any of the articles described herein is being substantially devoid of humidity.
  • any of the articles described herein is being substantially devoid of oxygen.
  • an amount of nitrogen- containing and/or oxygen containing reactive species in the article is lower than 1 ppm per cm 3 .
  • an amount of nitrogen- containing and/or oxygen containing reactive species in the article is lower than 1 ppb per cm 3 .
  • any of the articles described herein is a medical device, as described herein.
  • a tampon having sequestered therein gaseous nitric oxide.
  • an amount of the nitric oxide ranges from 1 ppm to 200 ppm per cm 3 .
  • an amount of the nitric oxide is at least 200 ppm per cm 3 .
  • a process of preparing a tampon having sequestered therein nitric oxide comprising: exposing a tampon to gaseous nitric oxide-containing environment, thereby preparing the tampon having sequestered therein nitric oxide.
  • the exposing comprises: placing the tampon is a chamber; and filling the chamber with the nitric oxide- containing environment.
  • the process further comprises, prior to the filling, generating a reduced pressure in the chamber.
  • a method of treating a vaginal medical condition in a subject in need thereof comprising placing a tampon having gaseous nitric oxide sequestered therein in a vagina of the subject.
  • a tampon having gaseous nitric oxide sequestered therein, identified for use in the treatment of a vaginal medical condition.
  • a method of treating a vaginal medical condition comprising intravaginally administering to a subject in need thereof gaseous nitric oxide.
  • intravaginally administering gaseous nitric oxide comprises placing a tampon having gaseous nitric oxide sequestered therein.
  • gaseous nitric oxide for use in the treatment of a vaginal medical condition.
  • gaseous nitric oxide in the manufacture of a medicament for treating a vaginal medical condition.
  • the gaseous nitric oxide is being sequestered in a tampon.
  • a tampon having gaseous nitric oxide sequestered therein, for use in delivering gaseous nitric oxide to a vagina of a subject.
  • a process of preparing a packaged article comprising: exposing a packaged article to a gaseous nitric oxide-containing environment, thereby preparing the packaged article.
  • the exposing comprises: placing the packaged article in a chamber; and filling the chamber with the nitric oxide- containing environment.
  • the process further comprises, prior to the filling, sealing the chamber.
  • the process further comprises, prior to the filling, generating a reduced pressure in the chamber.
  • generating the reduced pressure is such that a humidity in the enclosure is reduced by at least 50 %.
  • reducing the pressure is such that an amount of oxygen in the enclosure is reduced by at least 50 %.
  • filling is effected by flowing nitric oxide into the chamber.
  • the process further comprises, prior to the filling, absorbing humidity from the package.
  • filling is effected such that an ambient environment is provided within the package.
  • the nitric oxide-containing environment comprises at least 0.01 % nitric oxide.
  • the packaged article has gaseous nitric oxide sequestered within the article.
  • a process of preparing a packaged article wherein the packaged article comprises a non-gas permeable enclosure, the process comprising: positioning an intact article within the non-gas permeable enclosure, to thereby obtain a non gas-permeable enclosure having the article disposed therewithin; exposing the enclosure with a gaseous nitric oxide-containing environment, so as to introduce into the enclosure the nitric oxide-containing environment; and sealing the enclosure, thereby preparing the packaged article.
  • the exposing comprises: placing the enclosure in a chamber; and filling the chamber with the gaseous nitric oxide-containing environment.
  • the process further comprises, prior to the filling, sealing the chamber.
  • the process further comprises, prior to the filling, reducing a pressure in the chamber.
  • reducing the pressure is such that a humidity in the enclosure is reduced by at least 50 %.
  • reducing the pressure is such that an amount of oxygen in the enclosure is reduced by at least 50 %.
  • the filling is effected by flowing nitric oxide into the chamber.
  • the process further comprises, prior to the filling, absorbing humidity from the enclosure.
  • the filling is effected such that an ambient environment is provided within the enclosure.
  • the nitric oxide-containing environment comprises at least 0.02 % nitric oxide.
  • the packaged article has gaseous nitric oxide sequestered within the article.
  • a package comprising: a material configured to form an enclosure; a article disposed within the enclosure; and a gaseous nitric oxide-containing environment within the enclosure.
  • the package is a non-gas permeable package.
  • the enclosure is a sealed enclosure.
  • the environment is an ambient environment.
  • the environment comprises gaseous nitric oxide in an amount sufficient to sterilize an interior of the enclosure and the article.
  • the amount of gaseous nitric oxide is at least 200 ppm.
  • the article has gaseous nitric oxide sequestered therewithin.
  • the amount of the gaseous nitric oxide sequestered in the article is at least 1 ppm.
  • the gaseous nitric oxide sequestered in the article is releasable in an aqueous solution during at least 1 minute.
  • the package further comprises a desiccant.
  • the package further comprises a nitric oxide indicator.
  • the article is a medical device, as described herein.
  • a charging device comprising: a chamber comprising an inlet for receiving a gaseous nitric-oxide containing environment and an outlet for releasing the gaseous nitric-oxide containing environment; and an article disposed within the chamber.
  • the device further comprises a desiccant configured to absorb humidity from the gaseous environment.
  • the device further comprises a nitric oxide indicator configured to undergo a color change suitable for visual assessment of whether the article has been exposed to the nitric oxide.
  • the device further comprises an outlet for generating a reduced pressure in the chamber.
  • the device further comprises a package enclosing the article.
  • package is a non-gas permeable package.
  • the article includes at least a portion of a surface configured to sequester the nitric oxide.
  • the surface is configured to sequester at least 1 ppm nitric oxide.
  • the surface is configured to sequester about 1 ppm nitric oxide to about 20,000 ppm nitric oxide.
  • a charging device comprising: a sealed chamber having a reduced pressure therewithin; and an article disposed within the chamber.
  • the chamber further comprises an outlet for generating the negative pressure within the chamber.
  • the device further comprises an inlet configured for receiving a gaseous nitric oxide-containing environment and an outlet for releasing the gaseous nitric oxide-containing environment.
  • the device further comprises a package enclosing the article.
  • the package is selected from the group consisting of a gas permeable package and a non gas-permeable package.
  • FIG. 1 is a bar graph demonstrating the antimicrobial effect of an exemplary polymeric medical device sample (a catheter section) dosed with nitric oxide for 1, 5, 15, 30, 60 or 120 minutes, upon incubation in a bacterial suspension of 10 3 , 10 5 or 10 8 CFU/ml for 8 hours at 37 °C, while showing the number of CFU/ml remaining in the suspension with respect to dose time and starting culture CFU/ml.
  • FIG. 2 is a chart presenting the total accumulation of nitrites and nitrates, produced from an exemplary medical device sample (a catheter section) dosed with nitric oxide and immersed in water, and showing a time-dependent nitric oxide dosing.
  • FIGs. 3A-B present a micrograph (FIG. 3A) and bar graphs (FIG. 3B) demonstrating the effect of catheter storage conditions on the antimicrobial activity of control and nitric oxide-eluting samples after storage and upon incubation with suspensions of 10 3 CFU/ml E. Coli for 1 minute followed by 24 incubation in PBS.
  • FIG. 3A presents images of representative three-compartment petri plates of control and nitric oxide-eluting samples after both sets were stored for 7 days in 50 ml sterile air (I) or 5 ml sterile water (II);
  • FIG. 3B presents the viable counts of the triplicate CFU with the hatched bars are data from the control, and the checkered bars are data from the NO- impregnated catheters. The error bars indicate standard deviations.
  • FIGs. 4A-B present a bar graph (FIG. 4A) and a micrograph (FIG. 4B) showing the biofilm formation and biofilm-embedded bacteria on the inner lumen of control and NO-dosed catheter samples subjected to 24 hours of urine flow.
  • FIG. 4A presents biofilm formation as the absorbance at 595 nm of the crystal violet attached to the catheter pieces after extraction with ethanol. The hatched bars are data from the control, while the checkered bars are data from the impregnated catheters.
  • FIG. 4B presents representative images of biofilm-embedded bacteria determined in 1 ⁇ (bottom) and 10 ⁇ (top) of water surrounding a selected catheter piece and incubated for 24 hours.
  • FIGs. 5A-B present a photograph showing the effect of nitric oxide gas on biofilms of Escherichia coli after exposure for 5, 10, 30, 60 or 120 minutes, relative to controls (FIG. 5A); and a photograph showing the effect of nitric oxide gas on biofilms of Acinetobacter baumanii after exposure for 5, 10, 30, 60 or 120 minutes, relative to controls (FIG. 5B).
  • FIG. 6 presents a bar graph showing the antimicrobial activity of nitric oxide- dosed catheter sections on the bacterial strains Enterococcus faecalis #29212 (E.f. #29212), Staphylococcus saprophyticus #15305 (S.s #15305), Staphylococcus epidermidis #35984 (S.e. #35984), Escherichia coli #25922 (E.c. #25922), Pseudomonas aeruginosa #14210 (P. a. #14210), Acinetobacter baumanii #BAA-747 (A.b. #BAA-747 and, Candida albicans (C.a. #14053).
  • FIG. 7 is a bar graph showing the antimicrobial activity of nitric oxide-dosed catheters the bacterial clinical isolates Enterococcus faecalis (E.f), Staphylococcus aureus (S.a.), E. coli (E.c), P. aeruginosa (P. a.), and Stenotrophomonas maltophilia (S.m.).
  • FIG. 8 is a bar graph presenting the relative biofilm formation on luminal surfaces of nitric oxide-sequestering catheter sections following 72 hour incubation in urine inoculated with 10 3 CFU/ml of Enterococcus faecalis #29212 (E.f. #29212), Staphylococcus saprophyticus #15305 (S.s #15305), Staphylococcus epidermidis #35984 (S.e. #35984), Escherichia coli #25922 (E.c. #25922), Pseudomonas aeruginosa #14210 (P. a. #14210), Acinetobacter baumanii #BAA-747 (A.b. #BAA- 747), and Candida albicans (C.a. #14053).
  • FIG. 9 presents a bar graph showing the data obtained for the growth of biofilm- embedded bacteria on nitric oxide-sequestering catheter sections following 72 hours incubation in urine inoculated with 10 3 CFU/ml of Enterococcus faecalis #29212 (E.f. #29212), Staphylococcus saprophyticus #15305 (S.s #15305), Staphylococcus epidermidis #35984 (S.e. #35984), Escherichia coli #25922 (E.c. #25922), Pseudomonas aeruginosa #14210 (P. a. #14210), Acinetobacter baumanii #BAA-747 (A.b. #BAA-747), and Candida albicans (C.a. #14053).
  • FIG. 12 is a bar graph presenting comparative data of E. coli growth in media containing pieces of an NO-sequestering catheter (NOX), a silver-alloy coated catheter (AG) and an antibiotic-coated catheter (NFC) versus media from control catheter, after immersion of the catheters for 24 hours in suspension comprising 10 3 CFU/ml and incubated for 24 hours at 37 °C.
  • NOX NO-sequestering catheter
  • AG silver-alloy coated catheter
  • NFC antibiotic-coated catheter
  • FIG. 13 presents images of representative three compartment LB agar petri plates showing E. coli growth in urine after 72 hours exposure to pieces of an NO- sequestering catheter (NOX; bottom left) , a silver-alloy coated catheter (AG; top right) and an antibiotic-coated catheter (NFC; bottom right) and control catheters (top left).
  • NOX NO- sequestering catheter
  • AG silver-alloy coated catheter
  • NFC antibiotic-coated catheter
  • FIG. 14 presents images of representative three compartment LB agar petri plates showing E. coli colonization on NO-sequestering catheter (NOX; bottom left) , a silver-alloy coated catheter (AG; top right) and an antibiotic-coated catheter (NFC; bottom right) and control catheters (top left), after immersion of catheters for 24 hours in suspension containing 10 3 CFU/ml of E. coli.
  • NOX NO-sequestering catheter
  • AG silver-alloy coated catheter
  • NFC antibiotic-coated catheter
  • control catheters top left
  • FIGs. 15A-B present bar graphs showing comparative data of colonized biofilm formation on NO-sequestering catheter (NOX), a silver-alloy coated catheter (AG) and an antibiotic-coated catheter (NFC) versus control after 72 hours of incubation, demonstrated by absorbance at 595 nm (FIG. 15A) and the bacterial growth from the biofilms from the different catheters (FIG. 15B).
  • NOX NO-sequestering catheter
  • AG silver-alloy coated catheter
  • NFC antibiotic-coated catheter
  • FIG. 16 presents comparative plots showing nitrite release over time from the NO-sequestering Tracheal tubes
  • Mallinckrodt Hi-Lo Tracheal tube 6.5 mm ID ref No. 861 10 (triangles) and Mallinckrodt: Hi-Contour Tracheal tube 4,5 oral/nasal 6,2, 1 1 mm ID ref No. 107-45.
  • FIG. 17 is a bar graph showing the amount of nitric oxide released after 30 minutes from four exemplary NO-sequestering tampons (see, Table 19), as measured by the respective total amount of nitrites released from the tested tampons Nitrites were measured using Griess reagent. The nitrites released were calculated per 1 tampon.
  • FIG. 18 presents comparative plots showing the accumulation profiles of nitric oxide production during the first 5 hours for four exemplary NO-impregnated tampons, as measured by the respective total accumulation of nitrites in water produced from the tested tampons. Nitrites were measured using Griess reagent. The nitrites released were calculated per 1 tampon
  • FIGs. 19A-D demonstrate the antimicrobial effect of four exemplary NO- sequestering tampons A (FIG. 19A), B (FIG. 19B), C (FIG. 19C) and D (FIG. 19D), as identified in Table 19 herein, by showing images of representative petri plates in which NO-treated tampons inoculated with C. albicans culture for 4 hours at 30 °C were rolled and upon incubating the plates overnight at 30 °C.
