Silver/water, silver gels and silver-based compositions; and methods for making and using the same

(54) Title: SILVER / WATER COMPOSITIONS, SILVER GEL COMPOSITIONS, SILVER-BASED COMPOSITIONS, AND THEIR METHODS OF PRODUCTION AND

Abstract: The invention relates to a colorless composition consisting of metal particles (eg silver nanoparticles) and water, said particles comprising a core of an elementary metal (eg. SILVER) AND AN ENVELOPE OF A METALLIC OXIDE (EG ONE OR MORE SILVER OXIDE (S)). METAL NANOPARTICLES ARE PRESENT IN WATER AT A LEVEL OF 5-40

MG / ι, AND THE MANIFEST COMPOSITION OF THE CONSEQUENTIAL ANTIMICROBIAL PROPERTIES. THE INVENTION RELATES TO METHODS OF USE OF THE COMPOSITION. THE COMPOSITION OF THE INVENTION MAY BE INCORPORATED IN A HYDROGEL WITHOUT ANY DEGRADATION OF ANTIMICROBIAL PROPERTIES. THE INVENTION FURTHER RELATES TO VARIOUS COMPOSITIONS CONTAINING METAL, WHICH SHOW UNEXPECTED BIOLOGICAL EFFICIENCY.

ΜΑ 29428Β1

SUMMARY The invention relates to a colorless composition consisting of metallic particles (eg nanoparticles of silver) and water, said particles comprising a core of an elemental metal (eg silver) and a shell of. a metal oxide (eg, one or more silver oxide (s)). The metal nanoparticles are present in water in an amount of 5-40 mg / l, and the composition exhibits consequent antimicrobial properties, the invention relates to methods of using the composition. The composition of the invention can be incorporated into a hydrogel with virtually no degradation of the antimicrobial properties, the invention further relates to various metal-containing compositions, which exhibit unexpected biological efficacy.

ΜΑ 29428Β1 ٠ ، 2 9 4 2 8 „٦٥١١١ ه MA ، 2008, Silver / water compositions, silver gel compositions, silver-based compositions, and their methods of production and use

[IJ Technical field

[2] The present invention generally relates to new silver / water mixtures (sometimes referred to as silver nanoparticles dispersed in water), and more particularly to new compositions and / or morphologies of silver / water mixtures, hydrogels of 'silver, new silver compositions combined with modern antibiotics and various ligands linked to silver ions, silver gels according to certain starting silver / water mixtures, silver ions and / or metals bound / contained in certain clathrates such as clays and / or zeolite materials, and methods of making and using these compositions as agents against various organisms (including including certain viruses) harmful to the health or well-being of humans and / or animals or other organisms. Further, other metals besides silver are also disclosed herein and can be used in many instances interchangeably with silver. Various combinations and concentrations of the inventive compositions are also disclosed.

Γ31 Description of the prior technique

[4] It is known that some silver preparations have exhibited germicidal properties. Silver was used as a germicide and as an antibiotic before modern antibiotics were developed. In previous centuries, users would shave silver particles in their drinking water, or submerge all silver coins in drinking water, in order to ingest the silver while drinking the water. It seems possible that the practice of feeding silver utensils (eg, silverware) may result from a belief in the healthful properties of silver.

[5] There can be many reasons why administering the suspended silver in a solution would increase the health of an individual. It is possible that such a solution will work to prevent the growth of bacteria, viruses, and other unwanted organisms, as well as eradicating existing bacteria, viruses and other organisms. It is also possible that a silver composition may have anti-inflammatory effects, sufficient to reduce, for example, hernia, lousy complications and certain symptoms of asthma.

[6] A first embodiment of the present invention describes the use of a composition of silver in water to treat certain human foods (or, for example, certain animals). One embodiment of the invention includes a silver composition comprising silver nanoparticles (eg, a majority of which are 10 to 50 nanometers in diameter) and which, in a preferred embodiment, may include an interior of 'metallic silver and an exterior coating or a different portion of said interior (eg, ionic silver coating, at least one silver oxide coating, (eg, different compositions and / or different phases, etc.) which particles are suspended in water (eg, purified water). In a further preferred embodiment, at least 90% of these particles are 10 to 50 nanometers in diameter. A preferred embodiment of the invention comprises a silver composition comprising silver particles (including some silver particles coated with silver oxide) where more than 50% of the number of particles is less than 0.015. micrometers in size and the particles are suspended colloidally (eg, not precipitating) in water. Another preferred embodiment of the invention includes similar particles where about 95٥/٥ of the particles are 10 to 40 nanometers in diameter. In a further preferred embodiment, about 95٥/٠ of the particles are 10 to 30 nanometers in diameter.

