{"adverse_reactions":["ADVERSE REACTIONS In a case series of a limited number of patients who had been treated for up to 6 weeks with topically applied Nitric Oxide at 0.02% there were no reported adverse reactions. Adverse reactions reported by others to Nitric Oxide donating creams at concentrations of up to 3 times the recommended dose concentration of gaseous Nitric Oxide have included irritation and burning sensation of the skin around the wound."],"carcinogenesis_and_mutagenesis_and_impairment_of_fertility":["Carcinogenesis, Mutagenesis, Impairment of Fertility No long-term studies in animals to evaluate the carcinogenic potential of Nitric Oxide have been performed. Nitric Oxide has demonstrated genotoxicity in Salmonella (Ames Test), human lymphocytes, and after in-vivo exposure in rats. There are no animal or human studies to evaluate Nitric Oxide for effects on fertility or harm to the developing fetus."],"clinical_pharmacology":["CLINICAL PHARMACOLOGY Nitric Oxide is a compound naturally produced by many cells of the body. It relaxes vascular smooth muscle by binding to the heme moiety of cytosolic guanylate cyclase, activating guanylate cyclase and increasing intracellular levels of cyclic guanosine 3',5'-monophosphate, which then leads to vasodilation. It also has dose dependent effects on the production of collagen and collagenase. It is also known to inhibit the reproduction of a broad range of pathogens. The bacteriocidal action of Nitric Oxide on a broad range of microorganisms is in response to its binding with all available reduced low molecular weight thiol compounds within the pathogens. This action results in NO attaching to iron enzymes in bacteria mitochondria respiratory chains causing cessation in oxygen consumption and mitochondrial asphyxiation. Additionally, subsequent intracellular changes result in reactive oxygen and reactive nitrogen oxide species damaging the bacterial DNA by deamination with associated damage from metal ion mobilization. Preliminary fibroblast in-vitro test data indicate that collagenase production increases, and collagen production decreases in the dose range 0.016% - 0.02% (160 to 200 ppm). Lower doses of Nitric Oxide in this test data indicate that collagen production increases in the dose range of 0.0005% (5 ppm). Topically delivered Nitric Oxide is under investigation for wound healing and its anti-infective properties. TOPICAL PHARMACOKINETICS (i) In-vitro bacteriocidal studies Evaluation of the bacteriostatic and bacteriocidal effects of topical Nitric Oxide gas on a range of gram positive and gram negative microorganisms was undertaken using an exposure incubator specifically designed for this study. The exposure incubator, containing both a control exposure chamber and a Nitric Oxide exposure chamber that was validated against a standard microbiology incubator for temperature and humidity during a 72 hour period and for supporting bacterial growth. The exposure chamber was also validated to assure correct and stable Nitric Oxide concentrations, lack of formation of excessive Nitrogen Dioxide levels and lack of significant alterations in pH. The exposure incubator was able to maintain stable temperature and humidity and supported the growth of bacteria equivalent to that of a conventional incubator. After dose ranging studies in solid media of ATCC samples of Pseudomonas aeruginosa and Staphylococcus aureus during which an optimal dose of 0.02% was determined for topical exposure, a series of experiments were performed on a range of ATCC and clinically obtained specimens (Table 1). The microorganisms selected for the preliminary studies were selected based on their prevalence of being found in hospitalized patients. Table 1. Microorganisms studied in exposure and control chambers Bacteria Gram staining Latent Period* (hrs) LD 50 (hrs) LD 100 (Hrs) S. aureus ( ATCC) Positive 3 3.3 4 P. aeruginosa (ATCC) Negative 1 2.1 3 MRSA Positive 3 4.2 5 Serracia sp. Negative 4 4.9 6 S. aureus (Clinical) Positive 3 3.7 4 Klebsiella sp. #1 Negative 3 3.5 6 Klebsiella sp.#2 Negative 2 4.1 5 Klebsiella sp. #3 Negative 3 5.1 6 S. maltophilia Negative 2 2.8 4 Enterobacter sp. Negative 4 5.3 6 Acinetobacter sp. Negative 4 5 6 E. coli Negative 3 4.2 5 Group B Streptococci Positive 1 1.5 2 Average N/A 2.77 3.82 4.77 SD N/A 1.01 1.17 1.30 These microorganisms were inoculated in saline wells at concentrations of 10 5 for exposure to either room air or 0.02% Nitric Oxide. The saline wells were sampled at frequent intervals until the NO exposed microorganisms were at zero survival levels. All of the control chamber microorganisms survived to the end of the exposure period of the NO exposed microorganisms (Figure 1). The average latency period before there was a change in microorganism survival for the NO exposed group was 2.77±1.01 hours. The LD 50 for the same set of microorganisms was 3.82±1.17 hours and the LD 100 was 4.77±1.30 hours. Figure 1 presents the survival curve of Klebsiella exposed to either room air (dotted line) or Nitric Oxide (solid line). As predicted by the described mechanism of bacteriocidal action, there was a latency period with little change in survival. Once the thiol (reduced glutathione) detoxification capability of each microorganism was depleted, this was followed by rapid death of all microorganisms, with zero (0) survival by 4.8 (SD=1.3) hours of exposure. Figure 1. Survival curve of incubated Klebsiella exposed to room air (- - -) or 0.02% nitric oxide (–) The conclusions reached