  • FIG. 20 is a bar graph demonstrating the anti-infective activity of four exemplary NO-sequestering tampons A-D, as identified in Table 19 herein, by showing the growth of C. albicans in media after immersion of the tampons for 6 hours in suspension comprising 10 1 CFU/ml of C. albicans (dark bars) compared with negative control tampons (white bars). Numbers represent viable counts of the triplicate CFUs. Error bars represent standard deviation.
  • FIG. 21 is a bar graph demonstrating the anti-infective activity of four exemplary NO-sequestering tampons A-D, as identified in Table 19 herein, by showing the growth of C. albicans in media after immersion of the tampons for 4 hours in suspension comprising 10 2 CFU/ml of C. albicans (dark bars) compared with negative control tampons (white bars). Numbers represent viable counts of the triplicate CFUs. Error bars represent standard deviation.
  • FIG. 22 presents representative photos demonstrating the zone of inhibition resulting from NO-treated tampons.
  • NO-treated tampon B top right
  • NO-treated tampon C bottom right
  • untreated tampons B and C top and bottom left, respectively
  • FIG. 23 presents a schematic illustration of the experimental setup used to test the zone of microbial inhibition in a vaginal model.
  • FIG. 24 presents representative photos demonstrating the zone of inhibition in a vaginal model.
  • a solution containing LB and 2 % (w/v) gelatin was inoculated with E. coli (10 6 CFU/ml), placed in a 250 ml Erlenmeyer flask and tampon B (see, Table 19) was suspended therein. The Erlenmeyer flasks were incubated at 37 °C overnight. Shown is a picture of NO-treated (right) and untreated (left) tampon B placed inside the inoculated solution after incubation.
  • FIG. 25 shows total accumulation of nitrites, produced in water during 4 hours, from tampons impregnated with nitric oxide outside their wrappers. Nitrites were measured using Griess reagent. The nitrites released were calculated per 1 tampon FIG.
  • 26 presents a schematic illustration of an exemplary charging device 300, according to some embodiments of the present invention, which is fitted with an inlet 345 for receiving gNO within chamber 337 to form gaseous nitric oxide-containing environment 306, valve 315 for closing outlet 345, an outlet 326 for releasing ambient environment or gaseous nitric oxide-containing environment from chamber 337, valve 336 for closing outlet 326, an article 301 disposed within chamber 337, and an optional moisture scavenger in the form of desiccant 302 disposed within chamber 337 and configured to absorb an amount of humidity from the within chamber 337;
  • FIG. 27 is a schematic illustration of an exemplary charging device 300, as presented in FIG. 26, wherein chamber 337 also includes a surface coating 370 which protects the material of chamber 337 from chemically interacting with gNO-containing environment 306, a gNO indicator 371 configured to undergo a color change when article 301 has been exposed to gNO in environment 306, and humidity indicator 374 for signaling exposure of article 301 to humidity;
  • a surface coating 370 which protects the material of chamber 337 from chemically interacting with gNO-containing environment 306, a gNO indicator 371 configured to undergo a color change when article 301 has been exposed to gNO in environment 306, and humidity indicator 374 for signaling exposure of article 301 to humidity;
  • FIG. 28 is a schematic illustration of an exemplary charging device 400, according to some embodiments of the present invention, which is fitted with an inlet 445 for receiving gNO within chamber 447 to form gaseous nitric oxide-containing environment 406, valve 415 for closing outlet 445, an outlet 426 for releasing ambient environment or gaseous nitric oxide-containing environment from chamber 447, valve 436 for closing outlet 426, and a non-gas permeable package 473 disposed within chamber 447 and housing article 401;
  • FIG. 29 is a schematic illustration of an exemplary charging device 400, as presented in FIG. 28, wherein chamber 437 includes surface coating 470 which protects the material of chamber 437 from chemically interacting with gNO-containing environment 406, a gNO indicator 471 configured to undergo a color change when article 401, having gNO-sequestering surface 472 and housed within non-gas permeable package 473 equipped with seal 490, has been exposed to gNO in environment 406, and humidity indicator 474 for signaling exposure of article 401 to humidity;
  • FIG. 30 presents a schematic illustration of an exemplary charging device 500 having a chamber 137, inlet 125 with valve 11, inlet 135 with valve 2, and inlet 145 with valve 15, inlets which may share a common tube 23 with valve 19 that connects to chamber 137; outlet 126 with valve 36 and connect to purge flow rotomoter 46, outlet 136 with valve 41 and connect to purge flow rotomoter 45, outlets which may share a common tube 33 that connects to chamber 137; and
  • FIGs. 31A-B present schematic illustrations of a package for charging and containing gNO-sequestering medical device, according to some embodiments of the present invention, such as a package 200, comprising non-gas permeable package 203 configured to be impermeable to gNO and optionally comprises seal 205 (Figure 3 IB), medical device 201 disposed within non-gas permeable package 203, gNO-containing environment 206 engulfing medical device 201 within non-gas permeable package 203 and comprising a predetermined concentration of gNO capable of sterilizing the interior of non-gas permeable package 203 and medical device 201, and further optionally comprising nitric oxide indicator 204 (Figure 3 IB), desiccant 202 disposed within non- gas permeable package and configured to absorb humidity from gNO-containing environment 206, and humidity indicator 208 ( Figure 3 IB) configured to indicate moisture saturation of the desiccant 202.
  • a package 200 comprising non-gas permeable package 203
  • the present invention in some embodiments thereof, relates to medical products and, more particularly, but not exclusively, to products having gaseous nitric oxide (NO) sequestered therein, to processes and systems for producing such products and to uses thereof.
  • NO gaseous nitric oxide
  • impregnation of gaseous nitric oxide (gNO) in medical devices is regarded highly beneficial for sterilizing articles such as medical devices, by imparting the medical devices with the gNO capabilities to reduce or prevent growth of a variety of pathogenic microorganisms, as well as biofilm formation.
  • gaseous nitric oxide sequestered in the solid substance of medical devices can be used to kill bacteria in an established biofilm and in the surrounding environment; be used as an effective sterilizing agent of such devices; and prevents adherence and growth of various strains or species of bacteria and fungi over an extended period of time following sterilization.
  • medical devices comprising one or more polymers and sequestering gaseous nitric oxide are self-sterilizing at a sufficient level so as to prevent microbial adherence and growth on the medical device or in the liquid media in its immediate vicinity for an extended period of time.
  • polymeric material composing the device as exemplified by silicone, or silicone-coated medical devices, are not substantively altered by treatment with nitric oxide, however, in some cases, a color change is observed that may be useful as an indicator of NO load of the device.
  • tampons can be effectively charged with gNO so as to result in gNO-sequestering tampons; and (ii) tampons having gNO sequestered therewithin efficiently reduce microbial growth and thus can be used to treat or prevent various common vaginal infections.
  • the present inventors have searched for an improved process for impregnating gNO within medical devices.
  • the present inventors have thus designed and successfully practiced a process which involves exposing the article to be impregnated (e.g., a medical device) to reduced pressure, so as to substantially evacuate ambient oxygen and humidity prior to exposure to gNO.
  • the present inventors have contemplated that by removing ambient atmosphere from the article to be loaded, and thereby evacuating voids within the article, the gNO sequestering efficiency of the article would be improved due to enhanced free void volume to be occupied by gNO.
  • the enhancement in void volume can be translated in some cases to enhanced affinity of NO, as is discussed in detail hereinunder.
  • the present inventors have also contemplated that removal of ambient atmosphere would prevent gNO degradation and would thus substantially reduce the production of undesired reactive nitric oxide species associated with gNO degradation.
  • the present inventors have further contemplated a process for producing sterilized and even self-sterilizing packages, which takes advantage of the sterilizing effect imparted by gaseous NO.
  • the present inventors have designed and successfully practiced a process in which medical devices are packaged in a non-gas permeable enclosure which is loaded with gaseous NO.
  • Such a packaging process allows, for example, storing and/or transporting packaged articles while maintaining the packaged articles sterilized.
  • a process of preparing an article having gaseous nitric oxide (gNO) sequestered therewithin is effected by:
  • the term "sequestering" and any inflections thereof refer to a state of an article having a foreign substance, such as a gas, incorporated therein; a state which exists substantially from the time the substance is introduced into the article from an external source to the time the substance leaves the article. According to some embodiments of the present invention, in the chemical sense the sequestered substance being released from the article is essentially the same substance that was charged into the article.
  • releasably sequestering is meant to define an article having gNO absorbed therein in a reversible manner, wherein the gNO can be released to the ambient environment from the article under certain conditions.
  • gNO is released from the article releasably sequestering gNO at an essentially controllable manner.
  • gNO gaseous NO sequestered within the article, namely, in or on at least a portion of the article or in or on any surface thereof.
  • therewithin and “therein: are used herein intgerchnageably.
  • gNO is releasably sequestered in an article by charging the article with gNO.
  • the amount of sequestered gNO depends, among other factors, on the size, volume, surface area and composition of the article.
  • the article may be composed and assembled from various parts, layers and compositions, each having a different capacity to sequester gNO according to chemical composition, accessibility, surface area and the likes.
  • gNO is releasably sequestered within at least a portion of the surface of the article.
  • a part of the article which can sequester gNO is referred to herein as a "surface", however it is to be understood that the term "surface” as used herein is not limited to the exterior or upper boundary of the article, not to an external part or a layer of the article, and not to its outwardly expressed manifestations of the article, but rather to all of these features as well as all other internal and external features of the article which can sequester gNO, according to some embodiments of the present invention.
  • the article can be disassembled to expose these components, or be assembled from raw materials and/or components that were charged with gNO according to some embodiments of the present invention.
  • the article to be charged with gNO in the process presented herein preferably does not sequester gNO in advance, prior to effecting the process.
  • the article is untreated with gNO at the time it is first placed in the chamber of the gNO charging device.
  • the article which is exposed to gaseous nitric oxide-containing environment can be seen as an intact article, whereby the phrase "intact article” is meant to describe an article that has not been exposed to gNO-containing environment.
  • the process according to embodiments of this aspect of the present invention starts by placing an article in a chamber.
  • the article placed in the chamber can therefore be regarded as an intact article.
  • the chamber can be any tank, canister, vat, barrel, cask, hogshead, drum, case, wrapper, sheath, bag, compartment, vessel, container or receptacle which can serve as encasement for the article in terms of size (internal size) and capacity to contain gases, and particularly gNO containing environment.
  • capacity to contain it is meant that the chamber is sealed to an extent that allows charging the article, and mechanically fit to sustain both negative and positive pressure.
  • Such requirements typically translate to mechanical integrity for maintaining impermeability to gases, rigidity and/or durability to maintain negative and positive pressure, and the ability to be fitted with inlets and outlets without losing containment of gases, while maintaining the integrity of the article disposed therein.
  • the chamber forms a part of a charging device, which is designed to carry out the process presented herein and includes inlets and outlets (a single or a plurality of each); tubes for connecting the inlets and outlets to the chamber, to external sources, pumps and exhausts and therebetween; various inlet and/or outlet valves; various optional inlet and/or outlet gauges for monitoring inflow and outflow of gases; and various optional absorbers and scavengers of undesirable contaminants, as well as gauges and indicators for monitoring the environment within the chamber at various steps of the process, as is further detailed hereinunder.
  • the process involves placing an article of interest (an article to be impregnated) into the chamber with the intention of loading the article with gNO.
  • the article disposed within the chamber can be any article-of-manufacture, any part of an article-of-manufacture or a stock or raw material for manufacturing an article- of-manufacture or manufacturing a part of an article-of-manufacture.
  • the process may be carried out by placing the entire medical device in the chamber, placing some of the parts of the medical device, such as tubing or surface- forming parts of the medical device in the chamber, or placing in the chamber the pre- processed, pre-shaped, pre-formed or pre-cut raw materials constituting parts of the medical device.
  • the process involves generating a reduced pressure in the chamber.
  • reduced pressure is used synonymously with the terms “negative pressure” "under pressure” and/or “vacuum”.
  • Evacuating the chamber from the ambient environment substantially evacuates (removes) gaseous substances and/or volatile substances found in the chamber as well as gaseous substances and/or volatile substances found in the article, at least to the extent of parts of the article which are exposed to the negative pressure.
  • gaseous refers to a state of a substance being a gas under certain conditions of pressure and temperature.
  • the melting point of nitric oxide at atmospheric pressure is -164 °C and the boiling point of nitric oxide is -152 °C, hence nitric oxide is a gaseous substance at ambient conditions of pressure and temperature (i.e., room temperature).
  • Oxygen, nitrogen and CO2, present in an ambient atmosphere are also gaseous substances.
  • Humidity and moisture forming-water is essentially a mixture of vapor (gaseous water) and liquid water at ambient conditions of pressure and temperature, however under reduced pressure the equilibrium of gas-liquid of water would essentially shift towards the gaseous state.
  • reducing the pressure in the chamber facilitates the removal of moisture and humidity from the chamber prior to introducing gNO therein.
  • Generating a reduced pressure in the chamber can be effected by connecting any inlet or outlet of the charging device to an external source of reduced pressure, such as a vacuum pump, or a vacuum reservoir.
  • a vacuum reservoir can be in the form of, for example, a container which has been evacuated from its content to posses a volume under reduced pressure which is substantially larger than the volume of the chamber, thereby being capable of taking-in (sucking in) at least a part of the ambient atmosphere of the chamber.
  • the level of the reduced pressure which is reached in the chamber can be at any level of vacuum which is reasonably attainable in the charging device resented herein.
  • the depth of the vacuum can range from low vacuum levels (about 100 kPa to 3 kPa) to high vacuum levels (about 100 mPa to 100 nPa).
  • vacuum levels can be expressed as a negative value, namely by the value representing the difference in pressure relative to atmospheric pressure (which is 760 Torr, 101.23 kPa, 14.7 psi or 1 atmosphere).
  • the reduced/negative pressure attained in the chamber can range from about -50 psi to -0.5 psi.
  • the unit "psi" is used in the context of some embodiments of the present invention, to denote a pressure difference, typically as recorded by a gage or device, from a reference pressure, typically atmospheric pressure. "0 psi” therefore denotes atmospheric pressure (e.g., ambient pressure), whereby "-X psi” is used to express negative pressure (under pressure).
