Patent Application
20070298126
The formation of I2 in an aqueous composition is preferably generated in accordance with this invention from a reaction by an oxidant and a reductant in a manner such that at least 40% of the total iodine present in the aqueous composition is I2 at a minimum concentration of I2 above about 25 ppm. The concentration of I2 contemplated in this application ranges from 25 ppm up to 250 ppm with at least 40%, but preferably at least 50%, of the total iodine being in the form of 12. The preferred concentration of I2− is from 25 ppm to 150 ppm. The most preferred concentration of molecular iodine is from 50 ppm to 75 ppm.
The concentration of total iodine contemplated in this application ranges from 25 ppm up to 500 ppm. The preferred concentration of total iodine is from 25 ppm to 300 ppm. The most preferred concentration of total I2 is about 50 to 150 ppm.
The ratio of I2 contemplated in this application ranges from 40 to 100%. The preferred ratio of molecular iodine is from 70 to 100%. The most preferred ratio of molecular iodine is 100%.
The rate of iodine generation is defined herein, as the time required for the generation of I2 to reach a maximum value. The rate of iodine generation contemplated in this application ranges from seconds for diffusion controlled reactions such as that between iodide and iodate in an aqueous environment to a high of 15 minutes. The most preferred rate of I2 generation contemplated in this application is 2-5 seconds. It is possible to practice this application by diluting stable compositions of iodine and then immediately applying the product of said dilution. This approach is also contemplated under the current application and would be considered to have an instantaneous rate of I2 generation.
Numerous methods known in the art can be utilized to generate I2 as contemplated in this application. For in situ generation of I2 from iodide the most common oxidants are active chlorine compounds and hydrogen peroxide. The preferred oxidant to generate I2 from iodide is iodate. Iodate can be introduced as a salt from the following group: calcium iodate, sodium iodate, potassium iodate, magnesium iodate, zinc iodate, ammonium iodate, and the like. Molecular iodine can also be generated by dilution of formulations that contain complexed iodine or by dissolution of elemental iodine as is done in several devices utilized for water disinfection.
Suitable dry sources of iodide anion include sodium iodide, calcium iodide, ammonium iodide, magnesium iodide, zinc iodide and potassium iodide as well as other salts of iodide. Any compound that yields iodide anion upon dissolution in an aqueous environment is suitable for this application. The simple salts of iodide are preferred and have the advantage of being less costly. Additionally, they have a long shelf life in solid and liquid form
The types of compositions contemplated under this application include liquids, gels, creams, ointments and emulsions with the proviso that oil-based creams and emulsions are not contemplated in this application. The type of composition is not a determinative aspect of this application rather the absolute and relative concentration of I2 and complexed I2 are the two most critical aspects of this invention. Examples of the different types of compositions are provided, by way of example, in the Examples section of this application. It is clear from these experiments that many different types of compositions are compatible with the teachings of this application.
The thickeners useful in the context of the invention are preferably taken from the group consisting of alkyl celluloses, the alkoxy celluloses, xanthan gum, guar gum, polyorgano sulfonic acid and mixtures thereof. The thickeners are chosen based on compatibility with the other formulation ingredients and desired viscosity. Generally speaking the thickener should be present at a level of from about 0.01-10% by weight, and more preferable from about 0.1-1% by weight.
Cyclodextrins are crystalline, water soluble, cyclic, non-reducing, oligosaccharides comprised of glucopyranose units. I2 is substantially less hydrophilic than water, and therefore has the potential to be included in the cyclodextrin cavity in the presence of water. Such complexation of I2 with a cyclodextrins reduces its capacity to evaporate. There are three classes of cyclodextrins (i.e., α, β and γ) comprised of 6, 7 and 8 glucopyranose units respectively. Cyclodextrins are potentially useful for the formulations contemplated in this application since they can bind I2 and thereby reduce the effective vapor pressure of I2 in a formulation.
Suitable buffers for the compositions contemplated in this application include water and hydroalcoholic mixtures buffered with glycine, phthalic acid, citric acid, phosphates, dimethylglutaric acid, acetate, succinic acid, phthalic acid, malic acid, boric acid, and the like. The commonly available salts of these agents, e.g. sodium citrate, potassium phosphate, calcium maleate, are equally suitable for use in this compositions contemplated in this application.