from these experiments indicate that Nitric Oxide gas in the 0.02% range has an effective bacteriocidal action on a broad range of potentially pathogenic microorganisms. Since the detoxification mechanism involves one to one binding with thiols, it was postulated that higher concentrations of nitric oxide would deplete available thiols quickly and lead to faster bacteriocidal activity, see Table 2 . Table 2. Bacteria studied with higher NO concentrations Nitric Oxide Exposure Bacteria (starting at 10 6 CFU/mL) Conc. Time Kill MRSA 0.1% 2 hrs. none 0.5% 45min complete 1% 15min complete Pseudomonas aeroginosa 0.1% 2 hrs 1 log 0,5% 45min complete 1% 15min complete Acinetobacter baumanii 0.1% 2 hrs none 0,5% 45min complete 1% 15min complete Streptococcus pyogenes 0.1% 2 hrs none 0,5% 45min complete 1% 15 min complete These microorganisms were inoculated in saline wells and were exposed to 1% NO for the times listed. The bacteria were then assayed. Several species of fungus were treated to the same exposure of NO in supplemented wells (Table 3) Table 3. Fungus studied with higher NO concentrations Nitric Oxide Exposure Fungus (controls were 100%) Conc. Time Kill Trichophyton Rubrum 0.5% 30min 80% 1% 30min complete Aspergillus niger 0.5% 30min 65% 1% 30min complete Trichophyton Mentagrophytes 0.5% 30min complete 1% 30min complete Figure (ii) In-vivo animal studies In an independent animal study, full thickness skin wounds (four punch-biopsies 8.0mm in diameter on each side of dorsal medial) infected with Staphylococcus aureus received topical application of Nitric Oxide at 0.02% for duration of 8 hours per day for six consecutive days. Bacterial load as measured from tissue biopsies at day 4 was used to evaluate the anti-infective properties. Diffusion of Nitric Oxide through dermal wounds into the systemic circulation of the animals was studied by observing common end metabolites, namely methemoglobin (MetHb) and nitrates. In blood, Nitric Oxide combines with oxyhemoglobin to produce methemoglobin and nitrates. MetHb levels in fresh arterial blood samples from animals were studied at Day 0 and 6 post treatment. At 0.02% topical application of Nitric Oxide, no significant change in blood MetHb levels was observed between control and treated group. Maximum MetHb levels did not exceed 0.8%. Figures 2 to 4 summarizes the data: Figure 2. Bacterial load in control and inoculated wounds in rabbits exposed to 0.02% for 8 hours per day (* p<0.05). Figure 3. Blood Methemoglobin (MetHb) levels Following Topical Exposure to 0.02% Nitric Oxide. Treated groups were exposed to 0.02% Nitric Oxide and control only to medical grade air at a flow of 10 L/min over the skin. Changes in MetHb are statistically insignificant when compared with control results (p > 0.05), meaning only minute amount of nitric oxide gas diffused through the dermis into animal's circulatory system under above conditions. MetHb levels were analyzed using a Radiometer ABL 700 Series Blood Gas Analyzer. Other predominate end products of Nitric Oxide in systemic circulation, including endogenous NO produced in response to infection, are nitrates and nitrites (NO 3 and NO 2 ). Therefore, measurements of oxides of nitrogen (NO x ) in the blood serum were used as a marker for systemic bioavailability of Nitric Oxide. Animals exposed to 0.02% Nitric Oxide showed no increase in NO X content of blood serum in comparison to the control group, suggesting that only a small amount of Nitric Oxide gas diffused into the systemic circulation through the open wounds. Data are summarized in Figure 4. Figure 4. NO x Levels in Blood Serum – 0.02% Nitric Oxide. Animals in the treatment group (Nitric Oxide) were exposed daily to 0.02% Nitric Oxide for 8 hours daily for six consecutive days. The control group was exposed to medical air. NO x levels were measured using High Pressure Liquid Chromatography techniques. In a separate study, following 72 hours of continuous exposure of animals' full thickness wounds to 0.04% (400 ppm) Nitric Oxide gas, only a small increase in systemic circulation levels of NO x was observed (Figure 5: p > 0.05). No toxicity to Nitric Oxide treatment was detected, as MetHb levels remained unchanged (p < 0.05). Figure 5. NO x Levels in Blood Serum – 0.04% Nitric Oxide Animals in treated group were exposed continuously for 72 hours to 0.04% Nitric Oxide. Control group was exposed to medical air only. NO x levels were measured using High Pressure Liquid Chromatography techniques. Bacterial load in the wounds that were exposed to 0.04% of NO (figure 6) for 4 days were significantly reduced, reaching statistical significance (p <0.05). Figure 6. Bacterial load in control and inoculated wounds in rabbits exposed to 0.04% NO for 72 hours (* p<0.05). In all studies, no animal had discontinued treatment for topical Nitric Oxide toxicity. Topical Nitric Oxide had no detectable effect on the health of the animals. NO was tested for antimicrobial activity at 1% (10,000ppm) in a porcine study performed at an independent pre-clinical testing laboratory. Three biopsies were taken from each of three similarly treated (or non-treated) wounds and assayed in duplicate after growth on two different types of media. 