Î7) Summary of the invention

[8] The present invention is generally directed to the use of silver, at a level of 5 to 40 ppm in water (but in some cases less than 5 ppm), to kill or disrupt microorganisms (including some. viruses) which are hazardous to humans and / or animals or other living organisms. In addition, the present invention is directed specifically to compositions comprising silver nanoparticles, said particles, in a preferred embodiment, comprising, for example, an interior of elemental silver and an exterior coating of a partial coating or a coating. layer, for example, of at least one silver oxide (for example, ionic silver oxide, silver oxides such as AgiO, AgO, AgO, etc.), said oxide coatings being in various phase states (eg, AgiO being monoclinic and / or tetragon) and water, where the silver particles are suspended (eg, colloidal suspension) in water at a total level of 5 to 40 ppm. One embodiment of the present invention includes silver nanoparticles (prior to this point in the specification, it should be understood that the use of the term silver particles, or the like, when processed according to the electrochemical techniques disclosed herein. present, relates only to elemental silver, but also to elemental silver particles which may have a partial or substantially complete coating of at least one composition below, your coatings comprising at least one silver oxide in

/٢١١

ΜΑ 29428Β1 minus a portion thereof) being present in water (preferably purified water, discussed later herein), at a concentration of 5 to 40 ppm, where more than 50% of the particles d silver have a maximum dimension of less than 0.015 micrometers. In a preferred embodiment, most of the particles are 10 to 40 nm in diameter. In a more preferred embodiment, more particles are 10 to 30 nm in diameter, the composition of silver in water (as well as the silver particles extracted as essentially discrete particles from the mixtures of. silver / water made according to the invention), as well as silver / water mixtures made according to the teachings of the invention and later shaped into a gel, powder, a clay or a zeolite (as discussed in the preferred embodiments hereinafter) according to the teachings of this invention is / are, for example, highly effective antimicrobial agents and antiviral agents (and in some cases also anti-parasitic), the present invention is also directed to silver compositions, silver from 5 to 40 ppm in water and, according to the methods of using said silver / water compositions disclosed herein, are highly effective as antimicrobial agents using said compositions as follows: (1) internally in living organisms; (2) externally on living organisms as well externally (or internally) in a variety of surfaces, both hard and porous (eg, countertops, food preparation surfaces, food preparation equipment, , hospital surfaces, medical instruments, water flows (metal and / or plastic), air filtration devices, etc.): and (3) kneading silver or silver water compositions with contaminated water (eg, sewage treatment, pond water, contaminated water containers, water pipes, etc. ., which have preferably had large solids removed therein prior to said mixing) to result in a water purification process.

[9] A preferred embodiment of the present invention is directed to silver in water compositions made using a modification of the device and / or methods described in United States Patent No. 6,214,299 ('299 Patent), which is specifically incorporated herein by reference. In addition, the compositions of other metals such as, for example, copper (and copper alloy), zinc, platinum, and titanium and their alloys and mixtures) can be used to form other desirable metals / compositions, depending on the circumstances. the methods of the present invention, which are surprisingly efficient.

[10] The device and process of the '299 patent has been modified and improved to provide the silver composition of the present invention, which process is described in more detail hereinafter. Essentially, the common eight-silver / one common electrode device as disclosed in the '299 patent has been modified and scaled to accommodate a larger water chamber (eg, 75 to 85 gallons). To begin the process of making silver / water composition in a 75-85 gallon container, approximately 70-75 gallons of relatively high purity water (e.g., filtered water, reverse osmosis water, or water which does not contain large amounts of potential contaminants, etc.) typically containing less than 2 ppm of total dissolved solids, or also more preferably less than 1 ppm of total dissolved solids, are placed in the chamber. To this is added, in a preferred embodiment, approximately five gallons of a water / silver composition produced in the previous production run. This priming with approximately 5 gallons is useful, but not essential, the priming essentially provides a sufficient number of conductive silver particles to be present in the chamber so that current can flow between the various electrodes once a current is started. sufficient tension is achieved in a relatively short period of time, priming also results in small, initial, slightly smaller "Taylor cones", discussed later in this. The water chamber is fitted with the inlet air (typically placed near a lower portion of the water chamber) which allows a flow of air bubbles to pass through the water / silver liquid during its manufacturing. It has been discovered that this approach results in apparently improved mixing over a stirring paddle disclosed in the '299 patent, as demonstrated by certain increased efficiencies.

[11] The electrode system (s) is / are separated at voltages (at least initially) on the order of, or approaching approximately ten thousand volts alternating current (with each series of electrodes having an individual supply voltage) as described in the '299 patent. substantially higher voltages of ten thousand tend to produce a solution which may have significant amounts of the ionic silver dissolved therein. The present composition comprises a 97٥/٠ excess of metallic silver particles present at 5-40ppm, with essentially little to nonfree ionic silver present in the water / silver solution.

