CURE AIDS NOW

CURE AIDS NOW IS A NON PROFIT PLAN TO ERADICATE HIV / AIDS WITH AN INTERNET TECHNOLOGY SOLUTION PARTNER AND A USPTO UTILITY PATENT PARTNER APPROVED BY THE EPA AND FDA

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THE PATENTED SILVER “CURE” A TREATMENT TO REDUCE AND ELIMINATE VIRAL SERUM DETECTION OF HIV/AIDS

Retrovirus Infection (HIV). The method comprises the step of administering a silver composition, comprising 5 to 40 ppm silver one to five times a day orally area until there was a response. One patient exhibiting HIV (human immunodeficiency virus )was treated with about 5 ml (approximately one teaspoon) of a composition of the present invention two times per day. The patient’s symptoms resolved within five days.
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United States Patent 7,135,195
Holladay , et al. November 14, 2006
Treatment of humans with colloidal silver composition

CURE AIDS NOW PATENT # 7,135,195

Abstract
We disclose a colorless composition comprising silver particles and water, wherein said particles comprise an interior of elemental silver and an exterior of ionic silver oxide, wherein the silver particles are present in the water at a level of about 5 40 ppm, and wherein the composition manifests significant antimicrobial properties. Methods of use of the composition are described.

Inventors: Holladay; Robert J. (Logan, UT), Christensen; Herbert (Alpine, UT), Moeller; William D. (Alpine, UT)
Assignee: American Silver, LLC (Alpine, UT)
Family ID: 34317391
Appl. No.: 10/641,938
Filed: August 15, 2003
Prior Publication Data
Document Identifier Publication Date
US 20050061678 A1 Mar 24, 2005
Related U.S. Patent Documents
Application Number Filing Date Patent Number Issue Date
09946834 Sep 4, 2001 6743348
09323310 Jun 1, 1999 6214299
60475657 Jun 3, 2003
Current U.S. Class: 424/618; 424/405; 424/489; 424/490; 424/616; 514/849; 514/858; 514/894; 514/895; 514/924; 514/931; 514/932; 514/934; 514/951
Current CPC Class: A61K 9/0014 (20130101); A61K 9/0043 (20130101); A61K 9/0046 (20130101); A61K 9/0095 (20130101); A61K 33/38 (20130101); A61K 33/40 (20130101); A61K 45/06 (20130101); A61K 47/10 (20130101); A61K 47/32 (20130101); A61L 2/16 (20130101); A61L 2/238 (20130101); B01F 7/22 (20130101); B01F 13/0001 (20130101); B01F 13/0003 (20130101); B01J 13/0043 (20130101); B01J 19/087 (20130101); B01J 19/18 (20130101); C02F 1/4606 (20130101); A61K 9/0034 (20130101); A61K 33/38 (20130101); A61K 33/40 (20130101); Y10S 514/895 (20130101); A61L 2/186 (20130101); B01J 2219/082 (20130101); B01J 2219/0835 (20130101); B01J 2219/0841 (20130101); B01J 2219/089 (20130101); C02F 2303/04 (20130101); Y10S 514/932 (20130101); Y10S 514/858 (20130101); Y10S 514/934 (20130101); Y10S 514/951 (20130101); Y10S 514/931 (20130101); Y10S 514/894 (20130101); Y10S 514/849 (20130101); Y10S 514/924 (20130101); A61K 2300/00 (20130101); A61K 2300/00 (20130101)
Current International Class: A61K 33/38 (20060101); A01N 25/12 (20060101); A01N 25/26 (20060101); A01N 59/00 (20060101); A01N 59/16 (20060101); A61K 33/40 (20060101); A61K 9/10 (20060101); A61K 9/14 (20060101); A61P 11/00 (20060101); A61P 13/00 (20060101); A61P 15/00 (20060101); A61P 15/02 (20060101); A61P 17/00 (20060101); A61P 31/00 (20060101); A61P 31/02 (20060101); A61P 31/04 (20060101); A61P 31/10 (20060101)
Field of Search: ;424/618,405,489,490,616 ;514/849,858,894,895,924,931,932,934,951
References Cited [Referenced By]
U.S. Patent Documents
6214299 April 2001 Holladay et al.
6743348 June 2004 Holladay et al.
6890953 May 2005 Arata
6939568 September 2005 Burrell et al.
2002/0051823 May 2002 Yan et al.
2003/0185889 October 2003 Yan et al.
Primary Examiner: Pak; John
Attorney, Agent or Firm: Liner, Yankelevitz, Sunshine & Regenstreif LLP
Parent Case Text

The present application is a continuation-in-part of application Ser. No. 09/946,834, filed Sep. 4, 2001, now U.S Pat. 6,743,348 which is a continuation of application Ser. No. 09/323,310, filed Jun. 1, 1999, now U.S. Pat. No. 6,214,299. The present application claims priority to provisional application 60/475,657, filed Jun. 3, 2003, and incorporated by reference herein.
Claims

We claim:

1. A composition of silver in water comprising a total concentration of silver of between about 5 and 40 parts per million, said silver in the form of colloidal silver particles having an interior of elemental silver and a surface of silver oxide, wherein a majority of the colloidal silver particles have a maximum diameter less than 0.015 micrometers, wherein a majority of the colloidal silver particles have a minimum diameter greater than 0.005 micrometers, and wherein the composition exibits antimicrobial properties.