32 of the 36 samples showed zero bacteria present with an overall reduction of nearly 5 logs of bacteria. Figure 7 Antibacterial Activity at 1% NO Coagulase Negative Staphylcocci was inoculated into full thickness wounds on a Yorkshire pig. 24 hours later topical NO in 20% O 2 was administered to a bubble dressing over the wound at a flow rate of 500mL/min. Figure Figure Figure Figure Figure Figure (iii) In-vitro Human Skin Culture Study In additional studies, fibroblast cell cultures collected and harvested from human adult patients were exposed continuously to 0.016% Nitric Oxide for periods of 0, 24, and 48 hours. Effect of Nitric Oxide on fibroblast growth was monitored by taking pH readings on the medium following exposure. No significant difference in pH values of collected conditioned medium was observed between treatment and control group (Figure 6). Figure 8. Fibroblast Culture Medium pH– 0.016% Nitric Oxide Fibroblast culture growth media exposed to 0.016% Nitric Oxide gas (treated). Control group was only exposed to Medical Air and 5% carbon dioxide gas at flow of 10 L/min. p > 0.05 Visual microscopy and proliferation studies were conducted to verify viability of human fibroblast cells exposed to Nitric Oxide. Cellular morphology did not show any significant variation between treated and control (data not shown). Cell counts revealed no significant difference in fibroblast proliferation (cell division) between cultured cells exposed to Nitric Oxide and control group. Figure 9 summarizes the proliferation data. A better cellular growth was observed following 24 hours exposure to Nitric Oxide. Figure 9. Fibroblast Cell Growth Following Exposure to 0.016% Nitric Oxide Human fibroblast cultures exposed to 0.016% Nitric Oxide gas (treated). Control group was only exposed to Medical Air and 5% carbon dioxide gas at flow of 10 L/min. Higher cellular proliferation was observed following 24 hours exposure to Nitric Oxide. (T 0 and T 48 : p > 0.05 / T 24 : p < 0.05) Figure Figure"],"clinical_pharmacology_table":["
Table 1. Microorganisms studied in exposure and control chambers
Bacteria Gram staining Latent Period* (hrs) LD50 (hrs) LD100 (Hrs)
S. aureus ( ATCC) Positive 3 3.3 4
P. aeruginosa (ATCC) Negative 1 2.1 3
MRSA Positive 3 4.2 5
Serracia sp. Negative 4 4.9 6
S. aureus (Clinical) Positive 3 3.7 4
Klebsiella sp. #1 Negative 3 3.5 6
Klebsiella sp.#2 Negative 2 4.1 5
Klebsiella sp. #3 Negative 3 5.1 6
S. maltophilia Negative 2 2.8 4
Enterobacter sp. Negative 4 5.3 6
Acinetobacter sp. Negative 4 5 6
E. coli Negative 3 4.2 5
Group B Streptococci Positive 1 1.5 2
Average N/A 2.77 3.82 4.77
SD N/A 1.01 1.17 1.30
","
Table 2. Bacteria studied with higher NO concentrations
Nitric Oxide Exposure
Bacteria (starting at 106 CFU/mL) Conc. Time Kill
MRSA 0.1% 2 hrs. none
0.5% 45min complete
1% 15min complete
Pseudomonas aeroginosa 0.1% 2 hrs 1 log
0,5% 45min complete
1% 15min complete
Acinetobacter baumanii 0.1% 2 hrs none
0,5% 45min complete
1% 15min complete
Streptococcus pyogenes 0.1% 2 hrs none
0,5% 45min complete
1% 15 min complete
","
Table 3. Fungus studied with higher NO concentrations
Nitric Oxide Exposure
Fungus (controls were 100%) Conc. Time Kill
Trichophyton Rubrum 0.5% 30min 80%
1% 30min complete
Aspergillus niger 0.5% 30min 65%
1% 30min complete
Trichophyton Mentagrophytes 0.5% 30min complete
1% 30min complete
"],"contraindications":["CONTRAINDICATIONS There are no known contraindications for topically applied Nitric Oxide. However, caution should be taken when using gaseous Nitric Oxide in conjunction with Nitric Oxide donating or phosphodiesterase inhibiting compounds such as nitroglycerin, isosorbide dinitrate, amyl nitrate, erythrityl tetranitrate, pentaerythritol nitrate or sodium nitroprusside; or phosphodiesterase inhibitors such as sildenafil citrate, vardenafil HCl or tadalafil."],"description":["DESCRIPTION Nitric Oxide gas is a drug administered by topical application using an appropriate topical delivery device. Nitric Oxide is under investigation for use as a promoter of wound healing and as an anti-infective agent. Nitric Oxide is supplied as a gaseous blend of Nitric Oxide (1.0%) and Nitrogen USP (99.0%) or Nitric Oxide (2.0%) and Nitrogen USP (98.0%) in aluminum cylinders as a compressed gas under high pressure (750 pounds per square inch gauge [psig]). The structural formula of Nitric Oxide (NO) is shown below: Chemical Structure"],"dosage_and_administration":["DOSAGE AND ADMINISTRATION Dosage The recommended dose of Nitric Oxide 2% for topical anti-infective applications is 1% (10,000 ppm) or 2% (20,000 ppm) for a maximum of two (2) hours per day. Preliminary study data suggest that treatment should be maintained for 3 to 5 days or until the underlying cause of wound infection has resolved. On-going Phase II clinical studies for wound care applications require a dose of 0.02% for 8 hours a day for 6 weeks. There is no randomized controlled trial data on the appropriate dose for topical use as an anti-infective agent or for wound healing. Administration The Nitric Oxide delivery systems used in the case series of topically applied Nitric Oxide and the on-going Phase II clinical trials provided fixed concentrations of Nitric Oxide at fixed / controllable flow rates up to and including 1 Liter per minute. Nitric Oxide must be delivered through a system with these characteristics and which does not cause generation of excessive inhaled nitrogen dioxide. There is no clinical data using other concentrations or other flow rates."],"drug_interactions":["Drug Interactions No formal drug-interaction studies have been performed, and a clinically significant interaction with other medications used in the treatment of wounds or wound infections cannot be excluded based on the available data. In particular there are no data to evaluate