[12] The concentration of silver is determined according to the methods explained below. Essentially, the 75 gallon silver / water maker is operated essentially without interruption and the samples from the device are analyzed until the desired ppm silver concentration in the water is reached. It has been found that under the operating conditions described herein, the silver / water composition of 10 ppm requires approximately and days and a day and a half of operation; The 22 ppm silver / water composition requires approximately three days of operation, and the 32 ppm silver / water composition requires approximately six days of operation, the rate of formation of silver particles in the compositions of The silver / water appears to slow down as the highest concentrations of silver particles are sought. When silver concentrations in the silver / water compositions are desired to be above 50 ppm, they take a relatively long time to achieve, within the parameters of.

<img file="MA29428B1_D0001.tif" />

ΜΑ 29428Β1 treatment disclosed herein, with the highest concentration accomplished to date in a reasonable amount of time of about 50 ppm. The highest concentrations of silver particles are possible, if desired. However, the efficacy of the lower concentrations of silver particles against various pathogens has been so exceptional that higher concentrations of silver particles are not required to date.

(13] Silver nanoparticles in silver / water compositions all have very similar particle sizes in general and shape characteristics, described below in the characterization section in more detail, and unlike of many conventional colloidal silver compositions, these silver / water compositions are completely colorless and are substantially stable with respect to medium light and changes in temperature, without the need for the use of any additives to aid in stability (which many colloidal silver of the previous art require and / or use). It is believed that the components and business process steps used produce a silver / water composition which differs from other known colloidal silver products in such a way that it causes the silver / water compositions to be more effective. high. Some of the differences in the salient physical properties (e.g., particle size, composition, spectroscopy patterns, etc.) of the novel silver / water compositions of the present invention are discussed in more detail below in these.

[14] The silver / water compositions of the invention are also substantially unreactive to many materials added thereto including, for example, alone or in combination, (1) hydrogen peroxide, (2) DiSodium EDTA (disodium ethylene diamine acetic acid), which can effectively act as an enhancer of silver / water compositions (for example, can make silver / water compositions even more effective), (3) iodine (for example example, povidone iodine, which in some cases may show some mild reactivity), which may help silver / water compositions to also be more pathogenic against a variety of pathogens and (4) various antibiotics available in the market (which may actually result in certain effects cooperatives taking place between silver / water compositions and antibiotics, thus leading to the potential for new and very desirable combination therapies being realized). Accordingly, a variety of additional materials or substances can be used in combination with (eg, added to or supplemented with) the novel silver / water compositions of the present invention to synergistically enhance the desirable effects which either matter can show on its own. Specifically, in many cases (eg combination of antibiotics) the combined effects results are synergistic and have outweighed the individual additive effects of either the material or the substance alone, when combined (eg, 2 + 2 = 6). Obviously, some of the possible additives will make the new compositions suitable only as topical or surface treatments because of their potential for internal toxicity in biological organisms (eg, humans or animals), the amount of one. additive may vary depending on many circumstances including the particular affliction (e.g. virus, bacteria, parasite, etc.) or infection, the amount of other materials present in addition to additives, etc. However, the precise amount of the additive required would be ordinary experience to those skilled in the art. In addition, the concentrations of the silver / water mixture can also influence the amount of the additive required, also in ordinary experimentation for those skilled in the art.

[15] An example of a desirable additive is hydrogen peroxide. Hydrogen peroxide is a known disinfectant agent, hydrogen peroxide has been found to have a synergistic interaction with the inventive silver / water compositions of the invention, hydrogen peroxide is available in high concentrations, for example, 30٥/٠ by weight (٥/٥ of the weight by volume or percentage of the weight) or even greater. Although higher concentrations are suitable, the preferred concentrations for use with the silver / water compositions of the present invention appear to be 30% or less, and preferably more, in the range of about 1 to 5% by weight. weight.

[16] A preferred embodiment of the present invention is directed to compositions comprising particles of silver from 5 to 40 ppm, hydrogen peroxide from 1 to 3 by weight in%, and the remainder being water (by eg filtered or substantially purified water). Another preferred embodiment of the present invention is the use, and method of use, of compositions comprising 10 to 40 ppm of silver and 1 to 3 wt% hydrogen peroxide in water. as antimicrobial agents.

[17] Another example of an additive which works favorably with the silver / water compositions of the present invention is disodium ethylene diamine tetra acetic acid also referred to as Sodium EDTA or DiSodium EDTA (both of which are sometimes referred to as in the literature) and which may have a chemical formula as follows: (CINCHCQOHH ؛ COONa) 22H2O. in another embodiment of the invention, a small amount (eg 0.5 to 10 ppm, or preferably more 0.5 to 5 ppm. or more preferably more than about 0.5 ppm) of disodium EDTA is added, or supplemented by the silver / water compositions of the present invention. In this embodiment, it appears so like the addition of a small amount of disodium EDTA increases the potency (eg increases bactericidal, disinfectant and / or antimicrobial properties) of the silver / water composition. Without wishing to be bound by any theory or explanation

In particular, it is possible that disodium EDTA could increase your cell wall permeability, which increases the overall effectiveness of the silver / water compositions of the present invention. Another embodiment of the present invention is the use, and the method of use, of compositions comprising 10 to 40 ppm of silver and 0.5 to 10 ppm of disodium ED٢A in water as an antimicrobial agent. 'nt bactericidal, antiviral and / or disinfectant.