  • generating the reduced pressure in the chamber is effected for a time period of at least 1 minute, or for a time period that ranges from 1 minute to 60 minutes, or from 1 minute to 30 minutes, or from 1 minute to 20 minutes, or from 1 minute to 15 minutes, e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 minutes, or for 30, 40, 50 or 60 minutes.
  • the article is exposed to reduced pressure and in essence undergoes a purging process during which gases and volatile substances are substantially removed therefrom.
  • the generation of reduced pressure in the chamber can thus be regarded as a degassing process or as a gas-evacuation process.
  • gNO water and gNO tend to interact such that the capacity of a moist article to sequester gNO is reduced. It is further noted herein that gNO is a relatively hydrophobic substance and hence the presence of water (moisture ot humidity) can adversely interfere with sequestering gNO. Hence, it is desirable to reduce the humidity in the article prior to exposing the article to gNO-containing environment.
  • Oxygen and gNO also interact to produce reactive species such as nitrogen dioxide (nitrate), according to the reaction 2NO + O2 ⁇ 2 NO2, which is a toxic brownish gas.
  • Water, oxygen and gNO react to produce nitrite according to the reaction 4NO + O2 + 2H2O ⁇ 4HNO2.
  • Nitrates and nitrites are known to participate is various reactions in vivo, in which toxic reactive oxygen species are formed. These reactions and subsequent reactions involving products of these reactions are commonly referred to herein as gNO degradation, and the process presented herein attempts to minimize this gNO degradation, by minimizing the presence of reactive species other than gNO.
  • Nitrates, nitrites, and any other species that are formed directly or indirectly by a reaction of nitric oxide with oxygen and/or water or humidity are referred to herein as "reactive species other than nitric oxide” or simply as “reactive species” and are meant to include nitrogen and/or oxygen-containing reactive species.
  • generating reduced pressure in the chamber is effected so as to reduce the humidity level in the article/chamber by at least 50 % of its original (ambient) level.
  • generating reduced pressure is effected so as to effect a decrease the humidity level in the article by more than 50 %, more than 60 %, 70 %, 80 %, 90 % and up to 100 % reduction in humidity, which is essentially desiccating the article to a level of humidity of almost zero or essentially zero humidity.
  • the amount of humidity (relative humidity) in the ambient environment prior to introducing gNO into the chamber is reduced such that the relative humidity in the chamber prior and during the introduction of the gNO-containing environment can be from about 0 % to about 25 %, or any amount therebetween, for example, about 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 11 %, 12 %, 13 %, 14 %, 15 %, 16 %, 17 %, 18 %, 19 %, 20 %, 21 %, 22 %, 23 % or 24 %.
  • generating reduced pressure in the chamber is effected so as to effect reduction of oxygen level in the article/chamber by at least 50 % of its original (ambient) level.
  • generating reduced pressure may decrease the oxygen level in the article by more than 50 %, more than 60 %, 70 %, 80 %, 90 % and up to 100 % reduction in oxygen level, which renders the article essentially devoid of oxygen.
  • generating reduced pressure in the chamber is effected so as to effect reduction of both oxygen and humidity levels to below 50 %, or below 40 %, 30 %, 20 %, 10 % and down to essentially 0 % of the ambient levels of each of oxygen and humidity before the exposure to gNO-containing environment.
  • the chamber is sealed so as to allow the application of reduced pressure therein for any desired length of time.
  • the ambient environment in the chamber can be replaced with a gNO- containing environment as described hereinbelow.
  • the chamber is filled with a replacement environment which comprises gaseous nitric oxide.
  • the gNO-containing environment is an ambient environment comprising nitric oxide.
  • the phrase "ambient environment comprising nitric oxide” and the phrase “gaseous nitric-oxide containing environment” refer equally to pure gNO or to any mixture of gNO and a carrier gas.
  • a carrier gas can be any inert or otherwise biologically and chemically compatible gas such as, but not limited to, helium, argon, nitrogen gas and any combination thereof.
  • filling the chamber with a gNO-containing environment can be performed by allowing a gNO-containing environment to flow into the chamber, namely, by simply connecting the chamber, in which reduced pressure was generated, to a source of the gNO-containing environment. This gaseous environment will flow into the chamber due to pressure differences.
  • filling the chamber with a gNO-containing environment can be performed by pushing the gNO-containing environment into the chamber at an elevated pressure, or allowing the gNO-containing environment to sucked into the chamber by force of the vacuum present therein.
  • the gNO- containing environment which is introduced into the chamber contains a predetermined concentration of gNO which is capable of exerting the desired effect of NO on the article, as discussed herein.
  • the pre- determined concentration of gNO in the provided gNO-containing environment may range from about 0.05 % to about 10 %, or any amount therebetween, for example about 0.05 %, 0.1 %, 0.5 %, 1 %, 2 %, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 % or 10 % of the total amount of the gNO-containing environment, with the remaining environment being the carrier gas.
  • the amount of gNO can alternately be expressed in parts per million (ppm), wherein ppm and percent values are readily interconverted; for example 100 ppm is 0.01 %, 1,000 ppm is 0.1 %, 10,000 ppm is 1 %, and 100,000 ppm is 10 %.
  • the concentration of gNO in the gNO-containing environment ranges from 160 ppm to 50,000 ppm.
  • the gNO concentration within the gNO-containing environment can be determined by methods well known and widely practiced to those skilled in the art.
  • the gNO-containing environment may be provided (maintained, supplied as a continuous flow or as a single disbursement) for a pre-determined amount of time that ranges from about 1 minute to about 24 hours, namely from about 1 minute to about 24 hours, or any period therebetween, for example about 1, 2, 5, 10, 15, 20, 30, 40, 50 or 60 minutes, or about 1, 2, 4, 6, 8, 10, 12, 16, 18 or 24 hours or higher, and any period of time therebetween.
  • a continuous flow of gNO-containing environment can be effected at a flow rate that ranges from about 1 cubic centimeter or milliliter per minute (cc/min) to about 2,000 cc/min or any rate therebetween, for example 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 250, 500, 750, 1,000 or 2,000 cc/min, or any rate therebetween. According to some embodiments, the flow rate ranges from about 250 cc/minute to 1,700 cc/min.
  • the ability to control the flow rate, concentration of gNO in the gNO-containing environment and the duration of exposure serve as three finely tuned means to adjust the amount of gNO sequestered in the article. For example, a higher concentration of gNO in the carrier gas will result in an article sequestering a higher amount of gNO. Maintaining the article exposed to the gNO-containing environment for a longer time will result in an article sequestering a higher amount of gNO. Similarly, increasing the flow rate of the gNO-containing environment through the chamber corresponds to increasing the amount of gNO which the article is exposed to, resulting in an article sequestering a higher amount of gNO. In some cases the article cannot sustain high vacuum levels to long or any duration of time, and in these cases controlling the amount of sequestered gNO will be achieved by concentration and flow rate of the gNO- containing environment.
  • the goal level of sequestered gNO in the article can be achieved in more than one way, using all possible variables presented herein (such as, for example, gNO concentration, duration of exposure, flow of gNO-containing environment, degassing by vacuum, desiccation and oxygen purging).
  • the mode by which the desired amount of sequestered gNO is achieved in the article depends on the nature and the composition of the article.
  • Articles of a higher volume and/or bulky articles and/or articles made of a material with lower affinity to NO and/or articles that require particularly high level of sequestered NO typically require higher loading time and/or higher duration and/or extent of reduced pressure and/or higher flowing rate of gaseous NO. Undershooting the optimal parameters of the aforementioned variables will result in an article with sub-effective level of sequestered gNO, and overshooting may damage the article or some of its components and/or the chamber.
  • the concentration of gNO in the gNO-containing environment ranges from 150 ppm to 60,000 ppm.
  • the concentration of gNO in the gNO-containing environment ranges from 800 ppm to 20,000 ppm; the period of time of exposure to the environment in the chamber ranges from 1 to 600 minutes or overnight (10-24 hours); and/or the flow rate at which the environment is introduced into the chamber ranges from 0.5 liter/minute to 5 liter/minute.
  • the article or at least a portion thereof may sequester gNO at a range of 1 ppm to about 20,000 ppm.
  • the result of the process presented herein is essentially an article (releasably) sequestering gNO in at least a portion thereof, as discussed hereinbelow.
  • the process results in an article sequestering from 1 ppm to 200 ppm gNO per cm 3 of the article.
  • the process results in an article sequestering from 1 ppm to 200 ppm gNO as the total amount of gNO.
  • higher amounts of gNO are also contemplated, as well as any value between the 1 ppm and 20,000 ppm.
  • the amount of gNO sequestered in an article impregnated upon generating a reduced pressure is higher by at least 5 %, 10 %, 20 %, 30 %, 40 % and even 50 % compared to the gNO sequestered in an article without prior generation of reduced pressure.
  • the process involves evacuating the residual gNO-containing environment from the chamber, in preparation to opening the chamber and retrieval of the treated article from the charging device.
  • a step can be effected by purging the chamber by flowing a carrier gas or ambient atmosphere into the chamber, by pumping the gNO-containing environment from the chamber with a vacuum pump or by a combination of pumping and purging.
  • the amount of gNO in the environment, the duration of exposure to the environment during dosing and on various other factors as described hereinabove, the resulting gNO-sequestering article or at least portions thereof, are dosed with a predetermined concentration of gNO which is sufficient to sustain release of an effective amount of gNO (e.g., for maintaining sterility) at storage conditions for a time period ranging from about 1 day to about 5 years, or from about 1, 2, 3, 4, 5, 6, 7 days, 2 weeks, 3 weeks, 4 weeks, about 2, 3, 4, 5, 6, 12, 18, 24, 30, 36, 42, 48, 54 or about 60 months, or any period therebetween.
  • storage conditions it is meant that the article is kept under conditions which maximize the length of time that gNO can be released therefrom at an effective amount once it is removed from storage conditions.
  • storage conditions encompasses any form of maintaining a gNO-containing environment in the immediate vicinity of the article, either in the charging device, the sealed chamber or in a non-gas permeable container or package, as these are discussed hereinbelow. Storage conditions also include maintaining an environment which is low in oxygen, moisture, heat or other factors which may reduce the levels of gNO sequestered in the article.
  • the amount of sequestered gNO depends, among other factors, on the size, volume, surface area and composition of the article.
  • Articles can be selected to be made from materials having a high intrinsic capacity to sequester gNO, or alternatively, one can select parts of the article which are made from materials having a high intrinsic capacity to sequester gNO, to be assembled into a complete article.
  • Materials having a high intrinsic capacity to sequester gNO include polymers, co-polymers and resins of various chemical compositions and other mechanical and morphological properties.
  • Articles are made primarily from materials which are most suitable to serve the intended use of the article, such as, for example, medical devices, which are generally made from polymeric materials that are compatible with the internal or external surfaces of tissues and organs of a living subject (biocompatible).
  • the article includes at least a portion of a surface configured to sequester nitric oxide, preferably a surface configured to sequester at least 1 ppm nitric oxide per cm 3 , and optionally, a surface configured to sequester at least 200 ppm nitric oxide per cm 3 , depending on the intended use of the article.
  • At least a portion of the article comprises a plurality of voids for accepting and sequestering nitric oxide.
  • Articles that comprise voids may be comprised of polymeric materials that have a certain degree of porosity due to e.g., chemical composition and manufacturing parameters of the polymeric material (e.g., cross-linked polymers) and/or due to physical parameters such as articles made of condensed layers or fibrous structures of a (e.g., polymeric) material or a plurality of materials.
  • articles that have at least a portion thereof which is gas-permeable are suitable for use in the context of these embodiments of the present invention.
  • polymeric materials, polymers, co-polymers and resins which are both suitable to serve the intended use of the article they comprise, and have a sufficient intrinsic capacity to sequester gNO are commonly referred to herein as "suitable polymeric materials”.
  • suitable polymeric materials for forming polymeric material-based medical devices include resins which may exhibit microporous structure, and which are generally gas-permeable, and are able to sequester gNO.
  • the sequestered gNO can be released controllably from these compatible polymers over an extended period of time, thereby creating a localized microbial-free environment surrounding the medical device, or a portion thereof, thus rendering the medical device self-sterilizing.
  • any gNO-permeable material wrapping or encasing the article would undergo gNO dosing at the same time as the article, or at least allow gNO to pass- through to impregnate the article therein
  • the term "article", as used herein, is meant to encompass an entire article and any appendage or supplemental part attached or connected thereto, including, without limitation, any gNO-permeable wrapping, coating and/or encasement material completely engulfing or partially encasing the article, and in particular any gNO-permeable wrapping or encasement which can also sequester gNO.
  • Chemical composition is one of the characteristics that influence a material's intrinsic capacity to sequester gNO.
  • suitable polymeric materials are permeable to gNO by virtue of their composition, degree of crosslinking, crystallinity and the likes.
  • gNO is substantially non-polar, it can diffuse more readily into non-polar media, such as, without limitation, hydrophobic polymers and in general, polymers composed of non-polar constituents.
  • Hydrophobic polymers have a high intrinsic capacity to sequester higher amounts of gNO and further to sequester gNO for extended periods of time, due to higher affinity of gNO to hydrophobic substances. Hence, hydrophobic polymers are suitable in applications where prolonged release of gNO is beneficial.
  • suitable polymeric materials in the context of embodiments of the present invention are not limited to hydrophobic polymers, and hydrophilic or amphiphilic polymeric material can also be impregnated with gNO effectively, according to some embodiments of the present invention.
  • hydrophobic and hydrophilic polymers and other substances may still be charged with gNO using the aforementioned factors of reduced pressure, gNO concentration in the environment, time of exposure to the environment and filled voids occurring in the impregnated substance.
  • hydrophobic, amphiphilic and hydrophilic polymers typically require exposure to gNO-containing environment for longer time periods and/or at higher flow rates and/or upon generating reduced pressure for longer time periods.