2. The composition according to claim 1, wherein at least 75% of the colloidal particles have diameters between 0.005 micrometers and 0.015 micrometers.

3. The composition according to claim 2, wherein at least 90% of the colloidal particles have diameters between 0.005 micrometers and 0.015 micrometers.

4. The composition according to claim 3, wherein at least 95% of the colloidal particles have diameters between 0.005 micrometers and 0.015 micrometers.

5. The composition according to claim 1, wherein the colloidal particles have an average diameter of about 0.0106 micrometers.

6. The composition according to claim 1 further comprising hydrogen peroxide.

7. The composition according to claim 6, wherein the hydrogen peroxide concentration is between about 1% wght/v and about 3.0% wght/v.

8. The composition according to claim 1, wherein the composition exhibits antimicrobial properties against microbes selected from the group consisting of Bacillus anthracis, Bacillus subtilis, Candida albicans, Mycobacteria bovis, Mycobacteria tuberculosis, Pseudomonas aeruginosa, Salmonella choleraesius, Staphylococcus aureus, Trichomonas vaginalis, and Yersinia pestis.

9. The composition according to claim 8, wherein Staphylococcus aureus is a methicillin-resistant strain.

10. The composition according to claim 1, wherein the composition exhibits antimicrobial properties against microbes associated with diseases selected from the group consisting of malaria, fungal infections of the skin, bacterial infections of the skin, vaginal infections, urinary tract infections, tonsillitis, pelvic inflammatory disease, pharyngitis, gonorrhea, conjunctivitis, otitis, respiratory tract infections, and nasal infections.

11. The composition according to claim 1, wherein the antimicrobial properties are antiviral properties.

12. The composition according to claim 11, wherein the antiviral properties are exhibited against a virus selected from the group consisting of human immunodeficiency virus and hepatitis B virus.

13. The composition according to claim 11, wherein the antiviral properties include inhibition of viral DNA polymerase.

14. The composition according to claim 11, wherein the antiviral properties include inhibition of viral reverse transcriptase.
Description

AREA OF THE ART

The present invention generally relates to colloidal silver, and more particularly to a composition of colloidal silver and a method for using said composition as an agent against organisms harmful to the health of humans.

DESCRIPTION OF THE PRIOR ART

It is well known that certain preparations of silver have germicidal properties. Silver was employed as a germicide and an antibiotic before modern antibiotics were developed. In previous centuries, users would shave silver particles into their drinking water, or submerge whole silver pieces in the drinking water, for the purpose of ingesting the silver by drinking the water. It seems likely that the practice of eating with silver utensils (i.e., silverware) resulted from a belief in the healthful properties of silver.

There may be many reasons why administering silver suspended in solution would enhance an individual’s health. It is possible that such a solution operates to inhibit the growth of bacteria, viruses, and other unwanted organisms, as well as eradicating such existing bacteria, viruses, and other organisms. It is also possible that a solution of silver can have an anti-inflammatory effect, sufficient to reduce symptoms of asthma.

The present invention describes the use of a silver composition in water to treat certain human ailments. An embodiment of the invention is a silver composition comprising small particles of silver which comprise an interior of metallic silver and an exterior of ionic silver which particles are suspended in water. A preferred embodiment of the invention is a silver composition comprising particles of silver wherein more than 50% of the number of particles are less than 0.015 micrometers in size and the particles are colloidally suspended in water.

SUMMARY OF THE INVENTION

The present invention is generally directed to the use of silver, at a level of 5 to 40 ppm in water, to kill or to disable microorganisms which are hazardous to human beings. The present invention specifically is directed to compositions comprising silver particles, said particles comprising an interior of elemental silver and an exterior of ionic silver oxide, and water, wherein the silver particles are placed in colloidal suspension in the water at a level of 5 40 ppm total silver. An embodiment of the present invention comprises silver particles in water, at a concentration of 5 40 ppm, wherein more than 50% of the silver particles have a maximum dimension less than 0.015 micrometers. The composition of silver in water of this invention is an effective antimicrobial agent. This invention is directed to silver compositions, of 5 40 ppm silver in water, which are effective antimicrobial agents, and to methods of using said silver compositions as antimicrobial agents.

A preferred embodiment of the present invention is directed to compositions of silver in water made using a modification of the device and methods described in U.S. Pat. No. 6,214,299, which is a parent of the instant application and is incorporated herein by reference.

The device and process of Pat. No. 6,214,299 have been modified and improved to provide the silver composition of the present invention. Essentially, the eight-silver/one common electrode device as disclosed in the patent has been modified and scaled to fit a 75-gallon water chamber. To start the process approximately 70 gallons of high purity water are placed in the chamber. To this is added approximately five gallons of silver composition produced in a prior production run. This is necessary because the high purity water is insufficiently conductive for the process to occur properly. The water chamber is equipped with an air input that allows a stream of air bubbles to be streamed through the liquid during the processing. It has been discovered that this approach gives improved mixing as compared to the impeller mixer described in the patent.