the possibility that topically applied Nitric Oxide may have an additive effect to the actions, including vasodilation, of pharmaceutical Nitric Oxide donor compounds such as sodium nitroprusside and nitroglycerin. They may have an additive effect with Nitric Oxide on the risk of developing methemoglobinemia. Caution should be taken when using gaseous Nitric Oxide in conjunction with Nitric Oxide donating compounds such as nitroglycerin, isosorbide dinitrate, amyl nitrate, erythrityl tetranitrate, pentaerythritol nitrate or sodium nitroprusside; or phosphodiesterase inhibitors such as sildenafil citrate, vardenafil HCl or tadalafil."],"effective_time":"20091130","how_supplied":["HOW SUPPLIED 1% Nitric Oxide is available in the following sizes: Size 33a Portable aluminum cylinders containing 550 liters at 70°F of Nitric Oxide gas in 1% concentration in nitrogen (delivered volume 550 liters) (NDC 43402-103-05) 2% Nitric Oxide is available in the following sizes: Size 22a Portable aluminum cylinders containing 209.6 liters at STP of Nitric Oxide gas in 2% concentration in nitrogen (delivered volume 209.6 liters) (NDC 43402-101-02) Cylinders should be kept in a cool, well-ventilated area. Cylinders should be stored upright and firmly secured to prevent falling or being knocked over. Cylinder temperatures should not exceed 52°C (125°F)."],"how_supplied_table":["
Size 33a Portable aluminum cylinders containing 550 liters at 70°F of Nitric Oxide gas in 1% concentration in nitrogen (delivered volume 550 liters) (NDC 43402-103-05)
","
Size 22a Portable aluminum cylinders containing 209.6 liters at STP of Nitric Oxide gas in 2% concentration in nitrogen (delivered volume 209.6 liters) (NDC 43402-101-02)
"],"id":"68adc464-5eb3-48bb-a0e6-e101ba9edd72","indications_and_usage":["INDICATIONS Nitric Oxide gas is currently under investigation for use in numerous indications including topically for wound healing and as an anti-infective agent. Please consult your own local Institutional Review / Ethics Board approved protocol, and contact your study Principal Investigator for further information. Nitric Oxide / Nitrogen gas mixes, in conjunction with ventilatory support and other appropriate agents, have received Food and Drug Administration approval for products manufactured by others for the treatment of term and near-term (>34 weeks) neonates with hypoxic respiratory failure associated with clinical or echocardiographic evidence of pulmonary hypertension, where it improves oxygenation and reduces the need for extracorporeal membrane oxygenation."],"nursing_mothers":["Nursing Mothers Nitric Oxide is not indicated for use in the adult pregnant population, including nursing mothers. It is not known whether Nitric Oxide is excreted in human milk."],"openfda":{},"overdosage":["OVERDOSAGE Over dosage with topical Nitric Oxide may be manifested by elevations in NO 2 production. Elevated NO 2 may cause acute lung injury. In clinical studies of inhaled Nitric Oxide, NO 2 levels >3 ppm were treated by reducing the dose of, or discontinuing, Nitric Oxide. If there is a possibility of an overdose the patient should be moved to a well-ventilated area."],"package_label_principal_display_panel":["PRINCIPAL DISPLAY PANEL - 2% Tank Label NITRICbio Advanced Healing Systems Nitric Oxide 2% v/v (20,000 ppm) CAUTION: Contains an Investigational Drug – Limited to Clinical Trial Use Only by a Qualified Investigator (Nitric Oxide 2% v/v (20,000 ppm)) COMPRESSED GAS, N.O.S. (NITROGEN, NITRIC OXIDE) UN 1956 Topical, Rx Only, NOT FOR INHALATION Product Code: NDC 43402-101-02 Airgas Product Code: X02NI98EA22C2T4 Cylinder Contents: 209.6 liters @ 70°F 2% v/v (20,000 ppm) Nitric Oxide CAS Numbers: 10102-43-9 NO 7727-37-9 N 2 Read and understand Material Data Sheet before use. Indications and Directions: Use only in accordance with your local Ethics Committee approved protocol. WARNING: Administration of this gas mixture may be hazardous or contraindicated. For use only by or under the supervision of a licensed practitioner who is experienced in the use and administration of gas mixtures and is familiar with the indications, effects, dosages, test methods, frequency and duration of administration, hazards, contraindications, side effects, and precautions to be taken. CAUTION: HIGH PRESSURE GAS. CAN CAUSE RAPID SUFFOCATION WITHOUT WARNING. Store and use with adequate ventilation. Use with equipment rated for cylinder pressure. Secure cylinder in use and during storage. Open valve slowly. Close valve after each use and when empty. Cylinder temperature should not exceed 125°F (52°C) FIRST AID: If Inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Get medical help. RETURN WITH 150 PSIG. To be refilled only by a pharmaceutical facility authorized by Nitric BioTherapeutics, Inc. Manufactured for Study Sponsor: Nitric BioTherapeutics, Inc. , 2 Canal's End Rd, Suite 201-A, Bristol, PA 19007, USA. For product inquiry: 1-215-788- 6200 Manufactured by: Airgas Specialty Gases, 12722 South Wentworth Avenue, Chicago, IL 60628-7251, USA PRINCIPAL DISPLAY PANEL - 2% Tank Label","PRINCIPAL DISPLAY PANEL - 1% Tank Label NITRICbio Advanced Healing Systems Nitric Oxide 1% v/v (10,000 ppm) CAUTION: Contains an Investigational Drug – Limited to Clinical Trial Use Only by a Qualified Investigator (Nitric Oxide 1% v/v (10,000 ppm)) COMPRESSED GAS, N.O.S. (NITROGEN, NITRIC OXIDE) UN 1956 Topical, Rx Only, NOT FOR INHALATION Product Code: NDC 43402-103-05 Airgas Product Code: Z02NI99E33AC103 Cylinder Contents: 550 