  • a hydrophobic material is characterized by an oil-in-water partition coefficient (LogP) greater than 0, greater than 1 , greater than 2, greater than 5 or higher.
  • LogP oil-in-water partition coefficient
  • Non-limiting examples of suitable polymeric materials useful in making gNO- charged articles include silicone, polyacetal, polyurethane, polyester, polytetrafluoroethylene, polyethylene, polymethylmethacrylate, polyhydroxyethyl methacrylate, polyvinyl alcohol, polypropylene, polymethylpentene, polyetherketone, polyphenylene oxide, polyvinyl chloride, polycarbonate, polysulfone, acrylonitrile-butadiene-styrene, polyetherimide.
  • polyvinylidene fluoride copolymers, polysiloxane, fluorinated polysiloxane, polyanhydride, ethylene vinyl acetate, methacrylic acid, ethylene oxide, propylene oxide, polystyrene, ethylene-propylene rubber, fluoroelastomer, silastic elastomers, polyethylene tetrephthalate, colloidion, carbothane, nylon, and combinations thereof.
  • suitable polymeric materials include biodegradable and bioresorbable polymers exemplified by polyglycolic acid (PGA), polylactic acid (PLA), poly(L-lactide), poly(DL-lactide), poly(DL-lactide-co-glycolide), polycaprolactone, poly(e-caprolactone), polydioxanone, polyparadioxanone, polytrimethylene carbonate, polyoxalate, a polyanhydride, a poly(phosphoester), or copolymers and/or combinations thereof, as well as biologically derived materials such as rayon, cotton, catgut, collagen, silk, elastin, chitin, chitosan, cellulose, cellulosic polymers, coral, hyaluronic acid, bone, hydroxy apatite, bioabsorbable calcium phosphate and any combinations thereof.
  • PGA polyglycolic acid
  • PLA polylactic acid
  • PDA poly(L-lactide), poly(DL-
  • suitable polymeric materials can be used as coating on an article such as a medical device, or serve as encasement of the medical device, whereby the coating or encasement provides gNO-sequestering capacity to the medical device.
  • an implantable pump which forms a part of an implantable drug delivery system can be encased within one or more layers comprising one or more of the suitable polymeric materials exemplified herein.
  • suitable polymeric materials can be prepared such that they possess a porous microstructure, ranging from solid compositions having microscopic voids dispersed sparsely therein to low density foams having more voids than substance.
  • suitable polymeric materials may range widely on the scale of degree of porosity.
  • polymers and resins having a porous microstructure to any degree of porosity referred to herein as "porous substances"
  • voids act as receptacles for gNO, and hence in the context of a substance having voids the term "sequestering” is regarded also as “accepting” or “containing” gNO in said voids.
  • the texture of the polymeric material can range from rigid to soft, and be elastic, plastic, pliant and resilient.
  • the degree of rigidity, elasticity, plasticity, pliability or resilience of suitable polymeric materials can vary with the specific design and application of an article, such as a medical device. The choice of characteristics for a selected medical device will be apparent to and within the ability of a one of ordinary skill in the art. As the aforementioned properties correlate to certain chemical properties such as crosslinking, such mechanical properties may also correlate with the capacity of a polymeric material to sequester or accept gNO.
  • the article includes at least a portion of a surface configured to sequester gNO.
  • the portion of the article can be defined by its volume
  • the amount of gNO sequestered in that portion can be quantified by ppm of gNO per volume unit.
  • the article includes at least a portion of a surface configured to sequester at least 1 ppm nitric oxide per cm 3 .
  • Figure 27 presents an illustration of charging device 300, wherein article 301 further includes at least a portion thereof made from a suitable polymeric material, represented by surface 372, and configured to sequester gNO readily and effectively.
  • the methodology described herein is highly beneficial for preparing a medical device having gNO sequestered therewithin.
  • the article to be charged with gNO is a medical device.
  • medical devices which can be impregnated with gNO beneficially, include catheters, tubing, endotracheal tubing, tampons, prosthetic devices, medical implants, artificial joints, artificial valves, needles, intravenous access devices, cannula, stents, biliary stents, cardiovascular stents, cardiac surgery devices, nephrostomy tubes, vascular grafts, infusion pumps, adhesive patches, sutures, fabrics, meshes, polymeric surgical tools or instruments, intubation devices, orthopedic surgery devices, pacemakers, endoscope components, dental surgery devices, veterinary surgery devices, bone scaffolds, hemodialysis tubing or equipment, blood exchanging and transfusion devices, implantable prostheses, bandages, ophthalmic devices and breast implants.
  • the medical device is an implantable medical device, including medical devices that are implanted transiently or permanently.
  • medical devices that are implanted transiently or permanently.
  • examples include an indwelling catheter or an intubation device such as a tracheal tube.
  • indwelling catheters include urinary catheters, central venous catheters, biliary vascular catheters, pulmonary artery catheters, peripheral venous catheters, arterial lines, central venous catheters, peritoneal catheters, epidural catheters and central nervous system catheters.
  • CAB is one of the major causes of nosocomial infections
  • sterilization of urinary catheters is highly desired.
  • Preparing urinary catheters the releasably sequester gNO thus achieves this goal (amongst other goals that are met by the described methodology).
  • the medical device is such that is made of any of the suitable polymeric materials described hereinabove (e.g., at least a portion of the medical device comprises a hydrophobic polymeric material with an intrinsic capacity to sequester gNO).
  • the medical device is a tampon.
  • gNO has been shown to be bacteriostatic and bactericidal, and has been shown herein to be effective in eradicating microorganisms causing bacterial vaginitis.
  • gNO-charged tampons can serve also as a therapeutic medical device by delivering a medication (gNO) to an infected bodily site (vagina).
  • tampons are made essentially from fibers of less hydrophobic and/or even hydrophilic polymers and other substances, impregnating tampons with gNO can advantageously be effected under reduced pressure and/or using extended gNO exposure times and/or at higher gNO concentrations compared to an article made from hydrophobic polymers.
  • tampons are designed for absorbing liquids, their microstructure is essentially rich in voids and crevices which can be charged and filled with gNO, sequester and contain gNO, and effectively and controllably release sequestered gNO therefrom. Additional features related to gNO-impregnated tampons are discussed in further detail hereinbelow.
  • the article used in the methodology described in these embodiments of present invention encompasses bare (unwrapped) articles, coated articles, and wrapped articles, as long as the coating and/or wrapping does not impair the process of gNO loading to an extent that is not mitigated by means within the process (namely by process-controllable factors that can increase gNO uptake).
  • the process presented herein can be effected with a packaged article as well as with a bare (unpackaged) article.
  • the package is a gas-permeable package.
  • gas-permeable refers to a chemical attribute of a material, which allows gas molecules to pass therethrough by flux, conveyance, diffusion or transportation.
  • Gas-permeability also refers to a mechanical attribute of an article made from any material in the sense that the material can be shaped, designed, manufactured and processed to be impermeable, permeable and anywhere between the extremes (semi-permeable).
  • a material can be made porous or caulked, shaped thin or thick, processes glassy or rough and the likes, all of which are examples of mechanical propertied that affect gas-permeability.
  • semi-permeability in the sense of rate of permeation is regarded as gas- permeable
  • semi-permeability in the sense of selectivity towards one gas species to another is regarded as gas-permeable if it can permeate gNO, and gas-impermeable if it does not allow gNO to permeate therethrough.
  • Non-limiting examples of gas-permeable materials include low density polyethylene (LDPE), high density polyethylene (HDPE), medical grade paper, polycarbonate (PC), polyester, polyvinyl chloride (PVC), polyvinylidene chloride, perfluoroalkoxy (PFA), acrylobutylstyrene, polypropylene (PP) polytetrafluoroethylene (PTFE), polyacrylate, acrylic, polycarbonate, polyacrylonitrile-butadiene-styrene, polymethylpentene (PMP), polyacetal, polystyrene (PS) or the like.
  • LDPE low density polyethylene
  • HDPE high density polyethylene
  • PC polycarbonate
  • PVC polyvinyl chloride
  • PFA polyvinylidene chloride
  • PFA perfluoroalkoxy
  • acrylobutylstyrene polypropylene (PP) polytetrafluoroethylene (PTFE)
  • Pacrylate acrylic, polycarbonate, poly
  • the article can be housed in (e.g., enclosed by) a gas-permeable package when disposed in the chamber during the process of generating a reduced pressure and charging the article with gNO; wherein the presence of the gas-permeable package may or may not affect one or more parameters of the process.
  • a gas-permeable package when disposed in the chamber during the process of generating a reduced pressure and charging the article with gNO; wherein the presence of the gas-permeable package may or may not affect one or more parameters of the process.
  • the end result of the process would be a gNO-charged article that includes a gNO-charged gas-permeable package.
  • the process described herein is used for providing an article as described herein, with or without a gas-permeable package, within a non-gas permeable enclosure, in order to maintain the gNO-containing environment within the package for prolonged time and/or under various conditions.
  • non-gas permeable or the equivalent “gas-impermeable”, as used herein, refers to an attribute of a substance which is capable of preventing the passage of gas molecule therethrough by flux, conveyance, diffusion or transportation. In some embodiments of the invention, these terms refer to impermeability of gNO, however, this term is also used to indicate impermeability of the non-gas permeable to other gases such as water vapors and oxygen.
  • gNO has a lower diffusion cross-section then water vapors or oxygen, a gNO-impermeable substance forming the non-gas permeable container or package will also be impermeable to water vapors and oxygen, and that a substance that is impermeable to water vapors and oxygen may still be permeable to gNO.
  • gas-impermeable packaging examples include, without limitation, glass, glassy ceramics, metals, metallic foils, metallic -plastic composite foils and the likes and a combination thereof as composites or as parts in a complete gas-impermeable package.
  • the gas-impermeable material for packaging can also comprise more than one layer of a polymer, a metal, a resin or a plastic, and in some examples the packaging can comprise a plastic -backed metallic foil, such as used in many air-tight packaging.
  • a gas-permeable material can be combined with a gas- impermeable material to form a composite gas-impermeable package.
  • a gas-impermeable package is also referred to interchangeably as a non-gas permeable enclosure or gas- impermeable enclosure.
  • the article is housed in an unclosed (unsealed, open) sealable gas-impermeable package (a non-gas permeable enclosure that can be closed and sealed) when disposed in the chamber and during the generation of reduced pressure and charging it with gNO.
  • an unclosed (unsealed, open) sealable gas-impermeable package a non-gas permeable enclosure that can be closed and sealed
  • the process is carried out essentially as described hereinabove, and at the end of the process the non-gas permeable enclosure is closed and sealed so as to constitute an intact and complete gNO barrier with respect to the gNO in the gNO- containing environment and the gNO-sequestering article enclosed therein, insulating the article from ambient environment.
  • the sealable gas-impermeable package for insulating a gNO-sequestering article enclosed therein can further include a desiccant, a humidity indicator and a gNO indicator, as these are described herein, for monitoring the gNO-charging process and to indicate post-sealing integrity of the package and post-sealing level of gNO enclosed therein after manufacturing.
  • a desiccant for monitoring the gNO-charging process and to indicate post-sealing integrity of the package and post-sealing level of gNO enclosed therein after manufacturing.
  • These optional attachments may be placed inside the package and behind a transparent portion of the package so as to allow a visual signal generated therein to be detected without opening the sealed package.
  • the indicators may be designed to form a part of the gas-impermeable package such that the inner side is in communication with the inner environment and can interact therewith while the outer side of the indicator maintains gas-impermeability towards the outside ambient environment.
  • articles having gNO sequestered therein are provided. Such articles are advantageously characterized as comprising at least 1 ppm per cm 3 nitric oxide sequestered therewithin, optionally at least 200 ppm per cm 3 nitric oxide sequestered there within, and further optionally from 1 ppm to 20,000 ppm per cm 3 .
  • the gaseous nitric oxide sequestered in the article is releasable is an aqueous solution during a time period that ranges from 1 hour to 1 month.
  • the rate of release of NO depends on a variety of parameters, including, for example, the affinity of NO to the material composing the article (the material in which gNO is sequestered), the amount of sequestered gNO, the position of gNO within the article (being an external or internal surface), as well as the conditions at which gNO is released.
  • the amount of gNO charged into the article can be pre-determined, so as to impart a desired release rate of gaseous NO therefrom.
  • urine catheters are typically transiently implanted in patients for a period of from a few hours to a few days.
  • Such articles would therefore be loaded by a process that utilizes conditions for high loading of NO (e.g., longer duration of exposure to NO-containing environment).
  • Tracheal tubes are typically utilized for a few hours, yet require high load of gNO since are typically used during surgery or other emergency cases.
  • Tampons are intended to be replaced every few hours.
  • the amount of gNO sequestered in the articles described herein can be readily measured by measuring the amount of nitrites and/or nitrates, as gNO degradation products, in an aqueous solution in which the gNO-sequestering article is placed. Determining the amount of nitrites and nitrates is exemplified in Examples section that follows. Other methods for quantitatively determining nitrites and nitrates will be recognized by a person skilled in the art and are also contemplated herein.
  • the article disclosed herein is further characterized as being substantially devoid of humidity and/or oxygen.
  • the article described herein is characterized by no more than 25 % of humidity, no more than 20 % humidity, no more than 15 % humidity, no more than 10 %, 5 %, 4 %, 3 %, 2 %, 1 %, 0.5 %, 0.1 %, 0.05 %, 0.01 %, 0.005 %, 0.001 % humidity, and preferably by 0 % humidity.
  • the "%" represents weight percentages from the gaseous environment within the article.
  • the article described herein is characterized by no more than 20 % of oxygen, no more than 15 % oxygen, no more than 10 %, 5 %, 4 %, 3 %, 2 %, 1 %, 0.5 %, 0.1 %, 0.05 %, 0.01 %, 0.005 %, 0.001 % oxygen, and preferably by 0 % oxygen.