The electrode device is operated at approximately ten thousand volts alternating current (with each silver electrode having an individual voltage supply) as described in the patent. It has been found that voltages significantly lower than this produce a composition with larger particles not having the optimal properties described herein. Voltages significantly higher tend to produce a solution with significant ionic silver dissolved therein. The present composition comprises in excess of 97% metallic silver with essentially no free ionic silver in solution.

The silver concentration is determined according to the methods explained below. Essentially, the device is operated continuously and samples are analyzed until the desired silver concentration is attained. The 10 ppm composition requires approximately one and one half days of operation. The 22 ppm solution requires approximately three days, and the 32 ppm composition requires approximately six days. The rate of the process appears to slow as the higher concentrations are attained. Higher concentrations take a prohibitively long time with the ultimate highest concentration being about 50 ppm, at least within the current parameters.

The compositions all have the size characteristics described below and unlike conventional silver compositions are completely colorless and stable to light and temperature changes without use of any additives. The compositions are unreactive towards added hydrogen peroxide.

Hydrogen peroxide, a know disinfecting agent, has been found to have a synergistic interaction with the inventive silver composition. Hydrogen peroxide is available in concentration of 30% wght/v (% weight per volume or weight percent) or higher. Although the higher concentrations are usable, the preferred concentrations are in the range of 1 to 5% wght/v.

A preferred embodiment of the present invention is directed to compositions comprising 5 to 40 ppm silver, said silver being primarily elemental silver, 1 to 3 wght % hydrogen peroxide, and water. A preferred embodiment of the present invention is the use, and method of use, of compositions comprising 10 to 40 ppm silver and 1 to 3 wght % hydrogen peroxide in water as antimicrobial agents.

DETAILED DESCRIPTION OF THE INVENTION

The following description is provided to enable any person skilled in the art to make and use the invention and sets forth the best modes contemplated by the inventor of carrying out his invention. Various modifications, however, will remain readily apparent to those skilled in the art, since the general principles of the present invention have been defined herein specifically to provide an improved colloidal silver product with significant abilities to kill human pathogens both in vivo and in vitro.

Generally, the present invention represents a novel approach to killing or disabling microorganisms which are hazardous to human beings by the use of silver particles in water, at a concentration of 5 to 40 ppm silver. Depending upon the application, the silver composition may be used internally or externally. Depending on the application, the silver composition may also contain hydrogen peroxide.

PREFERRED EMBODIMENTS

Non-limiting preferred embodiments are presented in the following:

A composition comprising silver particles, colloidally suspended in water, wherein the total content of silver is between 5 and 40 ppm, which composition kills or disables microorganisms which are hazardous to the human body.

A composition comprising silver particles, colloidally suspended in water, wherein the total content of silver is about 10.+-.2 ppm, which composition kills or disables microorganisms which are hazardous to the human body.

A composition comprising silver particles, colloidally suspended in water, wherein the total content of silver is about 22.+-.2 ppm, which composition kills or disables microorganisms which are hazardous to the human body.

A composition comprising silver particles, colloidally suspended in water, wherein the total content of silver is about 32.+-.3 ppm, which composition kills or disables microorganisms which are hazardous to the human body.

It will be appreciated that specifying the total amount of silver in a composition of particles does not completely specify the material. As the particles comprising the composition are made smaller, a given concentration of silver will represent a larger number of particles. In addition, the total surface area for a given silver concentration will increase. Therefore, particles size and range of particle size is an important parameter for defining an effective inventive composition.

A further class of embodiments is any of the above-described compositions, wherein more than 50% of the silver particles have a maximum dimension less than 0.015 micrometers.

A further class of embodiments is any of the above-described compositions, wherein more than 75% of the silver particles have a maximum dimension less than 0.015 micrometers.

A further class of embodiments is any of the above-described compositions, wherein more than 90% of the silver particles have a maximum dimension less than 0.02 micrometers.

A further class of embodiments is any of the above-described compositions, wherein more than 75% of the silver particles have a minimum dimension greater than 0.005 micrometers.

A further class of embodiments is any of the above-described compositions, wherein more than 90% of the silver particles have a minimum dimension greater than 0.005 micrometers.

A further class of embodiments is any of the above-described compositions, wherein the silver particles comprise both silver in the

A further class of embodiments is any of the above-described compositions, wherein the silver particles comprise both silver in the zero-valent, that is, metallic, oxidation state [Ag(O)] and a coating of silver in an ionic oxidation selected from the group consisting of Ag(I), Ag(II), and Ag(III).

A further class of embodiments is any of the above-described compositions, wherein the silver particles comprise both silver in the zerovalent, that is metallic, oxidation state [Ag(O)] and a coating of silver oxide with the stoichiometry AgO.

Experimental evidence shows that AgO in the particles of the present invention is at least partially in the form of Ag.sub.4O.sub.4–that is, silver II oxide. In a molecule of this material two of the silver atoms are in the 1.sup.+ state (silver I) while the other two silver molecules are in the 3.sup.+ state (silver III). Under certain conditions these molecules can give rise to silver atoms in the 2.sup.+ (silver II) state.