Liters @ 70°F 1% v/v (10,000 ppm) Nitric Oxide CAS Numbers: 10102-43-9 NO 7727-37-9 N 2 Read and understand Material Data Sheet before use. Indications and Directions: Use only in accordance with your local Ethics Committee approved protocol. WARNING: Administration of this gas mixture may be hazardous or contraindicated. For use only by or under the supervision of a licensed practitioner who is experienced in the use and administration of gas mixtures and is familiar with the indications, effects, dosages, test methods, frequency and duration of administration, hazards, contraindications, side effects, and precautions to be taken. CAUTION: HIGH PRESSURE GAS. CAN CAUSE RAPID SUFFOCATION WITHOUT WARNING. Store and use with adequate ventilation. Use with equipment rated for cylinder pressure. Secure cylinder in use and during storage. Open valve slowly. Close valve after each use and when empty. Cylinder temperature should not exceed 125°F (52°C) FIRST AID: If Inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Get medical help. RETURN WITH 150 PSIG. To be refilled only by a pharmaceutical facility authorized by Nitric BioTherapeutics, Inc. Manufactured for Study Sponsor: Nitric BioTherapeutics, Inc. , 2 Canal's End Rd, Suite 201-A, Bristol, PA 19007, USA. For product inquiry: 1-215-788- 6200 Manufactured by: Airgas Specialty Gases, 12722 South Wentworth Avenue, Chicago, IL 60628-7251, USA PRINCIPAL DISPLAY PANEL - 1% Tank Label"],"pediatric_use":["Pediatric Use There is no data on the use of topically applied Nitric Oxide in children. Topical Nitric Oxide is not indicated for use in the pediatric population."],"pharmacokinetics":["TOPICAL PHARMACOKINETICS (i) In-vitro bacteriocidal studies Evaluation of the bacteriostatic and bacteriocidal effects of topical Nitric Oxide gas on a range of gram positive and gram negative microorganisms was undertaken using an exposure incubator specifically designed for this study. The exposure incubator, containing both a control exposure chamber and a Nitric Oxide exposure chamber that was validated against a standard microbiology incubator for temperature and humidity during a 72 hour period and for supporting bacterial growth. The exposure chamber was also validated to assure correct and stable Nitric Oxide concentrations, lack of formation of excessive Nitrogen Dioxide levels and lack of significant alterations in pH. The exposure incubator was able to maintain stable temperature and humidity and supported the growth of bacteria equivalent to that of a conventional incubator. After dose ranging studies in solid media of ATCC samples of Pseudomonas aeruginosa and Staphylococcus aureus during which an optimal dose of 0.02% was determined for topical exposure, a series of experiments were performed on a range of ATCC and clinically obtained specimens (Table 1). The microorganisms selected for the preliminary studies were selected based on their prevalence of being found in hospitalized patients. Table 1. Microorganisms studied in exposure and control chambers Bacteria Gram staining Latent Period* (hrs) LD 50 (hrs) LD 100 (Hrs) S. aureus ( ATCC) Positive 3 3.3 4 P. aeruginosa (ATCC) Negative 1 2.1 3 MRSA Positive 3 4.2 5 Serracia sp. Negative 4 4.9 6 S. aureus (Clinical) Positive 3 3.7 4 Klebsiella sp. #1 Negative 3 3.5 6 Klebsiella sp.#2 Negative 2 4.1 5 Klebsiella sp. #3 Negative 3 5.1 6 S. maltophilia Negative 2 2.8 4 Enterobacter sp. Negative 4 5.3 6 Acinetobacter sp. Negative 4 5 6 E. coli Negative 3 4.2 5 Group B Streptococci Positive 1 1.5 2 Average N/A 2.77 3.82 4.77 SD N/A 1.01 1.17 1.30 These microorganisms were inoculated in saline wells at concentrations of 10 5 for exposure to either room air or 0.02% Nitric Oxide. The saline wells were sampled at frequent intervals until the NO exposed microorganisms were at zero survival levels. All of the control chamber microorganisms survived to the end of the exposure period of the NO exposed microorganisms (Figure 1). The average latency period before there was a change in microorganism survival for the NO exposed group was 2.77±1.01 hours. The LD 50 for the same set of microorganisms was 3.82±1.17 hours and the LD 100 was 4.77±1.30 hours. Figure 1 presents the survival curve of Klebsiella exposed to either room air (dotted line) or Nitric Oxide (solid line). As predicted by the described mechanism of bacteriocidal action, there was a latency period with little change in survival. Once the thiol (reduced glutathione) detoxification capability of each microorganism was depleted, this was followed by rapid death of all microorganisms, with zero (0) survival by 4.8 (SD=1.3) hours of exposure. Figure 1. Survival curve of incubated Klebsiella exposed to room air (- - -) or 0.02% nitric oxide (–) The conclusions reached from these experiments indicate that Nitric Oxide gas in the 0.02% range has an effective bacteriocidal action on a broad range of potentially pathogenic microorganisms. Since the detoxification mechanism involves one to one binding with thiols, it was postulated that higher concentrations of nitric oxide would deplete available thiols quickly and lead to faster bacteriocidal activity, see Table 2 . Table 2. Bacteria studied with higher NO concentrations Nitric Oxide Exposure Bacteria (starting at 10 6 CFU/mL) Conc. Time Kill MRSA 0.1% 2 hrs. none 0.5% 45min complete 1% 15min complete Pseudomonas aeroginosa 0.1% 2 hrs 1 log 0,5% 45min complete 1% 15min complete Acinetobacter baumanii 0.1% 2 hrs none 0,5% 45min complete 1% 15min complete Streptococcus