ΜΑ 29428Β1

[18] Another example of an additive which works favorably with the silver / water compositions of the present invention is Povidone iodine, iodine is a well known prophylaxis in medicine for the treatment of a wide range. Of pathogens, iodine is commercially available in various concentrations, but a commonly used, and preferred, concentration is 10%. In this preferred embodiment of the invention, a synergistic combination has about 25-50% by volume substitution of the silver / water mixture replacing 10% of the iodine solution. While certain reactions between the mixture of silver / water and iodine are possible, it appears from experimental results discussed hereinafter that the synergistic combination of silver / water with povidone iodine may. function as a topical disinfectant (eg, ointment) and / or prophylaxis against infection in dosages, burners and or scrapes, etc. another preferred embodiment of the present invention is the use, and the method of use, of compositions comprising 10 to 40 ppm of silver and povidone iodine in water as an antimicrobial agent, bactericidal agent, antiviral and / or disinfectant agent.

[19] Another preferred embodiment of the invention uses silver / water compositions of the present invention in combination with various commercially available antibiotics in a known approach by combination therapy, combination therapy has become of. '' of great interest because, in the last two decades, the progression of resistance to antibiotics has been widespread and therefore an overall subject of great responsibility, Infections caused by Gram-negative bacteria such as Escherichia coli, Klebsiella, Proteus, Shigella and Pseudomonas have become a growing cause of liability as these organisms have acquired multiple drug resistance to antibiotics. A recent study to investigate the resistance pattern of gram-negative clinical isolates causing hospital infections, showed that most isolates were resistant to common antibiotics such as ampicillin, gentamicin, chloramphenicol, cotrimoxazole, and the first and second generation of cephalosporins. Also approximately 70% of these isolates were resistant to ciprofloxacin. In this embodiment of the invention, the silver / water mixtures (either combined as a liquid or dried and added as a solid, thereby forming, for example, a powder, sometimes referred to herein as Sildust), when combined with various antibiotics, showed synergism, rather than just additive properties. Checkerboard trials have shown that some antibiotics when combined with silver / water mixtures resulted in antibiotics several times more effective than silver alone (e.g. silver / water mixtures combined with amikacin and cefoperazone showed an FIC index of about 0.1875. compared to them antibiotics used in combination of one another, which resulted in an FIC index of about 0.625, once both combinations were used eg against MRSA (Methicillin Resistant Staphylococcus aureus)) discusses in greater detail below herein. Another preferred embodiment of the present invention is the use, and method of use, of compositions comprising 10 to 40 ppm of silver and various antibiotics as an antimicrobial agent and / or a bactericidal agent and / or an antiviral agent in treatments designated by combination therapies, the precise amount (and concentration) of the silver / water mixtures according to the present invention which can be added to conventional antibiotic therapies is a matter of usual experimentation. In particular, the specific disease being treated with a course of specific antibiotic (as well as the effectiveness of the antibiotic against the pathogen) will influence the amount, and concentration, of the silver / water mixture required.

[20] Although a large number of tests using the silver / water solutions alone or in combination with various additives are presented below, it is also shown herein that certain vehicles can significantly improve performance. results that can be obtained with silver / water solutions in various situations, specifically, it has been discovered that the formulation of the aqueous silver / water composition as a semi-solid hydrogel (sometimes referred to herein as Silgel or another version referred to as Silderm), or alternatively sheets of such a material significantly increases its efficiency for certain applications. Hydrogels are typically hydrophilic gels produced by the addition of certain hydrophilic organic polymers to an aqueous solution - in this case a solution containing the inventive silver / water solution. However, it is anticipated that other colloidal silver solutions may also be formed into hydrogels, according to the teachings herein, and so long as such hydrogels cannot be effective like those of the present invention, these hydrogels may still have some degree of effectiveness. desirable utility. Accordingly, the present invention is intended to cover certain aspects of these hydrogels as well. As would be expected, the hydrogel improves the retention of silver on a surface, such as wound on the surface of the skin. For wound care, a hydrogel or sheet carrier also has the significant advantage of protecting the tissues surrounding the wound and preventing desiccation, which factors often increase wound healing. More significantly, the hydrogel does not appear to interfere, substantially, if any, with the antimicrobial properties of the silver nanoparticles of the present invention. In addition, these hydrogels work as excellent hand or skin cleansers, as well as protectors (for example, by placing the hydrogels on the hand so that the hand

ΜΑ 29428Β1 comes into contact with pathogens, the protective gel of the skin can assist in the prevention of infections due, for example, to cuts or abrasions, thus operating as a prophylactic), thus making the gels of great utility in the field of health care or welfare.