A further class of embodiments is the combination of any of the above-described embodiments with hydrogen peroxide, at a level of 1 3 wgt % hydrogen peroxide in the final product.

EXAMPLES

1. Formation of Composition

Compositions of silver in water can be made according to procedures set forth in U.S. Pat. No. 6,214,299, incorporated by reference herewith.

A preferred method for producing a composition comprising silver according to this invention utilizes a electrochemical cell comprising electrodes and comprises the steps

(a) placing a silver electrode in contact with a quantity of high purity water;

(b) conveying electrical current through the silver electrode to thereby separate particles of silver from said silver electrode in a manner sufficient to cause production of suspended silver particles within the water; and

(c) agitating the water during said production of suspended silver particles to thereby disperse the silver particles into a more uniform concentration within said water such that a higher quantity of suspended silver particles can be produced per batch.

Another preferred method for producing a composition comprising silver utilizes an electrochemical cell and comprises the steps of:

(a) establishing an electrical circuit comprising a current source, and a first conductor electrically connected to said current source and a second conductor electrically connected to said current source, wherein said first conductor is disposed spaced apart from said second conductor, and wherein at least one of the conductors is made of elemental silver;

(b) closing the circuit by placing the first conductor and the second conductor in communication with a fluidic resistor;

(c) operating the current source to supply alternating current simultaneously to the first conductor and the second conductor such that voltage is increasing and decreasing within the first and second conductors in alternating tandem to thereby cause silver particles to separate from the first electrode and enter the fluidic resistor and become disposed in suspension within said fluidic resistor; and

(d) selectively adjusting the electrodes by moving them toward the fluidic resistor to compensate for decrease in electrode length due to gradual separation of silver particles therefrom to thereby prevent arcing from occurring between the electrodes and said fluidic resistor.

The analysis of the silver content in the silver compositions of this invention may be done by atomic absorption (AA), inductively coupled plasma/atomic emission (ICP/AES), or other techniques known to one of ordinary skill in the art to be sensitive to silver in the appropriate concentration range. If the particles of the silver composition are small and uniformly sized (for example, 0.01 micrometers or less), a reasonably accurate assay may be obtained by running the colloid directly by AA or ICP/AES. This is because the sample preparation for AA ionizes essentially all of the silver allowing its ready detection.

If the compositions comprise particles as large as 0.2 micrometers, it is preferred to use a digestion procedure. The digestion procedure is not necessarily ideal for silver compositions that may have been manufactured or stored in contact with halides or other anionic species that may react with finely divided silver, or combined with protein or other gelatinous material. An embodiment of the digestion procedure is as follows:

1 Take a 10 ml aliquot of a thoroughly mixed or shaken silver composition to be analyzed, and place it in a clean polycarbonate bottle or other container of suitable material (generally, the bottle) with a tight fitting lid. A size of 30 100 ml is preferred.

2 With a micropipette or dropper, add 0.1 ml of nitric acid, reagent grade to the silver composition in the bottle.

3 With the lid of the bottle tightly in place, heat the silver composition to 80.degree. C. with mild agitation for a time sufficient to dissolve the silver-dissolution is essentially instantaneous.

4 Allow the resulting mixture to cool to room temperature with the lid in place. Shake the bottle thoroughly.

5 Utilize AA, ICP/AES, or equivalent means to analyze the silver content of the silver mixture. Preferably, one will utilize a freshly prepared standard or standards, preferably prepared according the equipment manufacturer’s instructions, with appropriate dilution as needed.

6 When reporting results, one must taken into account all dilutions during preparation, including the 1% dilution caused by addition of the nitric acid.

1. Analysis of Physical/chemical Form of Silver

A. Introduction

A sample of a composition, nominally containing 22 ppm silver in water, was analyzed by time-of-flight secondary ion mass spectrometry (TOF-SIMS) in order to determine the form of silver in the composition. The conclusion is that the bulk of the silver exists as silver (O) [that is, metallic silver] and that there is a surface coating which as on average a composition of silver (II) oxide [AgO]. As mentioned above silver (II) oxide is usually a stoichiometric combination of silver (I) and silver (III).

B. Experimental Procedure

A few drops of the 22 ppm inventive silver composition were evaporated to dryness on a silicon substrate at ambient temperature. The residue was analyzed by TOF-SIMS, and is denoted as the sample. A reference silver (II) oxide (AgO) material was analyzed by placing a few particles of the reference powder as received from the vendor on a silicon substrate, and is denoted as the reference.

The Time-of-Flight Secondary Ion Mass Spectrometry technique (TOF-SIMS) is based on the principle of bombarding a solid sample with a pulsed, finely focused beam of primary ions, and then analyzing the secondary ions produced from the surface of the sample via a time-of-flight mass spectrograph. This analytical technique is surface sensitive, deriving its information from a layer that extends to approximately 20 to 40 .ANG. (one Angstrom=1.times.10-4 micrometers) below the surface. The TOF-SIMS technique is normally used as a survey tool to identify the composition of unknown samples. It is capable of quantification if the appropriate microanalytical standards are available for calibration. This analysis was carried out using standard high mass-resolution conditions.