  • the "%" represents weight percentages from the gaseous environment within the article.
  • the article comprises no more than 1 ppm of oxygen and/or humidity.
  • the article comprises no more than 1 ppb of oxygen and/or humidity.
  • an amount of reactive species other than nitric oxide, as defined herein, in the article is lower than 1 ppm per cm 3 of the article, preferably lower than 1 ppb (part per billion) per cm 3 of the article. In some embodiments, an amount of reactive species other than nitric oxide, as defined herein, in the article, is lower than 1 ppm of the entire article, preferably lower than 1 ppb (part per billion) of the entire article.
  • the article may further comprise an enclosure (e.g., a non gas-permeable enclosure).
  • an enclosure e.g., a non gas-permeable enclosure.
  • the enclosure comprises a gaseous nitric oxide- containing environment.
  • the environment is an ambient environment (e.g., has an ambient pressure).
  • the enclosure contains a gaseous NO-containing environment with a concentration of NO therein that is sufficient to sterilize the interior of the package and/or the article, as is further discussed hereinunder.
  • the article and/or enclosure can further comprise a desiccant disposed within the enclosure, and/or a nitric oxide indicator disposed within the enclosure, as is further discussed in detail hereinunder.
  • an article having sequestered therewithin at least 1 ppm gaseous nitric oxide which is advantageously characterized as comprising less than 1 ppm, or less than 1 ppb, per cm 3 of the article, reactive oxygen-containing and/or nitrogen-containing species other than nitric oxide.
  • the article can be further characterized by any of the features described hereinabove.
  • the article is a medical device, as described herein.
  • the article is a tampon.
  • tampon refers to a medical device in the form of a plug made from a mass of absorbent materials which is inserted into a wound or a body site to absorb exuded fluids, such as blood.
  • tampons are regarded officially as medical devices in many courtiers around the world, and according to the United States Food and Drug Administration, tampons are a Class II medical device.
  • tampon describes tampons designed to be inserted into the vagina during menstruation to absorb the flow of menstrual fluid.
  • the tampon can be a commercially available tampon of any type, composition, absorption rate, size and/or blend.
  • one or more of the polymeric materials used for forming a tampon have a gaseous NO sequestered therewithin, and the tampon is made from these raw materials.
  • Tampons are typically made from cotton, rayon and blends thereof, and are available in different sizes for various conditions and absorbing rates. Tampons may include an applicator, which is a polymeric tube sheathing the absorbent plug for facilitating its insertion into the vagina.
  • an applicator which is a polymeric tube sheathing the absorbent plug for facilitating its insertion into the vagina.
  • Both cotton and rayon, comprising the major and absorbing part of a tampon, are cellulosic fibers, which are regarded in the context of embodiments of the present invention as hydrophilic polymers.
  • tampons releasably sequestering gNO are highly advantageous, particularly in the context of prevention and/or treatment of vaginal medical conditions such as vaginal infections.
  • the present inventors have successfully prepared tampons having sequestered therein gaseous NO and have shown that such tampons exhibit both an antimicrobial effect and a self- sterilization effect (by e.g., preventing bacterial growth, biofilm formation and/or bacterial growth within a biofilm).
  • a tampon having sequestered therein gaseous nitric oxide.
  • an amount of the sequestered gaseous nitric oxide ranges from 1 ppm to 200 ppm per cm 3 of the tampon.
  • an amount of the sequestered gaseous nitric oxide ranges from 1 ppm to 200 ppm of the tampon.
  • an amount of sequestered gaseous nitric oxide is 200 ppm per cm 3 .
  • an amount of the sequestered gaseous nitric is 200 ppm of the tampon.
  • Higher load of gaseous nitric oxide is also contemplated (e.g., 300 ppm, 400 ppm, 500 ppm, 1,000 ppm and up to 20,000 ppm).
  • a process for preparing a tampon having sequestered therein gNO which is generally effected by exposing the tampon to gNO-containing environment.
  • the process of preparing a gNO-sequestering tampon includes exposing the tampon to gNO-containing environment by placing the tampon in a chamber and filling the chamber with the gNO-containing environment, wherein the exposure is subject to variable exposure time periods and concentration of gNO in the gNO-containing environment for obtaining a predetermined gNO-load in the gNO-charged tampon.
  • the process of preparing a gNO-sequestering tampon may further include, subsequent to placing the tampon in the chamber, sealing the chamber and generating a reduced pressure (vacuum) in the chamber, prior to filling the chamber with the gNO-containing environment.
  • the depth of the vacuum and the duration of the time of maintaining the chamber under reduced pressure serve as additional means to control the amount of gNO sequestered in the tampon.
  • the process of preparing a gNO-sequestering tampon is effected without generating reduced pressure.
  • Such a process may comprise, prior to filling the chamber with NO, reducing the humidity within the tampon and/or within the chamber.
  • tampons comprise hydrophilic polymers, they require longer exposure to higher concentrations of gNO and optionally more negative pressure applied prior to exposure to gNO, compared to articles of similar mass and shape comprising hydrophobic polymers.
  • the principles of the process for impregnating tampons with gNO follow the same guidelines as the principles of impregnating other packaged or unpackaged articles with gNO, as described in detail herein.
  • tampon When subjected to exposure to gNO-containing environment, the tampon can be either wrapped or unwrapped.
  • tampons may also include an applicator which can be made from hydrophobic polymers as well as other polymers, and can be regarded as a "gNO-sequestering surface" or a "surface", as the term is defined hereinabove, and serves as an additional gNO-sequestering component in the gNO-sequestering tampon.
  • the term "tampon” also encompasses tampons sheathed in an applicator.
  • tampons can be charged with gNO as bare tampons or packaged tampons wrapped with a gas-permeable package. Both forms of tampons can also be charged with gNO while housed in a gas-impermeable enclosure, as described hereinabove for charging a general article with gNO.
  • the end result of such a process can be either a gNO-sequestering tampon, a gNO-sequestering tampon packed in a gas-permeable package, and/or a wrapped or bare gNO-sequestering tampon packaged in a gas-impermeable packaging material, either individually or as a plurality of gNO-sequestering tampons.
  • gNO-sequestering tampon is self-sterilizing, which adds a notable benefit for this medical device for insertion into a bodily site, however, a gNO-sequestering tampon may also serve for medicinal therapy of various illnesses associated with the vagina.
  • a gNO-sequestering tampon which is identified for use in treating or preventing (or preventing the recurrence of) a vaginal medical condition.
  • a method of treating a vaginal medical condition the method being effected by placing in the vagina of the subject the gNO-sequestering tampon as presented herein.
  • Such a method comprises intravaginal administration of gaseous NO.
  • a method of treating a vaginal medical condition which is effected by intravaginal administration of gaseous NO.
  • intravaginal administration of NO is effected by a tampon having (releasably) sequestered therein gaseous NO, as described herein.
  • a tampon having gaseous NO sequestered therein for use in a method of treating a vaginal medical condition, by intravaginal administration of gaseous NO.
  • a method of delivering gaseous nitric oxide into a vagina which is effected by placing in a vagina of a subject in need thereof a tampon having gaseous NO sequestered therewithin, as described herein.
  • Exemplary vaginal medical condition include, without limitation, bacterial vaginitis (BV), toxic shock syndrome (TSS), toxic shock-like syndrome (TSLS), streptococcal toxic shock syndrome (STSS), vulvovaginal candidiasis (VVC), chronic or persistent yeast infections (RVVC), a sexual dysfunction, a female reproductive system related disorder and a post-surgery vaginal related condition.
  • BV bacterial vaginitis
  • TSS toxic shock syndrome
  • TSLS toxic shock-like syndrome
  • STSS streptococcal toxic shock syndrome
  • VVC vulvovaginal candidiasis
  • RVVC chronic or persistent yeast infections
  • gaseous nitric oxide in the treatment or prevention of a vaginal medical condition.
  • the gaseous nitric oxide is used in the form of a gNO-sequestering tampon, such as described herein.
  • the present inventors have further contemplated using a methodology of impregnating gaseous NO for providing sterilized and self-sterilizing packages of articles.
  • a process of preparing a packaged article wherein the packaged article comprises a gas-permeable package, the process comprising exposing a packaged medical device to a gaseous nitric oxide-containing environment, as described herein.
  • exposing is effected by placing the packaged article in a chamber; and filling the chamber with a nitric oxide-containing environment.
  • the process is effected by generating a reduced pressure in the chamber, prior to filling the gNO-containing environment, as described in detail hereinabove.
  • filling is effected by flowing nitric oxide into the chamber, as described hereinabove.
  • the process further comprises, prior to filling the chamber, absorbing humidity from the package and/or chamber. In some embodiments, absorbing humidity is effected so as to reduce humidity by at least 50 %, as described hereinabove.
  • filling is effected such that an ambient environment (e.g., ambient pressure) is provided within the package.
  • an ambient environment e.g., ambient pressure
  • Such a process results in a packaged article having gaseous nitric oxide sequestered within the article and within the package.
  • Such a process can be advantageously utilized for sterilizing articles such as medical devices which are packaged in a gas-permeable package, without the need to open the sealed package.
  • the package can comprise a plurality of articles, individually packaged articles, and a package that comprises individually packaged articles.
  • the article is a medical device, as described herein.
  • a process of preparing a packaged article wherein the packaged article comprises a non-gas permeable enclosure.
  • the process according to this aspect of embodiments of the invention is effected by positioning an article (e.g., an intact article) within a non-gas permeable enclosure, to thereby obtain a non gas-permeable enclosure having the article disposed therewithin; exposing the enclosure to a gaseous nitric oxide-containing environment, so as to introduce into the enclosure the nitric oxide-containing environment; and
  • the (intact) article placed within the enclosure can be packaged with a gas- permeable package or be unpackaged, as described hereinabove.
  • Exposing the enclosure to a gaseous nitric oxide-containing environment is effected, according to some embodiments, following the guidelines described herein, with or without generating reduced pressure in the chamber.
  • the nitric oxide-containing environment comprises at least 0.02 % nitric oxide.
  • Such a process results in an article packaged in a non-gas permeable package, wherein the article has gaseous nitric oxide sequestered therewithin and/or the package comprises a nitric oxide-containing environment.
  • the package contains nitric oxide in an amount sufficient to sterilize an interior of the enclosure as well as to sterilize the article.
  • Such an amount is at least 1 ppm nitric oxide, and range from 1 ppm to 200 ppm nitric oxide. In some embodiments, the amount is higher than 200 ppm, as described hereinabove.
  • substantially encompasses a majority of a population of microorganisms, namely, at least 80 %, 85 %, 90 % 95 %, 98 %, 995, 99.9% and optionally 100 % of the microorganism population.
  • the process presented herein can be carried out so as to manufacture an article impregnated with gNO and encased in a non-gas permeable package as follows:
  • the package may include any one or more of the optional humidity indicator, gNO indicator or desiccant;
  • the process is carried out essentially as described hereinabove with an article which is not housed in a non-gas permeable package, and the process further includes, subsequent to opening the chamber and retrieving the gNO-charged article, a step of placing the gNO-charged article in a non-gas permeable package and sealing the package.
  • a bare article and an article in a gas-permeable wrapping, coating and/or packaging is regarded essentially the same in the context of the gNO-charging process, since a gas-permeable wrapping, coating and/or packaging does not substantially preclude or interfere with the process.
  • a package which comprises a material configured to form an enclosure; an article disposed within the enclosure; and a gaseous nitric oxide- containing environment within the enclosure.
  • the package is a non-gas permeable package.
  • the enclosure is a sealed enclosure.
  • the environment within the enclosure is an ambient environment, as described herein.
  • the article in the package has gaseous nitric oxide sequestered therein.
  • the package as described herein can further include desiccants, nitric oxide indicators and other components, as described herein.
  • the article is preferably a medical device, such as an indwelling catheter, an intubation device or a tampon, as described herein.
  • Figures 31A-B present schematic illustrations of a package according to some embodiments of the invention, such as a package 200, comprising non-gas permeable package 203 configured to be impermeable to gNO and optionally comprising seal 205 ( Figure 3 IB), medical device 201 disposed within non- gas permeable package 203, gNO-containing environment 206 engulfing medical device 201 within non-gas permeable package 203 and comprising a predetermined concentration of gNO capable of sterilizing the interior of non-gas permeable package
  • charging device which comprises:
  • a chamber comprising an inlet for receiving a gaseous nitric-oxide containing environment and an outlet for releasing the gaseous nitric-oxide containing environment;
  • Such a chamber can be utilized for practicing any of the processes described herein.
  • the chamber may be coated from within with a protective surface coating suitable for preventing gNO from reacting with the chamber's material.
  • a protective surface coating suitable for preventing gNO from reacting with the chamber's material.
  • Such protective coating may include, as non-limiting examples, glass, chromium, stainless steel and other gNO-resistant substances and mixtures thereof.
  • the device may further comprise a nitric oxide indicator configured to undergo a color change suitable for visual assessment of whether the article has been exposed to the nitric oxide.
  • a gNO indicator comprises one or more gNO-sensitive substances that can produce a detectable signal when exposed to a certain or any level of gNO in an environment.
  • the detectable signal may be a color change of the gNO-sensitive substance which occurs upon exposure to gNO.
  • a gNO indicator is suitable for a visual confirmation for sufficient exposure of the article to gNO.
  • Exemplary gNO-sensitive substances include dyes such as, for example, 4- amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM).
  • gNO-indicators are contemplated in the context of embodiments of the invention, such as other chemical indicators, electronic indicators, off-line indicators (a sample for measuring the exposure thereof to gNO at a site unrelated to the exposure site) and the likes.
  • a desiccant can be placed in the chamber to absorb humidity, as illustrated in Figures 26-30 (desiccants 202, 302, 402).
  • the desiccant can be disposed within the chamber and be configured to absorb humidity from the ambient environment.
  • Suitable desiccants include, but are not limited to, Drierite ® , silica gel, calcium sulfate, calcium chloride, montmorillonite clay, molecular sieves, etc.