pyogenes 0.1% 2 hrs none 0,5% 45min complete 1% 15 min complete These microorganisms were inoculated in saline wells and were exposed to 1% NO for the times listed. The bacteria were then assayed. Several species of fungus were treated to the same exposure of NO in supplemented wells (Table 3) Table 3. Fungus studied with higher NO concentrations Nitric Oxide Exposure Fungus (controls were 100%) Conc. Time Kill Trichophyton Rubrum 0.5% 30min 80% 1% 30min complete Aspergillus niger 0.5% 30min 65% 1% 30min complete Trichophyton Mentagrophytes 0.5% 30min complete 1% 30min complete Figure (ii) In-vivo animal studies In an independent animal study, full thickness skin wounds (four punch-biopsies 8.0mm in diameter on each side of dorsal medial) infected with Staphylococcus aureus received topical application of Nitric Oxide at 0.02% for duration of 8 hours per day for six consecutive days. Bacterial load as measured from tissue biopsies at day 4 was used to evaluate the anti-infective properties. Diffusion of Nitric Oxide through dermal wounds into the systemic circulation of the animals was studied by observing common end metabolites, namely methemoglobin (MetHb) and nitrates. In blood, Nitric Oxide combines with oxyhemoglobin to produce methemoglobin and nitrates. MetHb levels in fresh arterial blood samples from animals were studied at Day 0 and 6 post treatment. At 0.02% topical application of Nitric Oxide, no significant change in blood MetHb levels was observed between control and treated group. Maximum MetHb levels did not exceed 0.8%. Figures 2 to 4 summarizes the data: Figure 2. Bacterial load in control and inoculated wounds in rabbits exposed to 0.02% for 8 hours per day (* p<0.05). Figure 3. Blood Methemoglobin (MetHb) levels Following Topical Exposure to 0.02% Nitric Oxide. Treated groups were exposed to 0.02% Nitric Oxide and control only to medical grade air at a flow of 10 L/min over the skin. Changes in MetHb are statistically insignificant when compared with control results (p > 0.05), meaning only minute amount of nitric oxide gas diffused through the dermis into animal's circulatory system under above conditions. MetHb levels were analyzed using a Radiometer ABL 700 Series Blood Gas Analyzer. Other predominate end products of Nitric Oxide in systemic circulation, including endogenous NO produced in response to infection, are nitrates and nitrites (NO 3 and NO 2 ). Therefore, measurements of oxides of nitrogen (NO x ) in the blood serum were used as a marker for systemic bioavailability of Nitric Oxide. Animals exposed to 0.02% Nitric Oxide showed no increase in NO X content of blood serum in comparison to the control group, suggesting that only a small amount of Nitric Oxide gas diffused into the systemic circulation through the open wounds. Data are summarized in Figure 4. Figure 4. NO x Levels in Blood Serum – 0.02% Nitric Oxide. Animals in the treatment group (Nitric Oxide) were exposed daily to 0.02% Nitric Oxide for 8 hours daily for six consecutive days. The control group was exposed to medical air. NO x levels were measured using High Pressure Liquid Chromatography techniques. In a separate study, following 72 hours of continuous exposure of animals' full thickness wounds to 0.04% (400 ppm) Nitric Oxide gas, only a small increase in systemic circulation levels of NO x was observed (Figure 5: p > 0.05). No toxicity to Nitric Oxide treatment was detected, as MetHb levels remained unchanged (p < 0.05). Figure 5. NO x Levels in Blood Serum – 0.04% Nitric Oxide Animals in treated group were exposed continuously for 72 hours to 0.04% Nitric Oxide. Control group was exposed to medical air only. NO x levels were measured using High Pressure Liquid Chromatography techniques. Bacterial load in the wounds that were exposed to 0.04% of NO (figure 6) for 4 days were significantly reduced, reaching statistical significance (p <0.05). Figure 6. Bacterial load in control and inoculated wounds in rabbits exposed to 0.04% NO for 72 hours (* p<0.05). In all studies, no animal had discontinued treatment for topical Nitric Oxide toxicity. Topical Nitric Oxide had no detectable effect on the health of the animals. NO was tested for antimicrobial activity at 1% (10,000ppm) in a porcine study performed at an independent pre-clinical testing laboratory. Three biopsies were taken from each of three similarly treated (or non-treated) wounds and assayed in duplicate after growth on two different types of media. 32 of the 36 samples showed zero bacteria present with an overall reduction of nearly 5 logs of bacteria. Figure 7 Antibacterial Activity at 1% NO Coagulase Negative Staphylcocci was inoculated into full thickness wounds on a Yorkshire pig. 24 hours later topical NO in 20% O 2 was administered to a bubble dressing over the wound at a flow rate of 500mL/min. Figure Figure Figure Figure Figure Figure (iii) In-vitro Human Skin Culture Study In additional studies, fibroblast cell cultures collected and harvested from human adult patients were exposed continuously to 0.016% Nitric Oxide for periods of 0, 24, and 48 hours. Effect of Nitric Oxide on fibroblast growth was monitored by taking pH readings on the medium following exposure. No significant difference in pH values of collected conditioned medium was observed between treatment and control group (Figure 6). Figure 8. Fibroblast Culture Medium pH– 0.016% Nitric Oxide Fibroblast culture growth media exposed to 0.016% Nitric Oxide gas (treated). Control group was only exposed to Medical Air and 5% carbon