C. Results

Negative ion mass were obtained for the Ag(II)O reference material and the product sample, respectively. The mass spectral region for both spectra showed the presence of AgO- species. The data suggest that silver (II) is the average oxidation state of the silver present on the surface of the sample particles. The silver oxide (AgO) signals exhibit significantly higher intensity in the reference sample compared to the product sample which is probably because metallic silver is dominant in the sample. It will be appreciated that as the average particle size in the sample is decreased the ratio of silver to silver oxide will also decrease as more silver oxide will be present.

2. Size Analysis

It is likely that the unusual effectiveness of the silver preparations described herein is due to the relationship between the surface properties/inner properties (i.e., oxide/metal) of the particles and the size distribution of the particles. The smaller the average particle size, the greater the surface area and the greater the contribution of the particular surface chemistry. However, if the particles are excessively small there can be a loss of stability and/or other interactions that negatively affect the product. The silver compositions of the instant invention are remarkable because they are stable in essentially pure water without surf actants, etc. Also, the materials are essentially colorless while other colloidal silver preparations (particularly with larger particle sizes) usually show colors. These properties are a result of the exact manufacturing conditions as discussed above.

Digital analysis of the composition showed that there is an average particle diameter of 0.0106 micrometers with a range of 0.005 micrometer to 0.0851 micrometers. However, size distribution analysis shows that more than 95% of the particles were between about 0.005 micrometers and about 0.015 micrometers in diameter.

3. Evidence of Efficacy of 22 ppm Silver Composition Against Bacillus Subtilis

A. Purpose of Example

The purpose of this example is to demonstrate the antimicrobial activity of the silver-based composition of the present invention on bacterial endospores from the test organism Bacillus subtilis. This was accomplished by performing a standard kill-time assay using a suspension of B. subtilis endospores. Normally, bacterial endospores are resistant to killing.

B. Material and Methods

Test Organism. A test suspension containing endospores from Bacillus subtilis (ATTC #19659) was prepared from a culture grown on nutrient agar, to which additional sporulation enhancement ingredients were added. Plates were harvested with sterile water and endospores were purified by repeated centrifugations and resuspensions in water. The final wash was in 70% ethanol for 30 min, to ensure the destruction of all vegetative bacteria. The spores were resuspended in water containing 0.1% Tween 80 (brand of polysorbate surfactant) to prevent clumping.

Neutralizer. The Neutralizer mixture consisted of 12.7% Tween.RTM. 80 (brand of polysorbate), 6.0% Tamol.RTM. SN (brand of sodium salt of naphthalene-formaldehyde condensate), 1.7% lecithin, 1% Peptone, and 0.1% Cystine. This solution was intended to neutralize any chemicals so they would not affect subsequent growth of the bacteria.

Kill-Time Procedure:

a) A 9.9 ml aliquot of the disinfectant (inventive 22 ppm silver composition, in water) was placed in a sterile 20 mm.times.150 mm tube. The tube was equilibrated in a 20.degree. C. water bath.

b) A 9.9 ml aliquot of the disinfectant (inventive 22 ppm silver composition, in water) was placed in a sterile 20 mm.times.150 mm tube. The tube was equilibrated in a 20.degree. C. water bath.

c) At 30 min. 1 hr, and 4 hr, one ml of organism/disinfectant suspension was removed to a tube containing nine ml of Neutralizer. The tube was mixed thoroughly.

d) After two min, the neutralized suspension was serially diluted 1:10, in physiological saline solution (PSS).

e) The number of viable organisms in selected dilution tubes was assayed by membrane filtration. One ml aliquots were plated in duplicate. The membranes were washed with about 100 ml of sterile PSS and removed to Nutrient Agar plates. The plates were incubated at 37.degree. C. for 20 hr.

f) The number of colonies on each filter was counted and log reductions were computed.

Controls:

a) Titers of the test suspensions were computed by performing membrane filtration assays of selected 1:10 dilutions of the test suspensions in PSS.

b) A neutralizer control was performed by inoculating a mixture of 9 ml neutralizer and 1 ml of disinfectant with 100 .mu.l of a dilution of the titer containing 100 cfu. This produced about 10 cfu/ml in the tube, which was allowed to stand for 20 minutes prior to assay by membrane filtration using duplicate 1 ml samples.

C. Results

Bacillus subtilis Titer:

TABLE-US-00001 Dilution: 1:1 .times. 10.sup.6 1:1 .times. 10.sup.7 1:1 .times. 10.sup.8 Number of colonies: TNTC 75 7 TNTC 58 8 TNTC = too numerous to count

TABLE-US-00002 Dilution of B. subtilus spore/disinfectant suspension: Time 1:1 .times. 10.sup.1 1:1 .times. 10.sup.2 1:1 .times. 10.sup.3 1:1 .times. 10.sup.4 1:1 .times. 10.sup.5 1:1 .times. 10.sup.6 30 min — – TNTC TNTC 57 10 — – TNTC TNTC 51 7 1 hr — – TNTC TNTC 28 3 — – TNTC TNTC 55 3 2 hr — TNTC TNTC 126 23 — – TNTC TNTC 183 17 — 4 hr TNTC TNTC 88 12 — – TNTC TNTC 69 12 — – TNTC = too numerous to count Neutralization Control: 1:1 .times. 10.sup.8

D. Discussion

Results of the titer showed a viable B. subtilis spore concentration of 6.65.times.10.sup.8 spores per ml in the original suspension. Inoculation of 9.9 ml of disinfectant with 100 .mu.l of this suspension produced an initial concentration of 6.65.times.10.sup.6 spores per ml in the assay tube.