Generally, any dispersible conditioning agent, humectants and emollients, known to those of skilled in the art may be used in the present invention. Preferred emollients to be used in the invention are taken from the group consisting of glycerin, propylene glycol, sorbitol, lanolin, lanolin derivatives, polyethylene glycol, aloe vera, glucamate polyethoxylated glucose dioleates containing at least 100 ethoxy units in the polyethylene glycol moiety, available, polyethoxylated methyl glucose containing at least 10 ethoxy units, allantoin, alginates, monoester salts of sulfosuccinates, alphahydroxy fatty acids, esters of fatty acids, ceramides, and mixtures thereof. Broadly, the conditioning agents are used at a level of from about 0.5-20% by weight. The most preferred conditioning agents are sorbitol, mineral oil, glycerin and/or mannitol, and are usually employed at a level of from about 1-20% by weight, and more preferably from about 2-10% by weight.
Chelating agents or sequestrants can be useful stabilizing agents in the invention particularly when a complexed form of iodine is present. Commonly available chelating agents can be used in the invention including both inorganic and organic chelating agents. Organic chelating agents include alkyl diamine polyacetic acid, chelating agents such as EDTA (ethylenediamine tetracetic acid tetrasodium salt), acrylic acid and polyacrylic acid type stabilizing agents, phosphonic acid and phosphonate type chelating agents and others. Preferable organic sequestants include phosphonic acids and phosphonate salts including 1-hydroxy ethylidene-1,1-diphosphonic acid, amino [tri(methylene phosphonic acid)], ethylene diamine [tetra(methylene-phosphonic acid)], 2-phosphonobutane-1,2,4-tricarboxylic acid as well as alkali metal salts, ammonium salts, or alkyl or alkanol amine salts including mono-, di- or triethanol amino salts. Inorganic chelating agents include commonly available polyphosphate materials such as sodium pyrophosphate, sodium or potassium tripolyphosphate along with cyclic or higher polyphosphate species. Preferably, such a sequestering agent is used at a concentration ranging from about 0.05 wt % to about 0.5 wt % of the composition.
Commonly available organic acids that can be used in the invention include benzoic acid, mandelic acid, sorbic acid, citric acid, lower alkanoic acids and their food-grade salts, such as the sodium potassium or ammonium salts thereof. These organic acids, their salts, or mixtures thereof are present in the composition in an amount between about 0.010 to 0.5 percent by weight, preferably from 0.050 to 0.20 percent by weight. The presently preferred organic acids are mandelic acid, benzoic acid, citric acid and sorbic acid, with benzoic acid suitably present as sodium benzoate and sorbic acid suitably present as the free acid. Each of these acids, or their salts, and others, alone or in combinations, can be incorporated into the compositions contemplated in this invention.
The present invention demonstrates that a dose dependent application of molecular iodine reacts with Staphylococcus aureus enterotoxin superantigen and renders it incapable of binding to T-cell lymphocytes as measured by the failure of T-cells to synthesize and release various cytokines. The ability of iodine to interfere with T-cell binding of superantigen in a dose-dependent fashion is a novel observation of the present invention.
The teachings and examples in this application do not make any attempt to specifically enumerate the entire prior art in the area of topical iodine preparations. Excipients that are known to be compatible with iodine may also be of use with compositions and conditions described in this application. Such excipients include surfactants, thickeners, humectants, emollients, skin conditioning agents, stabilizing agents, opacifiers, wetting agents, essential oils, chelating agents, buffers, preservatives, organic acids and fragrances.
Nasal secretions were gathered from 10 volunteers (7 males and 3 female) after exercise in cold air (between 20 and 35° F.); four of the volunteers had colds. Dripping or blown secretions were collected in plastic graduated beakers (Fisher, Scientific) and the tops were covered with Parafilm M. The initial samples were frozen until all samples were collected. Samples were mixed with water (2 part sample to 1 part water (v/v)) and vortexed in a pulsatile manner until all samples were substantially homogeneous and uniform aliquots were able to be removed with a pipette.
Iodine crystals (ACS Reagent Grade, Sigma-Aldrich) were placed in a 1 liter volumetric flask and then 0.01N HCl was added to the flask QS to 1 liter; a rubber stopper was placed in the top of the flask to prevent evaporation. The rubber stopper had a small glass tube inserted through it; the top of this tube was sealed with parafilm. The I2 crystals were stirred at room temperature for 3 hours with a magnetic stir bar and magnetic stir plate. After two hours the glass tube was pushed down such that the bottom of the tube was located at a point about 3 inches above the bottom of the flask. Samples of the saturated I2 solution were withdrawn through this glass tube by using 50 mL glass syringe with an 18 gauge hypodermic needle that had a thin plastic tube (PVC ID 0.046″) attached to its end. The stopper was therefore maintained on the flask at all times. Samples of the stock I2 solution were withdrawn and the concentration of I2 was determined to be 330 ppm using the potentiometric method of Gottardi.