dioxide gas at flow of 10 L/min. p > 0.05 Visual microscopy and proliferation studies were conducted to verify viability of human fibroblast cells exposed to Nitric Oxide. Cellular morphology did not show any significant variation between treated and control (data not shown). Cell counts revealed no significant difference in fibroblast proliferation (cell division) between cultured cells exposed to Nitric Oxide and control group. Figure 9 summarizes the proliferation data. A better cellular growth was observed following 24 hours exposure to Nitric Oxide. Figure 9. Fibroblast Cell Growth Following Exposure to 0.016% Nitric Oxide Human fibroblast cultures exposed to 0.016% Nitric Oxide gas (treated). Control group was only exposed to Medical Air and 5% carbon dioxide gas at flow of 10 L/min. Higher cellular proliferation was observed following 24 hours exposure to Nitric Oxide. (T 0 and T 48 : p > 0.05 / T 24 : p < 0.05) Figure Figure"],"pharmacokinetics_table":["
Table 1. Microorganisms studied in exposure and control chambers
Bacteria Gram staining Latent Period* (hrs) LD50 (hrs) LD100 (Hrs)
S. aureus ( ATCC) Positive 3 3.3 4
P. aeruginosa (ATCC) Negative 1 2.1 3
MRSA Positive 3 4.2 5
Serracia sp. Negative 4 4.9 6
S. aureus (Clinical) Positive 3 3.7 4
Klebsiella sp. #1 Negative 3 3.5 6
Klebsiella sp.#2 Negative 2 4.1 5
Klebsiella sp. #3 Negative 3 5.1 6
S. maltophilia Negative 2 2.8 4
Enterobacter sp. Negative 4 5.3 6
Acinetobacter sp. Negative 4 5 6
E. coli Negative 3 4.2 5
Group B Streptococci Positive 1 1.5 2
Average N/A 2.77 3.82 4.77
SD N/A 1.01 1.17 1.30
","
Table 2. Bacteria studied with higher NO concentrations
Nitric Oxide Exposure
Bacteria (starting at 106 CFU/mL) Conc. Time Kill
MRSA 0.1% 2 hrs. none
0.5% 45min complete
1% 15min complete
Pseudomonas aeroginosa 0.1% 2 hrs 1 log
0,5% 45min complete
1% 15min complete
Acinetobacter baumanii 0.1% 2 hrs none
0,5% 45min complete
1% 15min complete
Streptococcus pyogenes 0.1% 2 hrs none
0,5% 45min complete
1% 15 min complete
","
Table 3. Fungus studied with higher NO concentrations
Nitric Oxide Exposure
Fungus (controls were 100%) Conc. Time Kill
Trichophyton Rubrum 0.5% 30min 80%
1% 30min complete
Aspergillus niger 0.5% 30min 65%
1% 30min complete
Trichophyton Mentagrophytes 0.5% 30min complete
1% 30min complete
"],"precautions":["PRECAUTIONS Methemoglobinemia Methemoglobinemia increases with the dose of Nitric Oxide when inhaled. Following discontinuation or reduction of Nitric Oxide the methemoglobin levels returned to baseline over a period of hours. Animal studies of topically applied Nitric Oxide have not demonstrated any increase in blood methemoglobin levels. Elevated NO2 levels Nitrogen Dioxide (NO 2 ) is a potentially toxic by-product of the interaction of Nitric Oxide with Oxygen (O 2 ). To minimize NO 2 production the O 2 must be incorporated into the flow immediately prior to the gas mix entering the topical application cover. The combined flow rate of Nitric Oxide and Oxygen needs to be high enough to clear the space of NO 2 . In the case series reported for topical applications using a lower limb covering, the concentration of Nitrogen Dioxide was maintained at safe levels using a flow rate of 1 liter per minute. The exit flow from topical application covers should be removed from the area around the patient's face should the Nitrogen Dioxide concentration rise above 0.0003% (3 ppm). The patient should be treated in a large room or a well-ventilated area. The exposure limit set by the Occupational Safety and Health Administration (OSHA) for inhaled NO 2 is 5 ppm (0.0005%). Drug Interactions No formal drug-interaction studies have been performed, and a clinically significant interaction with other medications used in the treatment of wounds or wound infections cannot be excluded based on the available data. In particular there are no data to evaluate the possibility that topically applied Nitric Oxide may have an additive effect to the actions, including vasodilation, of pharmaceutical Nitric Oxide donor compounds such as sodium nitroprusside and nitroglycerin. They may have an additive effect with Nitric Oxide on the risk of developing methemoglobinemia. Caution should be taken when using gaseous Nitric Oxide in conjunction with Nitric Oxide donating compounds such as nitroglycerin, isosorbide dinitrate, amyl nitrate, erythrityl tetranitrate, pentaerythritol nitrate or sodium nitroprusside; or phosphodiesterase inhibitors such as sildenafil citrate, vardenafil HCl or tadalafil. Carcinogenesis, Mutagenesis, Impairment of Fertility No long-term studies in animals to evaluate the carcinogenic potential of Nitric Oxide have been performed. Nitric Oxide has demonstrated genotoxicity in Salmonella (Ames Test), human lymphocytes, and after in-vivo exposure in rats. There are no animal or human studies to evaluate Nitric Oxide for effects on fertility or harm to the developing fetus. Pregnancy Category C Animal reproduction studies have not been conducted with Nitric Oxide. It is not known if Nitric Oxide can cause fetal harm when administered to a pregnant woman or can affect reproductive capacity. Although other Nitric Oxide products have been approved by the Food and Drug Administration for inhalation, Nitric Oxide products have not been previously approved by the Food and Drug Administration for topical use in adults; however, usage in adults is currently under investigation. Pediatric Use There is no data on the use of topically applied Nitric Oxide in children. Topical Nitric