Results from these procedures allowed log reductions (LR) and Percent Kill (PK) values to be calculated. They are listed in the table below. Values were computed using the formulae: LR=-Log(S/So) and PK=(1-(S/So)).times.100; where S=concentration of organisms at a specific time; and So=the initial concentration of organisms at time zero.

TABLE-US-00003 Time LOG REDUCTION PERCENT KILL 30 min 0.090 18.8 1 hr 0.205 37.6 2 hr 0.634 76.8 4 hr 1.928 98.8

Neutralization control data showed that the disinfectant was adequately neutralized. Actual counts correspond to those resulting from dilution without appreciable killing.

The disinfectant preparation tested here displayed good sporicidal activity against B. subtilis spores. B. subtilis is a common species used in sporicidal testing and belongs to the same genus as the organism that causes anthrax. Because of their genetic similarities, B. subtilis spores have been used as a non-pathogenic surrogate for Bacillus anthracis, the anthrax bacterium. Therefore, these results are applicable to anthrax. It is expected that longer exposure would result in additional killing.

4. Evidence of Efficacy of 10 ppm Silver and 1.0% h.sub.2O.sub.2 Compositions and 14 ppm Silver and 1.5% H.sub.2O.sub.2 Composition against Bacillus Subtilis

A. Purpose of Example

The purpose of this example is to demonstrate the antimicrobial activity of two silver-based compositions of the present invention on bacterial endospores from the test organism Bacillus subtilis. This was accomplished by performing standard kill-time assays using a suspension of B. subtilis endospores. Viewed relative to the previous example (employing 22 ppm silver), this example establishes the promoting effect of hydrogen peroxide (H.sub.2O.sub.2) on the antimicrobial properties of silver compositions. Hydrogen peroxide is stable in the presence of the silver compositions of the present invention. While hydrogen peroxide has significant antimicrobial properties itself, it is frequently broken down by catalase or other microbial enzymes. However, the hydrogen peroxide is capable of weakening bacterial cell walls and enhancing entry of the silver particles before any enzymatic destruction of the hydrogen peroxide can occur.

B. Material and Methods

1 Test Organism. A test suspension containing endospores from Bacillus subtilis (ATCC #19659) was prepared from a culture grown on Nutrient Agar, to which additional sporulation enhancers were added. Plates were harvested with sterile water and endospores were purified by repeated centrifugations and resuspensions in water. The final wash was in 70% ethanol for 30 min, to ensure the death of all vegetative bacteria. The spores were resuspended in water containing 0.1% Tween.RTM. 80 (brand of polysorbate) to prevent clumping.

2 Neutralizer. The Neutralizer mixture consisted of 12.7% Tween 80, 6.0% Tamol.RTM. SN (brand of sodium salt of naphthalene-formaldehyde condensate), 1.7% lecithin, 1% Peptone, and 0.1% Cystine. This solution was intended to neutralize any chemicals so they would not affect subsequent growth of the bacteria.

3 Kill-Time Procedure:

a) A 9.9 ml aliquot of each of the disinfectants (inventive colloidal silver compositions: one containing 14 ppm silver and 1.5% H.sub.2O.sub.2; the other containing 10 ppm silver and 1 .0% H.sub.2O.sub.2) was placed in a sterile 20 mm.times.1 50 mm tube. The tubes were equilibrated in a 20.degree. C. water bath.

b) Each tube of disinfectant was inoculated with 100 .mu.l of the test organism suspension at time zero.

c) At 10 min, 30 min, 1 hr, 2 hr, 4 hr, 6 hr, and 8 hr, one ml of organism/disinfectant suspension was removed to a tube containing nine ml of neutralizer. The tube was mixed thoroughly.

d) After two min, the neutralized suspension was serially diluted 1:10, in physiological saline solution (PSS).

e) The number of viable organisms in selected dilution tubes was assayed by membrane filtration. One ml aliquots were plated in duplicate. The membranes were washed with about 100 ml of sterile PSS and removed to Columbia Agar plates. The plates were incubated at 37.degree. C. for 20 hr.

f) The number of colonies on each filter was counted and log reductions computed.

4. Controls:

a) Titers of the test suspensions were computed by performing membrane filtration assays of selected 1:10 dilutions of the test suspensions in PSS.

b) A neutralizer control was performed by inoculating a mixture of 9 ml of neutralizer and 1 ml of disinfectant with 100 .mu.l of the 1:10.sup.3 dilution of the titer. This produced about 2,000 cfu/ml in the tube, which was allowed to stand for 20 minutes prior to diluting 1:10. Both tubes were assayed by membrane filtration using duplicate 1 ml. samples.