  • the desiccant can reduce the amount of humidity in the ambient environment by about 75 % to about 100 %.
  • the charging device may also include at least one humidity indicator (see, indicator 208, 374 and 474 in Figures 32, 27 and 29 respectively) configured to indicate moisture saturation of the desiccant or humidity level in the chamber.
  • a humidity indicator for indicating desiccant saturation include cobalt chloride (C0CI2), which undergoes a color change from blue (anhydrous state) to purple (( ⁇ ( ⁇ 3 ⁇ 4 ⁇ 2 ⁇ 2 ⁇ ) to pink(Co(H20)6]Cl2) as the absorption of water increases.
  • Devices which are configured for practicing any of processes as described herein while generating reduced pressure in the chamber further comprise an outlet for generating a reduced pressure in the chamber.
  • a package enclosing (or housing) the article is further included within the charging device.
  • the package is as non-gas permeable enclosure, utilized according to embodiments of the invention to provide a packaged gNO-sequestering article as described herein.
  • FIG. 26 is a schematic illustration of an exemplary charging device 300, according to some embodiments of the present invention, which is fitted with an inlet 345 for receiving gNO within chamber 337 to form gaseous nitric oxide-containing environment 306, valve 315 for closing outlet 345, an outlet 326 for releasing ambient environment or gaseous nitric oxide-containing environment from chamber 337, valve 336 for closing outlet 326, an article 301 disposed within chamber 337, and an optional moisture scavenger in the form of desiccant 302 disposed within chamber 337 and configured to absorb an amount of humidity from the within chamber 337.
  • inlet 345 can be used to connect to an external source of negative pressure, e.g.
  • FIGS. 27-29 present schematic illustrations of exemplary charging devices similar with respect to the chamber, the environment, the inlet, the outlet, the valves and the article, to the charging device presented in Figure 26.
  • Figure 27 is a schematic illustration of an exemplary charging device 300, as presented in Figure 26, wherein chamber 337 also includes a surface coating 370 which protects the material of chamber 337 from chemically interacting with gNO-containing environment 306, a gNO indicator 371 configured to undergo a color change when article 301 has been exposed to gNO in environment 306, and humidity indicator 374 for signaling exposure of article 301 to humidity.
  • a surface coating 370 which protects the material of chamber 337 from chemically interacting with gNO-containing environment 306, a gNO indicator 371 configured to undergo a color change when article 301 has been exposed to gNO in environment 306, and humidity indicator 374 for signaling exposure of article 301 to humidity.
  • FIG 28 is a schematic illustration of an exemplary charging device 400, according to some embodiments of the present invention, which is fitted with an inlet 445 for receiving gNO within chamber 447 to form gaseous nitric oxide-containing environment 406, valve 415 for closing outlet 445, an outlet 426 for releasing ambient environment or gaseous nitric oxide-containing environment from chamber 447, valve 436 for closing outlet 426, and a non-gas permeable package 473 disposed within chamber 447 and housing article 401.
  • inlet 445 or outlet 426 can be used to connect to an external source of negative pressure, e.g. a vacuum pump (not shown), for generating a reduced pressure in chamber 447.
  • Figure 29 is a schematic illustration of an exemplary charging device 400, as presented in Figure 28, wherein chamber 437 includes surface coating 470 which protects the material of chamber 437 from chemically interacting with gNO-containing environment 406, a gNO indicator 471 configured to undergo a color change when article 401, having gNO-sequestering surface 472 and housed within non-gas permeable package 473 equipped with seal 490, has been exposed to gNO in environment 406, and humidity indicator 474 for signaling exposure of article 401 to humidity.
  • surface coating 470 which protects the material of chamber 437 from chemically interacting with gNO-containing environment 406, a gNO indicator 471 configured to undergo a color change when article 401, having gNO-sequestering surface 472 and housed within non-gas permeable package 473 equipped with seal 490, has been exposed to gNO in environment 406, and humidity indicator 474 for signaling exposure of article 401 to humidity.
  • Figures 26-29 present exemplary charging devices fitted with only a single inlet and a single outlet
  • the charging device may include a plurality of inlets and a plurality of outlets, as illustrated in Figure 30.
  • Figure 30 presents a schematic illustration of an exemplary charging device 500 having a chamber 137, inlet 125 with valve 11, inlet 135 with valve 2, and inlet 145 with valve 15, inlets which may share a common tube 23 with valve 19 that connects to the chamber 137; outlet 126 with valve 36 and connect to purge flow rotomoter 46, outlet 136 with valve 41 and connect to purge flow rotomoter 45, outlets which may share a common tube 33 that connects to the chamber 137.
  • Separate inlets 125, 135, 145 and outlets 126, 136 allow for gases (e.g. gNO, carrier gases) to enter and exit the chamber 137 at separate locations.
  • gases e.g. gNO, carrier gases
  • Each of inlets 125, 135, 145 may also allow a vacuum creating mechanism (not shown) to have separate access to the chamber 137 for generating reduced pressure therein.
  • each inlet 125, 135, 145 may be connected to a different source of gases or vacuum.
  • inlet 145 may serve as a vacuum port
  • inlet 125 may serve as an inlet for gNO or nitrogen gas
  • inlet 135 may serve as a purging gas inlet for a carrier gas such as nitrogen gas, helium, argon, or any combination thereof.
  • the vacuum port 145 in Figure 30 may be used to create a vacuum within chamber 137 so that the gNO introduced to chamber 137 does not react with any gases or moisture already contained within chamber 137 or within the article disposed therein (not shown).
  • Chamber 137 may be pressured to any suitable pressure, for example, once the article has been disposed therein and chamber 137 has been sealed and evacuated substantially from the ambient environment therein, chamber 137 may be pressurized to 50 psig of gNO or of gNO mixed with a carrier gas such as helium, argon, nitrogen gas, and any combination thereof, helping to stabilize gNO.
  • Separate inlets 126, 136 in Figure 30 may be connected by tubes to two rotometer kits 45, 46 which are flow meters that indicate flow rate and can be operated in parallel.
  • the gaseous nitric oxide (gNO) sequestering articles presented herein can be prepared (charged with gNO) and be ready for use well in advance and kept in storage, or be prepared just prior to use, depending on the use, conditions and other preferences.
  • the article can be kept for extended lengths of time in a sealed chamber which is designed for charging its content with gNO, and then, prior to use, the device and article can be charged with gNO.
  • a charging device which includes a sealed chamber having a reduced pressure therewithin; and an article, as described herein (e.g., a medical device), disposed within the chamber.
  • Such a chamber can be utilized, for example, for charging the article with gNO, as described herein, and thus can further comprise an inlet configured for receiving a gaseous nitric oxide-containing environment and an outlet for releasing said gaseous nitric oxide-containing environment.
  • such a chamber further comprises an outlet used for generating the negative pressure within the chamber.
  • Such a chamber can further comprise a non-gas permeable enclosure in which the article is positioned, as described herein.
  • the device can further comprise desiccants and NO-indicators as described herein.
  • nitric oxide sequestering article is intended to include all such new technologies a priori.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • Articles are exposed to nitric oxide (Airgas Specialty Gases, Chicago, IL) in nitrogen (N 2 ) or argon (Ar) as a carrier gas (at a concentration ranging from 800 ppm to 20,000 ppm, unless otherwise indicated) for a period of 1-600 minutes or overnight (10- 24 hours) in a chamber, such as a stainless steel chamber.
  • a chamber such as a stainless steel chamber.
  • Articles are placed in a chamber and nitric oxide is introduced into the chamber at a flow rate of from 0.5 liter/minute to 5 liter/minute (unless otherwise indicated), depending on the parameters and intended use of the device to be impregnated.
  • NO impregnation of articles within a gas-permeable package is generally effected by placing the packaged article in the chamber and exposing the packaged article to NO, as described herein.
  • Preparation of gNO-impregnated packaged articles with a non-gas permeable package is generally effected by placing the article within an encasement made from the non-gas permeable packaging material while keeping the encasement open, exposing the article and the encasement to gNO as described herein and thereafter sealing the encasement containing the device within.
  • nitric oxide Airgas Specialty Gases, Chicago, IL
  • nitrogen N 2
  • argon Ar
  • a sealed chamber such as a stainless steel chamber.
  • Articles are placed in the chamber and the chamber is sealed.
  • a reduced (negative) pressure e.g., of -10 PSI; about -0.7 atmospheres
  • a vacuum pump connected to the outlet of the chamber for a period of e.g., 1- 15 minutes.
  • the pump is stopped and the outlet valve closed to maintain the negative pressure inside the chamber.
  • the inlet valve connected to a nitric oxide-containing cylinder is opened to allow the nitric oxide in the carrier gas to flow in and fill the chamber.
  • the gas influx is stopped when a pressure of zero PSI is reached.
  • the gas inside the chamber is purged with air for at least 5 minutes through nitric oxide absorbing filters.
  • the chamber is opened and the exposed article is kept at room temperature.
  • nitric oxide Airgas Specialty Gases, Chicago, IL
  • nitrogen N 2
  • argon Ar
  • Nitric oxide has a half-life of a few seconds; therefore stable metabolites of nitric oxide (nitrates and nitrites) were measured using the Griess test [Green et al.,
  • nitric oxide ppm (46 x [N0 2 ]) x 0.65 x 10 ⁇ 3 . Each ⁇ ( ⁇ / ⁇ ) was multiplied by the molecular mass of nitrite (46 grams/mo 1). This value was converted to ppm of nitric oxide, taking into account the difference in molecular weight (MW) between nitrites and nitric oxide (1 :0.65 ratio), and the multiplication factor between grams and milligrams (10 3 ).
  • MW molecular weight
  • ATCC American Type Culture Collection
  • E. faecalis #29212 Staphylococcus saprophyticus #15305, Staphylococcus epidermidis #35984, E. Coli #25922, P. aeruginosa #14210, Acinetobacter baumanii #BAA-747, Candida albicans #14053 were used for the described studies.
  • Bacteria were grown to 0.5 McFarland standard, and 1-ml aliquots of these preparations each containing approximately 2.5 x 10 CFU/ml were stored in vials at - 70 °C. On the day of the experiments, the fresh stock was removed from the freezer, thawed, and 2 ml of media was added (Luria Broth (LB) for E.coli, P. aeruginosa, A. baumanii; Sabouraud Dextrose Broth for C. albicans; Brain Heart Infusion (BHI) for E. faecalis, S. saprophyticus, S. epidermidis). Cultures were further diluted with same media to 10 3 cuff/ml in volumes appropriate to the experimental conditions.
  • LB Lia Broth
  • BHI Brain Heart Infusion
  • Biofilm formation assays were performed as described in O 'Toole et al, 1998,
  • Tensile strength measurements were conducted using a TA.XTPLUS texture analyzer, (Texture Technologies Corp), and then analyzed with the Exponent 32 software (version 3.0.4.0). Treated and untreated 3-cm samples of the studies object were stretched at a constant distance using the texture analyzer, following manufacturer's protocols. The maximum force that was used to stretch a specific piece was measured, using a "Miniature tensile grip" probe, and total area under the curve of force as a function of time was thereby determined.
  • Hardness and Springiness measurements were obtained using a TA-XT2 Texture analyzer (Texture Technologies Corp), and analyzed with the XTRAD software (version 3.7). Hardness and springiness (or elasticity) were determined following manufacturers' protocols, and the methods provided by Zhu J.H. et al., 2009, Effect of Guar gum on the rheological, thermal and textural properties of soybean ⁇ -conglycinin gel, Int J Food Sci and TechnoL, 44: 1314-1322 and by Bourne M.C., Food Texture and Viscosity Second Edition: Concept and Measurement, 2002, Academic Press, New York. Hardness is defined as the peak force during the first compression cycle of the sample.
  • Springiness also referred to as elasticity, is an indicator of how much the sample structure is broken down by an initial compression under a controlled force.
  • the sample is pressed between two plates for a set distance (e.g. 8 mm from initial contact of the top plate with the sample) at a constant rate, such as 2.5 cm per minute, for two compression cycles.
  • a curve with the applied force increasing from baseline to a maximum value was generated where the maximum compression distance is reached, followed by a reversal and reduction of applied force to a baseline value, and was plotted as a graph of force versus time.
  • Springiness is defined as a ratio of the time (in seconds) recorded between the initial application of compression force to the maximum value for the second compression cycle (L2), divided by the time (in seconds) recorded between the initial application of compression force to the maximum value for the first cycle (LI).
  • area is the area under the curve of force/time for first peak [Rincker et al, 2007, Evaluating an objective method to measure fresh pork loin firmness, Meat Sci., 77:213- 219].
  • Folysil ® silicone Foley catheters (catalog no. AA61 18; Coloplast ® Corp. Minneapolis, MN, USA) (Folysil ® and Coloplast ® are registered trademarks of Coloplast A/S, Humlebaek, Denmark) were aseptically cut into 2-cm sections and inoculated by immersion in 2 ml of one of 10 3 CFU/ml, 10 5 CFU/ml, or 10 8 CFU/ml E. coli. inocula for 30 minutes.
  • Inoculated catheter sections were subsequently dosed with 20,000 parts per million of nitric oxide (at a flow rate of 30 cc/minutes) during 1, 5, 15, 30, 60 or 120 minutes, using the general procedure I described hereinabove.
  • Control catheter sections were not impregnated with nitric oxide, but were stored in sterile sealed vials.
  • nitric oxide -treated and untreated catheter segments were then immersed separately in 2 ml media and then incubated for 8 hours at 37 °C. Aliquots of the incubation media were plated and numbers of CFU counted. Bacterial loads were calculated as CFU per ml.
  • Figure 1 shows the results of dosing the medical device samples with nitric oxide for 1, 5, 15, 30, 60 or 120 minutes, following incubation in a bacterial suspension of 10 3 , 10 5 or 10 8 CFU/ml for 8 hours at 37 °C. The CFU/ml remaining in the suspension are shown with respect to dose time and starting culture CFU/ml.