A 0.25 mL aliquots of the vortexed nasal secretions were placed in a 1 dram vial (15×45 mm) vial and a cap was placed on the top. Aliquots of a pH 5.0 citric acid buffer (100 mM) was placed in 7 dram vials (29×65 mm) and sealed by placing a thin plastic cap onto the vials. One mL of the stock I2 solution was injected into the vials containing 1, 3, 6, 10 or 15 mL of the 100 mM citric acid buffer; this yielded solutions containing 165, 83, 47, 30 and 21 ppm 12. A sample (0.25 mL) was withdrawn from each I2 solutions and injected through the plastic cap into the samples of vortexed nasal secretions; the combined samples were mixed by vortex. A control sample received 0.25 mL of citric acid buffer without any 12. All samples were allowed to incubate at room temperature for 10 minutes.
After ten minutes 1.0 mL of 0.5% sodium thiosulfate was added to each sample including the control. Trypticase Soya Agar (TSA) plates were inoculated by spreading 0.5 mL of each sample across the surface of the TSA plates. Plates were incubated for 24 hr at 37 degrees Centigrade. The plates were examined for the presence of bacterial colonies after 24 hours of incubation. The nasal cavity is conducive to bacterial replication since the mucopolysaccharides provide a source of nutrients; consequently, all bacteria need to be eliminated for an agent to be effective. Consequently, plates were scored as either positive (the presence of colonies) or negative (the absence of colonies). The results are shown in Table 1 and indicate that nasal secretions affect the ability of I2 to inactivate pathogens. This result is not surprising since the mucopolysaccharides that comprise nasal secretions contain a relatively high percentage of sulphydral groups.
The minimum concentration of I2 necessary to eliminate S. aureus from the nasal cavity was evaluated in human volunteers. Thirty-five adult volunteers were used to evaluate the ability of different concentrations of I2 to eliminate S. aureus from the nasal cavity. Specimens were taken from the anterior nares of adults by swabing the anterior 1.5 cm of each nasal vestibule with a BBL CultureSwab. The swab was rotated 4 times around the inner walls of each nasal opening and then placed into Stewart's medium and transported to the lab for evaluation. TSA II plates were inoculated with the swabs. The plates were inoculated at 37° C. in a non-CO2 incubator. Following incubation, the TSA II plates were examined for colonies suggestive of S. aureus. S. aureus were identified using standard methods including the Gram stain and coagulase testing. The volunteers were screened for nasal carriage of S. aureus on five separate occasions, 1 week apart. Only persistent carriers (i.e., at least 80% cultures positive) were used for the test.
The test article consisted of a two component get-liquid system. The gel and liquid were mixed prior to application in the nasal cavity with a swab. The gel was prepared using USP citric acid (10% w/v), NF glycerin (10% w/v), NF carboxymethylcellulose (0.75% w/v), NF and boric acid (0.3% w/v); the pH of the gel was adjusted to 3.0 with sodium hydroxide. An aqueous mixture of USP sodium iodide (0.354% w/v) and FCC potassium iodate (0.303%) in sodium carbonate (0.2% w/v) was prepared. The 12 treatment was prepared prior to use by mixing 9 parts of the gel with 1 part of the aqueous solution; this yielded a mixture with 300 ppm I2 as determined by the potentiometric method of Gottardi and by thiosulfate titration.
Seven different concentrations of I2 were used. The different I2 treatments were prepared by mixing different amounts of the carboxymethylcellulose (CMC) gel with the aqueous mixture of iodide/iodate. Table 2 identifies the concentrations of I2 and the relative volumes of gel-iodide/iodate solution used.
Chronically colonized volunteers were treated with test article for five (4) consecutive days. On each day of treatment the volunteers the activated gel was applied before the start of the work day and then 6 hours later. The CMC gel was mixed with the iodide/iodate mixture and then applied to each of the nostrils of each volunteer with sterile cotton tipped swabs. The CMC gel was activated and then applied within 5 minutes. To apply the gel the swabs were dipped into the activated gel and then rotated inside each nostril; this was done two times for each application with each nostril. Before treatment a BBL CultureSwab was taken to confirm the presence of S. aureus; a second BBL CultureSwabs was taken 24 hours after treatment had ended. Volunteers were also evaluated 1 and 2 weeks after the last treatment.