C. Results

TABLE-US-00004 Titer of Bacillus subtilis Spores: Dilution: 1:1 .times. 10.sup.6 1:1 .times. 10.sup.7 1:1 .times. 10.sup.8 Number of colonies: TNTC 36 5 TNTC 27 4 TNTC = too numerous to count.

TABLE-US-00005 Solution containing 14 ppm silver and 1.5% H.sub.2O.sub.2: Dilution of B. subtilis spore/disinfectant suspension: Time 1:1 .times. 10.sup.1 1:1 .times. 10.sup.2 1:1 .times. 10.sup.3 1:1 .times. 10.sup.4 1:1 .times. 10.sup.5 10 min — – TNTC TNTC 227 — – TNTC TNTC 265 30 min — – TNTC TNTC 258 — – TNTC TNTC 273 1 hr — – TNTC TNTC 55 — – TNTC TNTC 33 2 hr — TNC 207 29 — – TNC 237 24 — 4 hr 59 3 1 57 5 1 6 hr 0 0 0 3 0 0 8 hr 1 0 0 1 0 0 TNTC = too numerous to count.

Neutralization Control:

TABLE-US-00006 Undiluted 1:1 .times. 10.sup.1 TNTC 195 TNTC 210 TNTC = too numerous to count.

TABLE-US-00007 Solution containing 10 ppm silver and 1.0% H.sub.2O.sub.2: Dilution of B. subtilis spore/disinfectant suspension: Time 1:1 .times. 10.sup.1 1:1 .times. 10.sup.2 1:1 .times. 10.sup.3 1:1 .times. 10.sup.4 1:1 .times. 10.sup.5 10 min — – TNTC TNTC 230 — – TNTC TNTC 287 30 min — – TNTC TNTC 254 — – TNTC TNTC 260 1 hr — – TNTC TNTC 146 — – TNTC TNTC 124 2 hr — TNTC TNTC 64 — – TNTC TNTC 71 — 4 hr TNTC 72 5 TNTC 77 5 6 hr 0 0 0 2 0 0 8 hr 0 0 0 0 0 0 TNTC = too numerous to count.

Neutralization Control:

TABLE-US-00008 Undiluted 1:1 .times. 10.sup.1 TNTC 200 TNTC 184 TNTC = too numerous to count.

D. Discussion

The data showed a viable B. subtilis spore concentration of 2.59.times.10.sup.8 spores per ml in the original suspension. Inoculation of 9.9 ml of disinfectant with 100 .mu.l of this suspension produced an initial concentration of 2.59.times.10.sup.5 spores per ml in the assay tube.

Results from these procedures allowed log reductions (LR) and Percent Kill (PK) values to be calculated. They are listed in the following table. Values were computed using the formulae: LR=-Log(S/So) and PK=(1-(S/So)).times.100; where. S=concentration of organisms at a specific time; and So=the initial concentration of organisms at time zero. Since there was no significant kill within 30 min, the 10 min data was used for the So values. The 6 hr and 8 hr exposure times did not produce counts high enough to be reliable. Therefore, these data were not used in the linear regressions. Linear regressions were performed on the log reduction values using the `fitted line plots` command in the Minitab statistical software package. The regression equations produced, and the times required to effect a six-log reduction are shown along with the log reduction and percent kill values in the following table.

TABLE-US-00009 14 ppm SILVER + 10 ppm SILVER + 1.5% H.sub.2O.sub.2 1.0% H.sub.2O.sub.2 LOG PERCENT LOG PERCENT Time REDUCTION KILL REDUCTION KILL 30 min -0.03 -7.9 0.003 0.6 1 hr 0.66 78.0 0.28 47.8 2 hr 2.05 99.1 1.58 97.4 4 hr 4.63 99.998 3.54 99.97

Regression Analysis

Equation for 14 ppm calculated line: Y=-0.66704+1.32936x. Equation for 10 ppm calculated line: Y=-0.59690+1.03933x. These equations predict that the time for a 6-log reduction is 5.02 hrs for the 14 ppm composition and 6.35 hrs for the 10 ppm composition.

The neutralization control data showed that the disinfectant was adequately neutralized. Expected counts corresponded to those expected from the dilution.

The experimental disinfectant solutions tested exhibited significant sporicidal activity against B. subtilis spores. The B. subtilis strain used in these evaluations is the same one specified in the AOAC sporicidal test. Spores from this organism represent a significant challenge for most disinfectants. The times required to effect a six log reduction are in line with the sporicidal label claims of many cold sterilants.

5. Evidence of Efficacy of 10 ppm Silver Composition as a Broad Spectrum Antimicrobial

A. Methods

MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) tests were performed according to the standard broth microdilution method. The MIC is defined as the lowest concentration of an antibiotic that will inhibit the (in vitro) growth of an infectious organism. Results are reported in micrograms per ml. For medical antibiotics the interpretation of in vitro data is based on achievable serum concentrations of the drug, which may vary depending on dose, route of administration, degree of protein binding, site of infection, age and weight of the patient, and other factors. The MBC is defined as the lowest concentration of an antimicrobial agent needed to kill 99.9% of the initial organism inoculum.