  • Table 1 presents the colony formation following exposure of catheter segments to nitric oxide at various durations, wherein TNTC denotes "too numerous to count” or over 10 6 CFU/ml.
  • a commercially-available catheter such as a six-mm diameter Folysil ® silicone Foley catheters, (catalog no. AA61 18; Coloplast ® Corp. Minneapolis, MN, USA) is aseptically cut into 2-cm sections and inoculated by immersion in 2 ml of one of 10 3 CFU/ml, 10 5 CFU/ml, or 10 8 CFU/ml E. coli. inocula for 30 minutes.
  • Inoculated catheter sections are subsequently dosed with nitric oxide using the general procedure II described hereinabove. Control catheter sections are not impregnated with nitric oxide, but are stored in sterile sealed vials.
  • nitric oxide-treated and untreated catheter segments are then immersed separately in 2 ml media and incubated for 8 hours at 37 °C. Aliquots of the incubation media are plated and numbers of CFU counted. Bacterial loads are calculated as CFU per ml.
  • Catheters impregnated with NO using general procedure II are found to eradicate bacteria at all of the tested bacteria concentrations. Sterilization is thus achieved by NO-impregnation using general procedure II.
  • Figure 2 presents a plot of nitrite in ⁇ per cm of catheter relative to dosing time.
  • Table 2 presents the raw data of release of nitrates and nitrites from nitric oxide dosed catheter sample over varying dose times, wherein “Abs.” denotes absorbance, and “Abs average” denotes the average absorbance measured from duplicate sampling.
  • the amount of sequestered nitric oxide correlates to the time of exposure, showing a tendency for saturation at exposure periods over 240 minutes.
  • samples of each assay are then immersed in doubly-distilled water and after 1 hour are assayed for nitrates and nitrites, as described hereinabove, using the Griess reagent, and nitrites and nitrates concentrations are determined.
  • Folysil ® silicone Foley catheters were aseptically cut into 2- cm sections and dosed with nitric oxide overnight (10-24 hours) as described in general procedure I described hereinabove.
  • the catheter sections were immersed in suspensions of 10 3 CFU/ml E. coli for 1 minute. After transfer to PBS and incubation for 3 hours or 24 hours, no bacterial growth was observed in the aliquots prepared from nitric oxide sequestering catheter sections. However, significant bacterial growth (10 8 CFU/ml) occurred in the aliquots prepared from the control catheter.
  • Figures 3A-B show images of representative plates of control and nitric oxide - eluting samples ( Figure 3 A); and viable counts of CFU in triplicate ( Figure 3B), wherein the hatched bars represent data taken from control experiments, and the checkered bars represent data taken from the nitric oxide sequestering samples.
  • catheter section samples sequestering nitric oxide had absorbed sufficient nitric oxide to subsequently elute sufficient nitric oxide to demonstrate antimicrobial and sterilizing activity when exposed to bacterial cultures.
  • the catheter sections After a week of storage in air or water storage, the catheter sections are immersed in suspensions of 10 3 CFU/ml E. coli for 1 minute. After transfer to PBS and incubation for 3 hours or 24 hours, no bacterial growth is observed in the aliquots prepared from nitric oxide sequestering catheter sections.
  • Folysil ® silicone Foley catheters were dosed with 20,000 ppm nitric oxide at a flow rate of 30 cm/minute for 24 hours, according to the general procedure I described hereinabove.
  • urine (2 liters) was collected from male volunteers and placed in two 1 liter sterile plastic containers. Two ml of bacterial culture (10 2 CFU/ml) were inoculated into each container.
  • One container was circulated through nitric oxide-dosed catheter tubes and the control container was circulated using untreated catheter tubes.
  • Urine samples from each of the containers were re-circulated in a separate closed system using a flow rate of 1.5 ml/minute (Rabbit-Plus peristaltic pump; Rainin) through the catheters, for 24 hours at 37 °C.
  • Bacterial levels in the control and treated containers reached 10 8 and 10 7 CFU/ml after 24 hours, respectively.
  • the catheters were then aseptically removed, washed with sterile water, cut into 3-cm pieces, and processed as follows:
  • FIG. 4A shows the absorbance at 595 nm of the crystal violet that was attached to the catheter pieces after extraction with ethanol.
  • the hatched bars are data from the control, while the checkered bars are data from the impregnated catheters.
  • the error bars indicate standard deviations.
  • each piece was cut lengthwise, washed and put into 4 ml sterile water.
  • Figure 4B presents images demonstrating biofilm-embedded bacteria within the biofilms on control and impregnated catheters, grown in LB plates from the 1 ⁇ (bottom) and 10 ⁇ (top) of water surrounding a selected catheter piece.
  • Foley catheters are dosed with nitric oxide, according to either general procedure I or general procedure II described hereinabove.
  • urine (2 liters) is collected from male volunteers and placed in two 1 liter sterile plastic containers. Two ml of bacterial culture (10 2 CFU/ml) are inoculated into each container.
  • One container is circulated through catheter tubes dosed by nitric oxide using general procedure I, one container is circulated through catheters tubes dosed by nitric oxide using general procedure II, and the control container is circulated using untreated catheter tubes.
  • Urine samples from each of the containers are re-circulated in a separate closed system using a flow rate of 1.5 ml/minute (Rabbit-Plus peristaltic pump; Rainin) through the catheters, for 24 hours at 37 °C. Bacterial levels in the control and treated containers are then measured.
  • the catheters are then aseptically removed, washed with sterile water, cut into 3- cm pieces, and processed as follows:
  • a biofilm formation assay is performed as described hereinabove with each catheter piece.
  • samples Six-mm diameter Folysil ® silicone Foley catheters were cut into 3 cm sections, referred to as “samples” or “medical device samples", and were dosed with nitric oxide for 24 hours at a flow rate of 50 ml/minute as described in general procedure I hereinabove.
  • the physical properties of the samples were tested immediately upon NO dosing and after one-week storage either in water or in air (in a sealed plastic vial at room temperature (about 20 °C).
  • Table 3 presents the results of the discoloration assessment of NO-treated samples after 24-hour nitric oxide dosing, and of untreated control samples.
  • T-test of (-) denotes 'no statistical difference when P ⁇ 0.05'
  • (+) denotes 'statistical difference when P ⁇ 0.05'.
  • Table 4 presents the results obtained in the tensile strength measurements of NO-treated and untreated samples.
  • Table 5 presents the results obtained in the hardness and springiness measurements of NO-treated and untreated samples.
  • Table 6 presents the results of the discoloration assessment of NO-treated samples after 24-hour nitric oxide dosing and one week of storage in water, and of untreated control samples after one week of storage in water.
  • Table 7 and Table 8 present the results of the tensile strength and hardness- springiness measurements, respectively, as obtained for NO-treated and untreated samples after storage in water for one week.
  • Table 9 presents the results of the discoloration assessment of NO-treated samples after 24-hour nitric oxide dosing and one week of storage in air, and of untreated control samples after one week of storage in air.
  • Table 10 and Table 1 1 present the results of the tensile strength and hardness- springiness measurements, respectively, as obtained for NO-treated and untreated samples after storage in air for one week.
  • Folysil ® catheters (Coloplast ® AA61 18) were treated with nitric oxide (20,000 ppm) for 24 hours, at a flow rate of 1 liter/minute using general procedure I described hereinabove. After NO- treating, the packaged catheters were stored for one month at ambient temperature and humidity. Following storage, catheters were analyzed for color, tensile strength and hardness-springiness as described hereinabove.
  • Table 12 presents color measurements
  • Table 13 presents tensile strength
  • Table 14 presents results of the hardness-springiness measurements.
  • Exemplary catheter A is a silicone coated, latex based device: SILASTIC® by Dow Corning Corp., Midland, MI USA, Foley catheter 16 Fr. Bard® by C. R. Bard Inc., Murray Hill, NJ, USA.
  • Exemplary catheter B is a silicone coated, latex based device: bardia, Foley catheter, 2-way silicone elastomer coated, 20 Ch/Fr (6.7mm) by Bard®.
  • Exemplary catheter C is an all-silicone device: Catheter urethral drainage Foley 2 way 30 CC balloon all silicone, 18 Fr. Bard®.
  • Tables 15-17 The results are presented in Tables 15-17, whereas Table 15 presents color measurements, Table 16 presents tensile strength, and Table 17 presents results of the hardness-springiness measurements.
  • catheters such as six-mm diameter Folysil ® silicone Foley catheters and the catheters described in Example 1 1 hereinabove, are cut into 3 cm sections, and are dosed with nitric oxide either using general procedure I described hereinabove or using general procedure II as described hereinabove.
  • the physical properties of the samples are tested immediately upon NO dosing and after one -week storage either in water or in air (in a sealed plastic vial at room temperature (about 20 °C).
  • Color measurements and measurements of tensile strength, and hardness- springiness are performed after dosing and upon one week storage in air or water.
  • the packaged catheters are stored for one month at ambient temperature and humidity. Following storage, catheters are analyzed for color, tensile strength and hardness-springiness as described hereinabove.
  • a biofilm was established in each well of a 6-well plate and the time of treatment with nitric oxide gas for eradication of the biofilm was tested using the biofilm formation assay described hereinabove.
  • Figure 5 A shows the results of the 1, 10 and 100 ⁇ aliquots for E. Coli
  • Figure 5B shows the results of the 1 , 10 and 100 ⁇ aliquots for A. baumanii.
  • Control plates demonstrated bacterial growth proportional to the inoculum, while for all nitric-oxide treated plates (for either bacterial species), no colonies were observed.
  • Enterococcus faecalis #29212 E.f. #29212
  • Staphylococcus saprophyticus #15305 S.s #15305)
  • Staphylococcus epidermidis #35984 S.e. #35984
  • Escherichia coli #25922 E.c. #25922
  • Pseudomonas aeruginosa #14210 P.a. #14210
  • Acinetobacter baumanii #BAA-747 A.b. #BAA-747
  • Candida albicans C.a. #14053
  • the following bacterial clinical isolates were tested: Enterococcus faecalis (E.f), Staphylococcus aureus (S.a.), E. coli (E.c), P. aeruginosa (P.a.), Stenotrophomonas maltophilia (S.m.)
  • Figures 6 and 7 present the data obtained for selected bacterial strains obtained from the ATCC ( Figure 6) and for selected bacterial clinical isolates ( Figure 7) in nitric oxide-dosed vs. control catheter sections.
  • NO-dosed catheters are prepared as described in general procedure I or II hereinabove. Untreated catheters are used as control.
  • Catheter sections were immersed for 24 hours in 10 3 CFU/ml of each of the ATCC and clinical strains of bacteria or fungi described in Example 15 hereinabove. Catheter sections were washed twice with sterile distilled water and then aseptically transferred to agar plates containing the appropriate growth media for the particular strain. Each section was rolled once on the plate and the plate incubated at 37 °C overnight.
  • Table 18 presents the data inspected for the presence of bacteria on the surface of nitric oxide-treated catheter compared to untreated control samples, wherein sections denoted “2" exhibited the same growth density as that of the control; “ 1 " exhibited less growth density compared to control; and "0" exhibited no growth.
  • NO-dosed catheters are prepared as described in general procedure I or II hereinabove. Untreated catheters are used as control.
  • Catheter sections are immersed for 24 hours in 10 3 CFU/ml of a bacterial or fungal strain, washed twice with sterile distilled water and then aseptically transferred to agar plates containing the appropriate growth media for the particular strain. Each section is rolled once on the plate and the plate incubated at 37 °C overnight.
  • Urine was collected from male volunteer and placed into sterile vial (1.8 ml in each vial). 200 ⁇ of 10 3 of bacteria were inoculated into each vial to reach a final concentration of 10 2 CFU/ml.
  • NO-treated and untreated control catheters (2 cm length) were placed into each of the vials. Vials were incubated for 72 hours at 37 °C. Thereafter catheters were aseptically removed, washed with sterile water, cut into 1 cm pieces, and the formation of a biofilm was assessed as described hereinabove.
  • Figure 8 presents the relative biofilm formation on luminal surfaces of nitric oxide-sequestering catheter sections following 72 hour incubation in urine inoculated with 10 3 CFU/ml of the tested bacteria. As can be seen in Figure 8, biofilm formation was reduced for all tested strains where catheters were treated with nitric oxide before incubation in urine.
  • each lengthwise-cut piece of catheter was washed and put into 4 ml sterile water. Following sonication for 30 seconds, samples were plated on compatible agar plates in triplicates and incubated at 37 °C for 24 hours.
  • Figure 9 presented the data obtained for the growth of biofilm-embedded bacteria on nitric oxide-sequestering catheter sections following 72 hours incubation in urine inoculated with 10 3 CFU/ml bacteria.
  • Figures lOA-C present scanning electron micrographs Staphylococcus epidermidis biofilms on NO-sequestering and untreated control catheter sections, wherein untreated control sample at magnification 2.5k (inset at 20.0k) is shown in Figure 10A, untreated control sample at magnification 2.5k (right inset at 15k, left inset at 20k) is shown in Figure 10B and nitric oxide treated catheter sample at magnification 1.5k is shown in Figure IOC.
  • Figures 1 1A-C present scanning electron micrographs of A. baumanii (ATCC
  • Antiseptic urinary catheters have recently become commercially available and others are in the development stage.
  • the efficacy of both commercially available and emerging urinary catheter technologies in relation to their effects on bacteriuria caused by E. coli in vitro has been studied.
  • NOX the same catheter as control after impregnation with NO as described in general procedure I hereinabove;
  • BARDEX I.C silver-coated Anti-Infective Foley Catheter Ch/Fr 18 (Catalog No. 0165SI18; Bard, Inc. Covington, GA);
  • NFC Release NF - Nitrofurazonecoated silicone Foley catheter Ch/Fr 16 (Catalog No. 95216; Rochester Medical, Stewartville, MN).
  • E. coli bacterial culture was obtained from American Type Culture Collection (ATCC #25922).