[21] In particular, clean hands are thought to be the single most important factor in preventing the proliferation of dangerous germs and antibiotic resistance in health care options. Most of the hygienic hand washes used in modern medicine are alcohol based and have several limitations. The first among these limitations is the damage to the skin that is caused by repeated exposure to alcohol products. In some cases (1) irritant contact dermatitis as well as (2) allergic contact dermatitis have also been reported. This reduces the compliance of many health care workers in the use of hand hygiene products.

[22] Another factor causing non-observance of effective hand hygiene practices is the fact that, being liquid, hand hygiene products are, typically, permanently fixed over sinks or wash basins. sinks. This results in healthcare personnel having to move from the patient bed to the sink and back to the next patient. If a hand wash is available as a rag this problem could be overcome, thus ensuring better compliance, the hydrogel products of the present invention have been shown to reduce the number of germs of indicator organisms by significant amounts, over time. Extended periods of time, as discussed in more detail herein, thereby resulting in an alternative viable hand hygiene product. Accordingly, the hydrogel products of the present invention have also shown great utility as skin protectors, normally protecting healthy skin from various pathogenic materials in a prophylactic manner.

[23] In another preferred embodiment of the invention, a silver product can be at least partially, or in some cases substantially completely, substituted by the silver compositions of the present invention. Specifically, EDTA silver (or AgEDTA) itself has been discovered to have very intriguing antimicrobial characteristics. In particular, as discussed above, DiSodium EDTA is a useful additive to the water-silver compositions of the present invention. However, EDTA (ethylenediaminetetraacetic acid) is an excellent synthetic chelating agent. EDTA (C10-H16-N2-08) is permitted for use in tium foods and is often added to non-alcoholic beverages as a preservative. EDTA has also been used in heavy metal chelation therapy for humans. However, what has not been considered is the use of AgEDTA as an antimicrobial (e.g., by itself or in combination with other therapies, such as those disclosed herein), wide use applications such as meat or protein production and processing industry, soap industry, detergent industry (e.g. personal products and household care), The agriculture or the harvesting industry and the health care industry may be well suited for a stable powdered form of silver which can provide many powerful health and wellness benefits (eg. example, therapeutic and prophylactic at the same time). In particular, AgEDTA is readily available and it is relatively simple to manufacture, store and transport. This embodiment of the invention identifies a novel use for AgEDTA, namely, using powdered AgEDTA for the health and welfare of humans, plants and / or animals and / or the treatment of certain diseases. disorders in animals and humans (eg, can be used as a therapeutic treatment and / or as a prophylactic). Akzo-Nobel is currently manufacturing an acceptable AgEDTA. Other chelating or complexing agents such as, for example, silver EDDS, silver curcuminate, silver berberine, and silver tetracycline also show antimicrobial properties and the use of these materials for the health and well-being of humans or animals. is also new and unknown in the prior art. Several other organic structures can be used to carry and / or deliver silver and / or silver ions to various effective locations in or on biological structures. Again, the amount of AgEDTA required will change depending on the particular biological issues surrounding the need (eg, treatment conditions and / or prophylaxis).

[24] In another preferred embodiment of the invention, the additional inorganic silver products can be at least partially, or in some cases, substantially, completely substituted by the silver / water compositions herein. invention.

Specifically, silver (e.g. silver ions, silver metal, Ag +) can be attached or fixed in a controllable manner, e.g. in or between layers of clay and / or in cages in them. zeolites. Such attachment can take place by controlling the charge of, for example, the silicate layer, the charge of the zeolite, as well as the distances between the layers or the size of the zeolite cage. In this regard, silver may be tied or tied closely or relatively loosely, depending on the particular application of health or welfare and the point of interaction between silver and biological material (for example, on the surface of the biological part, or of an internal part, or in combination of the internal parts, etc.). Accordingly, the resulting products can include those products which are completely fluid and thus are drinkable or sprayable; as well as products which are gel or paste-like and are spreadable on surfaces such as gels or pastes.

ΜΑ 29428Β1

[25] Any of the metals discussed herein may be held in a crystalline or amorphous clathrate of at least one atomic layer of oxygen-containing or oxygen-containing molecules. Certain clathrate metal structures have been shown to have unexpected efficacy. In addition, in addition to silver being incorporated into a structure of an oxide layer (eg clays) and networks (eg zeolites) silicates, phosphates, and oxides such as hydrotalcytes can also be used. Still further, desirable families of clay or mica which are capable of being used with the present invention (and which are capable of having different surface loads and / or different distances between layers) include, for example, illites, montmorillonites, chlorites, and vermiculites.