The test was preformed by growing pure cultures of each of the test organisms in liquid culture. Turbidometric measurements were used to control the concentration of the culture. Serial dilutions of each test antibiotic were made in nutrient broth. The dilutions were calculated to cover the susceptible ranges for each organism for each agent. A standard amount of the test culture was added to each tube and the tube returned to an incubator (37.+-.2.degree. C.) for growth. The tubes were checked turbidometrically to determine bacterial growth. Below the MIC concentration the tubes showed an increase in optical density with time indicating bacterial growth. The lowest concentration of the antibiotic that showed no growth was the MIC. The “no growth” tubes were then subcultured in fresh medium. The “no growth” tube with the lowest concentration of antibiotic that showed no growth on subculturing was the MBC.

TABLE-US-00010 B. Results: Antimicrobial (ppm) Organism Tetracycline Ofloxacin Penicillin G Cefaperazone Erythromycin Silver S. pyogenes 0.625/>5 1.25/2.5 >5.0 0.313/1.25 0.003/0.019 2.5/5.0 S. mutans 0.625/>5 2.5/>5.0 0.521/>5 1.25/>5 .sup. 0.009/0.019 2.5/10.0 S gordonii 0.156/0.625 2.5/5.0 0.009/0.039 1.25/1.25 0.005/0.019 2:5/10.0 S. pnuemoniae 0.078/0.625 2.5/2.5 0.019/0.019 0.313/0.313 0.002/0.004 2.5/- 2.5 S. faecalis 0.313/>5 1.25/5.0 5.0/>5.0 >5.0 0.009/1.25 10.0/10.0 S. aureus 0.313/>5 0.417/0.625 2.5/>5.0 5.0/5.0 0.039/>5.0 .sup. 5.0/5.0 P. aeruginosa 0.078/5 0.156/0.313 0.13/>5.0 2.5/5.0 2.5/>5.0 1.67/5 E. coli .sup. 1.67/>5 0.104/0.156 >5.0 0.625/>5.0 .sup. 5.0/>5.0 2.5/2.5 E. aerogenes >5 0.078/0.156 >5.0 2.92/>5.0 >5.0 2.5/2.5 E. cloacae .sup. 1.67/>5 0.156/0.156 >5.0 >5.0 >5.0 2.5/5.0 S. typhimurium .sup. 1.25/>5 0.078/0.156 >5.0 1.25/2.5 5.0/>5.0 2.5/5.0 S arizona 0.625/>5 0.078/0.078 >5.0 0.833/>5.0 .sup. 4.17/>5.0 2.5/5.0 S. boydii .sup. 1.25/>5 0.078/0.156 >5.0 0.625/0.625 5.0/>5.0 1.25/1.25 K. pneumoniae 2.5/>5 0.417/0.625 >5.0 >5.0 >5.0 2.5/2.5 K. oxytoca .sup. 1.25/>5 10.104/0.156 >5.0 1.25/>5.0 >5.0 1.25/1.25

Data are presented as MIC/MBC (minimum inhibitory concentration/minimum bactericidal concentration) in parts per million (ppm)); “>” denotes that the concentration needed to obtain the MIC or the MBC was higher than test parameters measured for the test. For example, the highest concentration of tetracycline used on S. pyogene was 5 ppm. At that concentration there was still growth upon subculturing of the “no growth” tubes. Therefore, the MBC must be >(greater than) 5 ppm.

The MIC/MBC of E. coli strain 0 157:H7, which has been associated with outbreaks of hemorrhagic diarrhea and colitis, was determined in a subsequent study. The MIC was determined to be 2.5 ppm and the MBC was determined to be 5 ppm.

C. Conclusion

The 10 ppm silver composition of the present invention was tested and found to be both bacteriostatic and bactericidal for all organisms tested. In other studies, this composition was compared to other commercially available colloidal silver products and found to have a superior activity to all other preparations tested (data not shown). The most interesting observation was the broad spectrum that the 10 ppm silver composition possesses. The antimicrobial activity that was observed was fairly constant independent of the particular organism tested. With the exception of Streptococcus faecalis and Streptococcus aureus (which had MIC values of 10 ppm and 5 ppm, respectively), MIC values ranged between 1.25 ppm and 2.5 ppm for both gram positive and gram negative organisms. The MBC values behaved similarly with values ranging from 1.25 ppm to 5 ppm with the exception of Streptococcus mutans, Streptococcus gordonii, and Streptococcus faecalis (which all had MBC values of 10 ppm). The data suggest that 10 ppm silver embodiment of this invention exhibits an equal or broader spectrum of activity than any one antibiotic tested. Antibiotics generally have restricted antibacterial spectra limited to susceptible organisms, but as the data demonstrate, the silver composition of the present invention is equally effective against both gram positive and gram negative organisms. The data suggest that with the low toxicity associated with silver, in general, and the broad spectrum of antimicrobial activity of this silver composition, this preparation can be effectively used as an alternative to antibiotics.

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