A gel of cross-linked acrylic acid was used to evaluate the yield of I2 versus several formulation variables. Cross-linked acrylic acid polymers have rheological properties that may render them useful for use in the nasal cavity. In addition, cross-linked acrylic acid polymers are odor free and have a high number of carboxylic acids groups on the polymer, which helps to maintain a stable acidic pH. Three different gels (B182, NoE-026, NoE-004) were prepared to explore the yield of I2 versus different concentrations of the excipients in the formulation.
A beaker was charged with about 400 ml of water; the polyacrylic acid polymer of interest, e.g. Carbopol 980 NF, was then added and stirred on a Lightnin LabMaster mixer at 800-1000 rpm for 1-2 hour until the Carbopol was hydrated and then the glycerin was added. A solution containing EDTA, boric acid, and 10 N NaOH in 80 ml of water was then added to the mixture. The mixture was then stirred for 1 hour at 600 ppm and stored at room temperature and then QS to 1 liter. A stock solution of sodium iodide and sodium iodate was prepared for admixture with the gel in order to generate defined amounts of 12. Sodium iodide (0.60 grams) and sodium iodate (2.0 grams) was dissolved in 120 ml of water that contained 1.4 ml of 10 N NaOH. The final pH of the gels was between 4.5 and 6.0.
One ml of gel was mixed with 1.0 ml of the stock iodide/iodate mixture. The reactions were stopped at 0.5, 1.0, 2.0, and 4.0 minutes. The gels were then extracted with 10 ml of chloroform and 50 ml of a 1.0 N phosphate buffer pH 4.8 that contains 300 grams of sodium sulfate per liter. The absorbance at 520 nm was measured in a Schimadzu UV-1602 spectrophotometer. Gel NoE-026 was tested prepared freshly and compared to NoE-026 gel stored at 40° C. for 4 months. The yield of I2 was above 50% in all instances; storage of the gels did not appear to impact the yield of 12. The rate of the reaction between iodide and iodate is known to be diffusion controlled and it is not surprising that the yield of I2 was not a function of time.
The perceived I2 odor of the formulations contemplated in this application bear directly on utility. The actual potential to generate a detectable odor from I2 in a particular formulation must be measured and cannot be predicted for most formulation matrices other than water. This experiment provides a quantitative means of characterizing the perceived odor from the complex compositions contemplated in this application based upon the I2 odor perceived by humans in an aqueous medium.
Several different concentrations of pure I2 in 0.1N HCl were prepared by dissolution of I2 crystals (Sigma-Aldrich Cat No.266426-250G) in a glass volumetric flask with stopper in place. A 1 inch strip of potassium iodide starch paper (Whatman International, Ltd, Cat No. 2602-500A) was completely moistened with distilled water and vertically aligned flush to the surface of the inside of a 50 mL self-standing graduated plastic tube (Corning Cat No. 430897). Once the start paper was adhered to the inner side of the wall 150 μl of the I2 solutions were transferred into the bottom of the plastic tube; the bottom section of these tubes are conical in shape and hold this volume of fluid in a relatively well defined area. Once the samples were transferred into the graduated plastic tube the top was immediately screwed on and a stopwatch was started. The time required for the starch paper to turn blue was recorded in seconds.
Five volunteers (3 male; 2 female) were used to evaluate the odor from the aqueous solutions of I2. An odor free room not adjacent to a laboratory, cafeteria or bathroom was used for the evaluation. The individuals selected to evaluate the iodine odor did not have colds or allergies. Tests were conducted in the morning and volunteers were instructed to shower on the morning of the test and not to use lotions or after shave on that morning. The volunteers were instructed to identify any sample that provided an unpleasant odor. None of the volunteers detected any odor at I2 concentrations of 22.5 ppm or less. All of the volunteers were able to detect the presence of a aroma above 55 ppm but this odor was not deemed to be objectionable (4 out of 5 volunteers) until the concentration of I2 was 275 ppm and above.
This experiment demonstrates that a dose dependent application of I2 reacts with superantigens, such as Staphylococcus aureus enterotoxin B (SEB), rendering them incapable of binding to T-cell lymphocytes. Stimulation of T-cells by superantigen binding is required for cytokine synthesis. The inventors were investigating methods of disinfecting cultured mammalian cells to kill bacteria or fungi/yeast without killing the mammalian cells. The mammalian cells chosen were human peripheral blood leukocytes (PBL) collected fresh using BD vacutainer CPT cell preparation tubes with sodium citrate. Previously described procedures were used to yield highly enriched lymphocytes, which include both B- and T-cells. Staphylococcus aureus was obtained from a local hospital on an agar slant. The S. aureus isolate was from a patient suffering from food poisoning. The S. aureus isolate expressed SEB.