[26] Clays or mica, as well as zeolites, are very desirable because ion carriers of metals for several reasons are naturally occurring or easily derived, the particles can be maintained within a collo size range. i'dale desirable which make them, for example, suspended in liquids (eg, water) and are typically very familiar biologically (eg, little or no side effects). In this regard, once the silver is placed, for example, in a clay or zeolite clathrate, the molecules are then heated to mild temperatures (for example, 100 to 2OO٥C) to fix the silver on or on it. inside of clathrate. All of these materials can be made in a wide range of viscosity since being very fluid to very viscous.

[27] In general, the electronic levels in elements such as cations, in any given valence state, can be changed when the cation of that element is coordinated by various anions. In particular, the more covalent the bond, the more energy levels can be changed. It is very likely that small to moderate changes in the electronic structure of silver will take place once silver is surrounded (or coordinates) by different numbers of oxide ions. This change in the electronic structure for cations, such as silver cations, is expected to occur in any of the silver oxide structures. Still more, there is a general way in which silver can be placed in a clathrate or oxygen cage. In this regard, by exchanging, for example, the sodium cation in a structure, with a silver cation, then the sodium ions placed in the cavities or in the exchangeable spaces (for example, either on or between the sheets of clay or within zeolite arrays) can occur. In general, a material's ability to exchange cations is known by its CEC or cation exchange capacity these units for CEC are typically referred to as meq / 100 grams or milliequivalents per one thousand grams. In general, the higher the number of CECs, the greater the capacity of a material to accept cations (eg silver cations). As a result, many compounds of silver combined with oxygen can serve the role of a carrier of silver (or other metals) and thus are able to act as therapeutic agents on their own, or in combination with other therapeutic agents.

[28] Still further, the silver metal or silver ions incorporated onto the silica by diffusion and drying are also desirable mechanisms for delivering the metal or metals of the present invention.

[29] In another preferred embodiment of the combinations of the invention of the above-indicated particles organic and / or inorganic structures can be used to positively affect the health and welfare of humans and animals. Specifically, the metal particles, according to the invention, can be used alone, as discussed above. In addition, the metal particles can be combined alone, eg, with the organic compounds discussed above (eg, AgEDTA). Still further, the metal ions according to the present invention can be combined with any of the inorganic compounds (eg, clays or zeolites). Still further, the metal ions of the present invention can be combined with all organic molecules (eg, AgEDTA) and inorganic molecules (eg, clays or zeolites). This combination of silver metal or silver ion delivery systems can be constructed so that, for example, domestic consumption of any of the above indicated silver delivery systems can result in money being delivered. to different portions, for example, an organism. In particular, for example, in humans, some silver may have been absorbed through the mouth, through the intestine, as well as through the colon and / or the small intestine, etc. In addition, depending on, for example, the amount of clays or zeolites relative to water (as well as various gelling compounds disclosed herein) the resulting products of the present invention can be very liquid (low viscosity) to. very viscous (high viscosity). In this regard, in general, the more clay or zeolite supplied relative to the water (as well as the gelling agent) the more viscous the end product.

Γ3٠1 Detailed description of the invention

[31] The following description is provided to enable any person skilled in the art to make and use the invention and describes the best modes contemplated by the inventor for carrying out his invention. Various modifications will be. However, 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 silver / water composition (sometimes referred to as nanoparticles dispersed in water) which, may be used by itself or in combination with (e.g., mixed with or substantially provided adjacent with cel) other disclosed materials, and which can be formed into various compositions

ΜΑ 29428Β1

[48] ​​Figure 23a shows two Raman spectra corresponding to inventive silver / water compositions; and Figure 23b shows three Raman spectra which correspond to three colloidal silver products available on the

[49] Figure 23c shows another Raman spectrum corresponding to the inventive silver / water compositions.

[50] Figure 24a shows a Raman spectrum of a silver / water composition of the present invention; and Figure 24b shows three Raman spectra of the compositions silver / water, zinc / water, and copper / water.

[51] Figure 25 shows a diagram of potential interactions in a disk method for bacterial synergy.

[52] Figure 26 shows microtiter plates and graphs showing additive, synergistic and antagonistic effects in combination therapy.

[53] Figure 27 shows photographs of the sensitivity of MDR isolates at 10 ppm of silver / water mixtures.

[54] Figure 28 shows photographs of the antibiotic combinations for MRSA.

[55] Figure 29 shows photographs of antibiotic combinations for E. coli.

[56] Figure 30 shows photographs of antibiotic combinations for Pseudomonas.

[57] Figure 31 shows a diagram of instantaneous applied voltage, and instantaneous concentrations of silver as a function of treatment time during the process of forming the silver / water composition.

[58] Figure 32 shows a diagram of an instantaneous silver concentration as a function of treatment time using atomic absorption spectroscopy and electrical conductivity measurement techniques, respectively. This figure also shows the concentration of silver after 32 hours of production, and before homogenization.

[59] Fig. 33 shows a diagram of instantaneous applied voltage, energy factor and silver concentration as a function of processing time during the process of forming the inventive silver / water composition.