  • Bacteria were grown to 0.5 McFarland standard. 1 ml aliquots of grown bacteria containing approximately 2.5 x 10 8 CFU/ml were stored at -70 °C. On the day of the experiments the fresh stock was removed from the freezer, thawed, and 2 ml of Luria Broth (LB) was added. Cultures were further diluted with LB to 10 3 CFU/ml.
  • E. coli culture (2 ml) at a concentration of 10 3 CFU/ml was added to a vial containing a 2 cm piece of catheter and incubated for 24 hours at 37 °C. After 24 hours, samples were vortexed and plated (using a 10 3 time dilution) on LB agar plates and were then incubated at 37 °C for 24 hours. The CFU was counted and calculated to represent the CFU per ml.
  • E. coli culture 200 ⁇ was added to 1.8 ml of urine (reaching a final concentration of 10 2 CFU/ml) containing a 2 cm section of a tested catheter and incubated at 37 °C for 72 hours.
  • Urine was collected in a sterile vial from a male volunteer on the day of the experiment. After 72 hours, samples were vortexed and plated onto LB agar plates and incubated at 37 °C overnight. The CFU were counted and calculated to represent the CFU/ml.
  • Catheter sections (2 cm) were immersed in tubes containing 3 ml of E. coli culture at 10 3 CFU/ml and incubated at 37 °C for 24 hours. After 24 hours, catheter sections were washed twice using 3 ml of sterile saline (0.9 % wt/v NaCl) and transferred aseptically to a LB agar plate. Each catheter section was rolled once on the plate then incubated at 37 °C for 24 hours.
  • Catheter pieces (2 cm each) were added, aseptically, to 1.8 ml of urine (collected as stated above) plus 200 ⁇ of E. coli culture at 10 3 CFU/ml and incubated at 37 °C for 72 hours. After 72 hours, catheter pieces were washed twice in 3 ml sterile saline then cut into two pieces of equal size, aseptically. One half of each catheter section was added to 1.5 ml of Crystal Violet dye in water (1 % wt/v) for 15 minutes then washed twice in 4 ml distilled water (dH20). Washed catheter pieces were then added to 2 ml of 95 % ethanol to relinquish crystal violet bound to the surface of the catheter.
  • the absorbance at 595 nm of each ethanol sample was measured using a spectrophotometer and used as an indicator of biofilm formation.
  • each catheter section was added to 2 ml of sterile distilled water (dF ⁇ O) and sonicated at a level of 5 for 30 seconds.
  • the extent of colonial E. coli released from each catheter was determined by plating each sample on LB agar plates and incubating at 37 °C overnight. The CFU were counted and calculated to represent the CFU per ml.
  • Figure 12 presents the comparative data obtained for E. coli growth in media containing pieces of the NOX, AG and NFC catheters versus media from control catheter, after immersion of the catheters for 24 hours in suspension comprising 10 3 CFU/ml and incubated for 24 hours at 37 °C.
  • Figure 13 presents the comparative data obtained for E. coli growth in urine after 72 hours exposure to pieces of NOX, AG and NFC and control catheters. Within each three compartment LB agar petri plate 1, 10, and 100 ⁇ of each sample were plated and incubated overnight at 37 °C.
  • the solution containing the AG catheter had a similar bacterial concentration to the control, 2.0 x 10 8 and 5.0 x 10 7 respectively, while the solutions containing the NOX and NFC catheters completely eradicated bacteria in urine.
  • Figure 14 presents the comparative data obtained for E. coli colonization on NOX, AG and NFC catheters versus control after immersion of catheters for 24 hours in suspension containing 10 3 CFU/ml of E. coli. In each LB Petri dish a catheter was rolled over the surface and then incubated at 37 °C overnight.
  • FIG 14 both the control and AG catheters contained extensive colonization on the surface of the catheter, whereas for catheters NOX and NFC, no bacterial colonization was observed.
  • Crystal Violet studies indicate the extent of biofilm formation on each catheter type. Each experiment was repeated three times with three replicates in each one of the experiments.
  • Figures 15A and 15B present comparative data of colonized biofilm formation on NOX, AG and NFC catheters versus control after 72 hours of incubation, demonstrated by absorbance at 595nm of the Crystal Violet that was attached to the catheter pieces after extraction of color with ethanol ( Figure 15A) and the bacterial growth from the biofilms from the different catheters ( Figure 15B).
  • the AG catheter showed the highest average absorbance indicating extensive biofilm formation.
  • the AG catheter rendered an average absorbance 8-10 times greater than all other catheter types, even the control.
  • the NOX catheter had half the absorption of the control and the NFC catheter had 40 % less. This indicates that both the NOX and NF catheters had less biofilm formation on their surface when compared to the control.
  • Comparative ANOVA test (p ⁇ 0.005) shows that all three tested catheters were significantly different from the control and NF and NOX were found to be significantly different from AG.
  • Figure 15B shows the amount of colonized bacteria released from the catheter surface.
  • the NOX and NFC catheters proved effective at eradicating bacteria embedded within the biofilm whereas the control and AG catheters produced bacteria concentration of 7.5 xlO 4 and 9.8 x 10 2 CFU/ml, respectively.
  • NO impregnated catheters are shown to be comparable to antimicrobial coated urinary catheters in their level of antimicrobial activity. NO coated catheters prevented E. coli growth in urine for 72 hours, same as the NF catheters.
  • a whole pack of individually-packaged Tracheal Tubes was placed in a NO chamber as described herein.
  • the chamber was subjected to a reduced pressure (- 10 psi) atmosphere for 5 minutes and was thereafter filled up with 20,000 ppm NO during 16 hours, following general procedure II described hereinabove.
  • a NO-impregnated tube was thereafter cut into 1 cm sections which were put into 2.5 cm of dd3 ⁇ 40 in screw-capped vials.
  • NO release from the NO-impregnated tube was assayed for nitrates and nitrites, as described hereinabove, using the Griess reagent, during 1 week.
  • Table 19 presents the compositions of the tampons selected for testing in this example.
  • Tampons were put in a petri dish in their individual packaging and impregnated with nitric oxide using a 20,000 ppm cylinder, using general procedure I described hereinabove. Tampons were charged overnight (12-16 hours) at a flow rate of 0.05 liter/minute. Control tampons were used directly out of their packaging.
  • Nitric oxide impregnated tampons were removed from their individual wrappers and cut in half into equally weighted pieces. Each piece was placed inside a separate 50 ml vial to which 25 ml of distilled water (dF ⁇ O) was added. At time points of 10, 30, 60, and 120 minutes, nitrites were measured using Griess test, using absorbance at 543 nm, essentially as described hereinabove. The concentration of nitrites was determined using a standard curve. The results are presented in Table 20 below.
  • Figure 17 presents a bar-graph showing the amount of nitric oxide released after 30 minutes from four exemplary NO-impregnated tampons, as measured by the respective total amount of nitrites released from the tampons impregnated with nitric oxide, wherein the letters under each bar denote the tampon type presented in Table 19 hereinabove.
  • Figure 18 presents comparative plots showing the accumulated of nitric oxide production during the first 5 hours for four types of NO-impregnated tampons, as measured by the respective total accumulation of nitrites in water produced from tampons impregnated with nitric oxide, wherein nitrites released were calculated per 1 tampon.
  • the difference in release rates is assumed to be a function of tampon composition, penetration of NO through the packaging, absorption capacity, and other substance related factors. All four tampons tested reached a peak in their respective release profiles within the first three hours of immersion in water. The total amount of nitric oxide released from a single tampon in 25 ml water varied from 200 to 450 ⁇ (reaching concentrations of 8 to 18 mM). All four tampon brands tested released between 46-60 % of the charged NO within the first 30 minutes (out of 4 hours tested). Thus, some slow release was demonstrated, although it was more rapid than that of other medical devices such as Foley catheters.
  • Candida albicans yeast culture was obtained from the
  • a starter was prepared by overnight growth of the colony taken from a plate of C. albicans in DifcoTM Sabouraud Dextrose Broth (SAB) media at 30 °C. Once turbid, the Optical Dispersion at 600 nm (OD600) was measured and the culture was diluted with sterile SAB to give an OD600 of 0.1, representing a culture containing 10 6 CFU/ml. From the culture of 10 6 CFU/ml, two separate cultures containing yeast concentrations of 10 1 and 10 2 CFU/ml were prepared in sterile SAB inside 1 L sterile containers.
  • SAB DifcoTM Sabouraud Dextrose Broth
  • Presence of C. albicans on the surfaces of charged tampons In order to determine whether fungal growth could be prevented on the surface of impregnated tampons, treated tampons were inoculated with C. albicans culture for 4 hours at 30 °C. Growth was assessed by rolling the treated tampons on a petri dish incubated overnight at 30 °C.
  • Figure 19 shows representative plates from each brand of tampon. No growth of C. albicans in plates rolled with NO impregnated tampons was observed. In contrast, controls from all brands showed growth of C.
  • Tampons were aseptically removed from their individual wrappers and inserted into separate sterile 50 ml vials. The string of each tampon was left hanging on the outside of each vial. Twenty- five (25) ml of C. albicans culture at either 10 1 or 10 2 CFU/ml was added to each vial and vortexed thoroughly. Vials were then incubated at 30 °C for 4 (10 2 CFU/ml) or 6 (10 1 CFU/ml) hours and thoroughly vortexed every 2 hours. Following incubation, aliquots (100 ⁇ ) were plated onto SAB agar petri plates and incubated overnight at 30 °C. CFU were counted and calculated to represent the CFU/ml.
  • Figure 20 is a bar-graph showing the anti-infective activity of NO-impregnated tampons, according to some embodiments of the present invention, comparing the growth of C. albicans in media after immersion of the tampons for 6 hours in suspension comprising 10 1 CFU/ml of C. albicans for NO-impregnated tampons and for untreated control tampons, wherein numbers represent viable counts of the triplicate CFUs, white bars represent data from the control experiments and black bars represent data from the NO-impregnated tampons, with error bars representing standard deviation.
  • Figure 21 is a bar-graph showing the anti-infective activity of NO-impregnated tampons, according to some embodiments of the present invention, comparing the growth of C. albicans in media after immersion of the tampons for 4 hours in suspension comprising 10 2 CFU/ml of C. albicans for NO-impregnated tampons and for control untreated tampons, wherein numbers represent viable counts of the triplicate CFUs, white bars represent data from the control experiments and black bars represent data from the NO-impregnated tampons, with error bars representing standard deviation
  • Table 20 summarizes the results of the abovementioned comparison of the obtained results.
  • NO charged tampons were shown to eradicate 100 % of C. albicans when inoculated with 10 3 - 10 5 CFU/ml and incubated for 4 hours (Table 21).
  • untreated tampons showed similar numbers of colonies, regardless of tampon type.
  • Untreated tampons contained average numbers of colonies of 3 x 10 3 , 2 x 10 4 , and 2 x 10 5 for staring concentrations of 10 3 , 10 4 , and 10 5 , respectively.
  • NO charged tampons were shown to either reduce, or eradicate E. coli when inoculated with 10 3 -10 5 CFU/ml after being incubated for 4 hours at 37 °C (Table 22).
  • Tampons B and D showed complete eradication of E. coli at concentrations 10 3 -10 5 CFU/ml whereas tampon A eradicated bacteria at concentrations of 10 3 and 10 4 CFU/ml, and showed a significant reduction in colonies at 10 5 CFU/ml.
  • Untreated tampons contained averages numbers of colonies of 3 x 10 4 , 4 x 10 5 , and 1 x 10 6 for staring concentrations of 10 3 , 10 4 and 10 5 CFU/ml, respectively.
  • Results shown in Figure 22 and Table 23, show that all charged tampons created a zone of bacterial growth inhibition with tampon B showing the highest zone of inhibition with 4.2 cm to the first shown colony. In all untreated tampons, no measureable zone of inhibition was observed.
  • Figure 25 shows a picture comparing the effect of a NO-treated tampon B against an untreated tampon when placed inside an Erlenmeyer flask containing LB, 2 % (w/v) gelatin, and E. coli at 10 6 CFU/ml. It can be readily seen that the NO treated tampon rendered the solid matrix inside the flask much clearer than the untreated tampon, indicating either a significant reduction or eradication of E.coli. In contrast, the matrix of the untreated tampon was evenly turbid throughout, indicating considerable bacterial growth. Additionally, the clear matrix of the NO-treated tampon in figure 25 suggests that the NO released from the tampon (as N0 2 ⁇ ) diffused over a great enough distance to create a large zone of inhibition in liquid.
  • VVC vulvovaginal candidiasis
  • BC vulvovaginal candidiasis
  • RVVC vulvovaginal candidiasis
  • Unwrapped tampons were treated with nitric oxide using general procedure I described hereinabove, except that the tampons were charged with nitric oxide outside their packaging material and additional concentrations of C. albicans were used during the experiment. The results are summarized in Figure 25 and Tables 24 and 25.
  • Figure 25 presents comparative plots of the total accumulation of nitrites produced in water during 4 hours of immersion as a function of time, as measured by the Griess test from NO-treated and untreated tampons, all outside their wrappers, wherein letters denote the type of tampon and the nitrite levels are calculated per a single tampon.
  • Table 24 presents a comparative summary of tampons' ability to release NO and efficacy in controlling yeast growth.
  • Table 25 presents a comparison of growth of C. albicans in media from tampons impregnated with nitric oxide without their wrappers versus media from control tampons, after immersion of the tampons for 4 hours in suspension comprising 10 1 - 10 5 CFU/ml of C. albicans. The numbers represent the averages and standard deviations of viable counts of the triplicate CFUs. "C” denotes negative control samples. Table 24
  • Nitrite levels of tampons impregnated with NO using general procedure II are higher than those impregnated with NO using general procedure I.
  • the tampons' efficacy in controlling yeast growth is determined by measuring growth of C. albicans in media from NO-treated and untreated, after immersion of the tampons for 4 hours in suspension comprising 10 1 — 10 5 CFU/ml of C. albicans.

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