Oxide is not indicated for use in the pediatric population. Nursing Mothers Nitric Oxide is not indicated for use in the adult pregnant population, including nursing mothers. It is not known whether Nitric Oxide is excreted in human milk."],"pregnancy":["Pregnancy Category C Animal reproduction studies have not been conducted with Nitric Oxide. It is not known if Nitric Oxide can cause fetal harm when administered to a pregnant woman or can affect reproductive capacity. Although other Nitric Oxide products have been approved by the Food and Drug Administration for inhalation, Nitric Oxide products have not been previously approved by the Food and Drug Administration for topical use in adults; however, usage in adults is currently under investigation."],"references":["Bibliography Kunimoto B, Cooling M, Gulliver W, Houghton P, Orsted H, Sibbald RG. Best practices for the prevention and treatment of venous leg ulcers. Ostomy Wound Management 2001; 47:34-46,48-50. Hannson C, Hoborn J, Moller A, Swanbeck G. The microbial flora in venous leg ulcers without clinical signs of infection. Acta Dermato-Venerologica 1995; 75:24-30. Consterton JW, Stewart PS. Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science 1999; 284:1318-1322 Consterton JW, Stewart PS. Battling Biofilms. Scientific American 2001;July:75-81 Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet 2001; 358:135-138 Moncada S, Palmer RMJ, Higgis EA. Nitric oxide. Physiology, Pathophysiology and Pharmacology, Pharmacol Rev 1991; 43:109-134. De Groote MA, Fang FC. NO inhibitions:antimicrobial properties of nitric oxide. Clinical Infectious Diseases 1995; 21(suppl 2):S162-165. Witte MB, Barbul A. Role of nitric oxide in wound repair. The American Journal of Surgery 2002; 183:406-412 Fang FC. Mechanisms of nitric oxide – related antimicrobial activity. American Society of Clinical Investigations 1997; 99:2818-2825. Vazquez-Torres A, Fang FC. Therapeutic applications of nitric oxide in infection in Nitric Oxide and Infection 1999 Plenum Publishers, New York pp 475-488. Ormerod A.D., et al. The inflammatory and cytotoxic effects of a nitric oxide releasing cream on normal skin. J Invest Dermatol 113:392-397, 1999. Bauer JA. Et al. Evaluation of linear polyethyleneimine / NO adduct on wound repair: therapy versus toxicity. Wound Repair Regen 1998; 6:569-577. Shabini M, Pulfer SK, Bulgrin JP, Smith DJ. Enhancement of wound repair with a topically applied nitric oxide-releasing polymer. Wound Rep Reg 1996; 4:353-362. Weller R, Ormerod AD, Hobson RP, Benjamin N. A randomized trial of acidified nitrite cream in the treatment of Tinea pedis. J. Am. Acad. Dermatol 1998; 38:559-563. Ghaffari A, Ardakani A, Neil DH, Miller C. In vitro antibacterial effect of gaseous nitric oxide on common bacterial strains contributing to wound infections: Pseudomonas aeruginosa and Staphylococcus aureus. 2nd International Conference of Nitric Oxide, Prague, Czech Republic, 2002. Ghahary A, Ghaffari A, Miller C. In vivo effects of exogenous gaseous nitric oxide on Staphylococcus aureus infection in a healing rabbit wound model (abstract). Wound Caring Symposium, Canadian Association of Wound Care, Vancouver, Canada; November, 2002: 23-24. Ghaffari A, Miller C, Ardakani A, Kilani R, Karami A, Ghahary A. Exogenous nitric oxide gas in chronic wound infection. Wound Repair & Regeneration 2003; 11(2): A11. Ghaffari A, Ardakani A, Miller C C, Ghahary A. Viability of Human Fibroblast Cells Exposed to Gaseous Nitric Oxide. 8th Annual Conference of the Canadian Association of Wound Healing, Vancouver, 2002. Data on file. Nitric BioTherapeutics, Inc. sponsored study conducted by Bridge PTS (Texas), May 2008."],"set_id":"71067c14-2a02-47cc-95b7-b9f62ea8066c","spl_product_data_elements":["Nitric Oxide Nitric Oxide Nitric Oxide Nitric Oxide Nitrogen Nitric Oxide Nitric Oxide Nitric Oxide Nitric Oxide Nitrogen"],"spl_unclassified_section":["COMPRESSED GAS, N.O.S. (Nitric Oxide, Nitrogen USP) UN1956 Rx Only Caution: New Drug--Limited by Federal law to investigational use. *Not for inhalational use.* Use only in accordance with your Institutional Review Board approved protocol.","Occupational Exposure The exposure limit set by the Occupational Safety and Health Administration (OSHA) for inhaled Nitric Oxide is 25 ppm (0.0025%), and for NO 2 the limit is 5 ppm (0.0005%).","CAUTIONS Caution: New Drug--Limited to investigational use. Not for Inhalational Use. Use only in accordance with your Institutional Review Board approved protocol.","Manufactured For: Nitric BioTherapeutics, Inc 2 Canal's End Road, Suite 201-A Bristol, PA 19007 USA Product Inquiry Phone: 215-788-6200 Manufactured by: Airgas Specialty Gases 12722 South Wentworth Avenue Chicago, IL 60628 USA Label No. 6/2008—2"],"storage_and_handling":["Cylinders should be kept in a cool, well-ventilated area. Cylinders should be stored upright and firmly secured to prevent falling or being knocked over. Cylinder temperatures should not exceed 52°C (125°F)."],"teratogenic_effects":["Category C Animal reproduction studies have not been conducted with Nitric Oxide. It is not known if Nitric Oxide can cause fetal harm when administered to a pregnant woman or can affect reproductive capacity. Although other Nitric Oxide products have been approved by the Food and Drug Administration for inhalation, Nitric Oxide products have not been previously approved by the Food and Drug Administration for topical use in adults; however, usage in adults is currently under investigation."],"version":"1"}