[60] Figure 34 is a diagram showing the moisture loss of SIIDERM.

[61] Figure 35 is a diagram showing the moisture absorption of SILDERM.

[62] Fig. 36 is a photograph showing antibacterial activity of silver chelates (Ag EDTA made by Akzo-Nobel) against pseudomonas aeruginosa (MDR).

[63] Figure 37 is a photograph showing the antibacterial activity of silver chelates (Ag EDTA made by Alpha Chemicals) against pseudomonas aerugimosa (MDR),

[64] Figure 38 is a photograph showing the susceptibility of SILDUST against E. coli (MDR).

[65] Figure 39 is a diagram showing the antiviral activity of SIIDUST as a function of exposure time.

[66] Figure 40 is a photograph of a central specimen, showing the development of plaques.

[67] Figure 41 is a photograph of a test tube, not showing plaques after three hours, and thus showing anti-bacteriophage activity of SILDUST.

[68] Fig. 42 shows four 200 ppm ray diffraction patterns of the inventive silver / water composition; four superimposed y-ray diffraction lanes (eg AgO, AgiO, Ag and Ag0 ؛).

[69] Figure 43 shows a TGA analysis of AgO, as well as the DTA analysis of AgC.

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ΜΑ 29428Β1

[JO] Figures 44a and 44b are SEM photomicrographs which correspond to kaolin / silver mixtures made according to the present invention.

[71] Figures 45a and 45b are EDS (EDAX) analyzes corresponding to photomicrographs of 44a and 44b, respectively.

72 ؛] Figure 46 is an SEM photomicrograph of a novel zeolite / silver mixture made according to the present invention.

[73] Figure 47 is an EDS (EDAX) analysis of linde 4Α zeolite containing silver substituted herein, and made in accordance with the present invention.

[74] Figure 48a shows a U٧-Vis spectrum of a lOppm silver / water solution and 32ppm of silver / water solution in a wavelength range of 190nm to 400nm (both made according to the present invention ); and Figure 48b shows a U٧-Vis spectrum of the same samples in a range of 190nm to 25٥nm.

[751 PREFERRED IMPLEMENTATION IODES

[76] Preferred non-limiting embodiments are present in the following:

[77] A composition comprising silver nanoparticles, in colloidal suspension in water, where the total silver content is between 5 and 40 ppm, which composition kills or invalidates microorganisms which are dangerous to humans and / or to human beings. animals.

[78] A composition comprising silver nanoparticles, in colloidal suspension in water, where the total silver content is approximately 10 + 2 ppm. which composition kills or disables microorganisms which are dangerous to humans and / or animals.

[79] A composition comprising silver nanoparticles, in colloidal suspension in water, where the total content of silver is about 22 + 2 ppm, which composition kills or disables microorganisms which are dangerous to humans and / or animals.

[80] A composition comprising nanoparticles of silver, in colloidal suspension in water, where the total content of silver is about 32 ± 3 ppm, which composition kills or disables microorganisms that are dangerous to humans and / or animals .

[81] A hydrogel composition made from a silver / water precursor composition comprising silver nanoparticles, colloidally suspended in water, wherein the total content of silver in the precursor material is, preferably about 32 + 3 ppm (but could be more or less), which hydrogel composition kills or disables microorganisms which are dangerous to the human body and acts, for example, as a skin cleanser, wound healers and / or skin protectant or skin disinfectant.

[82] It should be appreciated that the specification of the total amount of silver nanoparticles in a silver / water composition does not fully specify the material. Since the nanoparticles comprising the composition are made smaller, a given concentration of silver will represent a large number of particles. In addition, the total surface area for a given silver concentration will increase. Accordingly, particle sizes and particle size ranges are important parameters in defining an inventive silver / water composition. In addition, the coatings, such as oxide coatings (eg, partial or substantially complete) in said silver particles can also affect the effectiveness of the silver / water compositions of the invention, such coatings resulting from inherent in the processing conditions of the invention. However, similar coatings on silver particles made by other processes (as well as metals other than silver such as zinc, copper, copper alloys, titanium, platinum, and their alloys or mixtures) are also contemplated to be within the scope of this invention. Accordingly, when silver is referred to herein, the use of various other alternative metals discussed herein will also be considered to show possible efficacy, depending on the particular biological conditions (eg, the specific pathogens involved).

[83] A further class of embodiments are any of the above-noted compositions, where more than 50% of the silver nanoparticles have a maximum dimension of less than 0.015 microns.

[84] A further class of embodiments are any of the above-noted compositions, where greater than 75% of the silver nanoparticles have a maximum dimension of less than 0.015 microns.

ΜΑ 29428Β1 hydrogel or payroll, which show significant abilities to kill human and / or animal pathogens both in vivo and in vitro.