Lymphocytes (106/mL counted by hemocytometer) were suspended in Hanks' balanced salt solution with HEPES (3 mM) and 2% (v/v) fetal bovine serum and varying amounts of S aureus were added ranging from 103-105 cfu/mL and assays were incubated at 37° C. for one hour. After one hour 500 microliters of I2 was added to various cultures in various concentrations (0.1-100 ppm free molecular iodine, final concentration). After an additional 10 minutes 200 microliters of a 2N solution of sodium thiosulfate was added; 100 microliter aliquots were removed from each reaction vessel and streaked for isolation of S. aureus on nutrient agar plates; reaction tubes were then returned to the incubator (37° C.). The results were somewhat predictable. Lymphocyte-S. aureus (103-105) cultures treated with 0.1-10 ppm of the iodine had viable S. aureus that grew on the agar.
Reaction vessels with 10-100 ppm I2 had no viable S. aureus. However, the surprising discovery occurred three days later. Each reaction vessel was examined using the microscope/hemocytometer to observe and count the number of lymphocytes. Reaction vessels with 105 S. aureus and 10-ppm iodine had 100-fold more lymphocytes (˜108/mL) than similar cultures treated with 100 ppm I2. These results were confusing. We knew that the S. aureus strain used in these experiments expressed SEB, a known superantigen. We predicted that something triggered the lymphocyte proliferation at 10 ppm I2 but not at 100 ppm I2 and assumed that it was S. aureus SEB. The key assumption was that I2 blocked lymphocyte proliferation at higher I2 concentrations because somehow the I2 blocked a reaction between S. aureus and lymphocytes. These results prompted us to perform experiments that might explain the proliferation result.
We decided that the most direct way to test our hypothesis was to mix S. aureus SEB with I2; neutralize the mixture with sodium thiosulfate and then treat fresh lymphocytes with the neutralized solution. We could then measure cytokine synthesis following interaction with T-cells. We chose to measure interleukin 6 (IL-6) and interferon gamma (IFN-γ) as markers of T-cell stimulation based on published studies. All reagents including S. aureus SEB (US Biological, Swampscott, Mass.; cat# S7965-35A), purified mouse anti-enterotoxin B monoclonal IgG antibody, purified mouse monoclonal IgG to interferon IFN-γ and the cytokine IL-6 were purchased from commercial sources. The control experiments were conducted using fresh PBL and 1 pg/mL SEB. Two site capture ELISA immunoassays in 96 well microtiter plates were purchased as commercial kits IFN-γ (eBioscience, San Diego, Calif.; cat# 88-7314-76) and IL-6 (eBioscience, San Diego, Calif.; cat# 88-7066). The enzyme used was horseradish peroxidase (HRP) and the linkers were biotin-streptavidin, substrate was tetramethylbenzidine (TMB); color development was terminated with sulfuric acid and color was read at 570 nm with a Schimadzu UV-1602 spectrophotometer. The optical density of each unknown was determined and compared to the concentrations of IL-6 and IFN-γ obtained using standards supplied with each commercial kit.
Standard curves were prepared for both IL-6 (6-200 pg/mL) and IFN-γ (0.1-3.0 ng/mL). I2 was prepared fresh at a stock concentration of 330 ppm/mL and aliquots were added to various reaction tubes to achieve the desired final iodine concentration. SEB (10 microliters) was mixed undiluted with I2 (10 microliters) and buffer (IM citrate buffer pH 5.0) at room temperature for 30 minutes. After 1 hour, 5 μL of 2N sodium thiosulfate was added to all samples and gently agitated to insure complete neutralization of the I2. PBL cells, the iodinated SEB were gently mixed in reaction tubes and placed at 37° C. After 1 hour, cells were pelleted and 5RL was removed from the supernatant of each tube and analyzed for the presence of cytokines IL-6 and IF-γ in the cell-free fraction. Samples of the supernatant were also collected at 12, 24, 36 and 48 hours and analyzed for IL-6 and IFN-γ. The sample wells of the ELISA immunoassays 96 well microtiter plates were washed two times after binding of label to insure removal of all of the sodium azide used to preserve SEB. The results of these assays (shown below) demonstrate that at a concentration of >25 ppm I2 inhibits the ability of superantigens to activate T-cells synthesis of cytokines.