[32] Generally, (the present invention represents a new approach to kill or disable microorganisms which are dangerous to humans and / or animals by the use of silver nanoparticles in water, at a concentration of 5 to 40 ppm silver; or active silver particles contained, for example, in AgEDfA, and / or the other compounds discussed herein. Depending on the application, and / or the additives present, the silver / water composition can be used internally or externally. Depending on the application, the silver / water composition may contain various desirable additives which have not been specifically listed herein, but will become apparent to have utility to those skilled in the art.

Γ331 BRIEF DESCRIP ON OF THE FIGURES

[34] Figures 1-6 show TEM photomicrographs taken, at various magnifications, of the silver particles formed in the silver / water compositions formed according to the present invention.

[35] Figures 7a-7d show TEM photomicrographs generated from a different EM and using a different technique than that used to generate Figures 1-6: and Figure 7e shows an EDS (EDAX) spectrum of the particles of silver taken from the silver / water composition of the present invention.

[36] Figure 8 shows an electron diffraction pattern taken from a silver particle of a silver / water composition of the present invention.

[37] Figure 9 includes three SEM photomicrographs which simultaneously show the possible damage of the electron beam to silver particles taken from the silver / water compositions of the present invention.

[38] Figure 10 shows an SEM photomicrograph of a novel silver electrode prior to use in the process according to the present invention.

[39] Figures 11, 12 and 13 show elemental analyzes of EDS from parts 1, 2 and 3, respectively, shown in Figure 10.

[40] Figure 14 shows an SEM photomicrograph of the tip of the electrode used to make silver / water compositions according to the present invention.

[41] Figures 15 and 16 show elementary analyzes of EDS of parts 1 and 2 respectively, shown in Figure 14.

[42] Figure 17 shows an SEM photomicrograph taken approximately 3500Χ from the tip of the silver electrode.

[43] Figures 18a and 18b are TEM photomicrographs of silver particles taken from GNC liquid Silver Dietary Supplement (25ppm).

[44] Figures 19a and 19b are TEM photomicrographs of silver particles taken from a colloidal silver product referred to as Silverado ”.

[45] Figures 20a and 20b are TEM photomicrographs of silver particles taken from a colloidal silver product designated by Vitamin World Bioorganic Advanced Colloidal Minerals (3ppm).

[46] Fig. 21 is a coating comparison of five TEM photomicrographs of the silver particles, two of which are silver particles of the present invention and three of which are silver particles taken from colloidal silvers available in the market.

[47] Figures 22a and 22b show seven different Raman spectra, three of which correspond to silver / water compositions of the present invention, one corresponds to pure water, one corresponds to deionized water and two correspond to products of colloidal silver available in the market.

اج

ΜΑ 29428Β1

[85] Another class of embodiments is any of the compositions described above, where more than

90% of silver nanoparticles have a maximum dimension of less than 0.02 micrometers.

[86] Another class of embodiments is any of the above described compositions, wherein greater than 75٥/٥ of the silver nanoparticles have a minimum dimension greater than 0.005 microns.

[87] Another class of embodiments is any of the above described compositions, where greater than 90% of the silver nanoparticles have a size greater than 0.005 micrometers and less than 0.040 micrometers.

[88] Another class of embodiments are any of the above-described compositions, wherein the silver nanoparticles have both silver in zero valence, i.e., metallic, in an oxidation state. (Ag (٥)) in a core or in its central part, and at least one coating of silver in the ionic oxidation selected from the group consisting of Ag (I), Ag (II), and Ag (III), with a coating of AgO, Ag0 ؛, and / or AgO being more likely present on at least some (or substantially all) of the metallic silver core.

[89] Another class of embodiments are any of the above-described compositions, where my silver particles have both silver in zero valence, i.e. metallic, a state of. oxidation (Ag (0)) and a silver oxide coating with the stoichiometry AgO or Ag0 ؛ or other known stoichiometry, which is stable under the processing conditions used to make the novel Ag2O silver / water compositions of the invention.

[90] Further experimental evidence shows that silver oxide coatings inherently take place in at least part of the particles of the present invention is at least partially in the form of, for example, Ag O - c ' that is, silver II oxide. In a molecule of this material two of the silver atoms can be in the T state (silver I) while the other two silver molecules can be in the 3٠ state (silver III). In addition, under certain conditions, silver may be present in state 2 '(silver II), resulting at least in partial coatings, for example, of Ag₂0. These coatings are inherently a result of the processing conditions of the invention (e.g., those conditions created and around the electrode / water interface) and can be very important in the overall effectiveness of ؛ silver / water compositions. 'invention. The exact composition of the coatings has been difficult to determine to date, but experimental details have been provided in the characterization section below herein.

[91] A further class of embodiments is the combination of any of the above mentioned embodiments of silver / water with hydrogen peroxide, at a level of 1 to 3% hydrogen peroxide in. the final product.

[92] Another class of embodiments is the combination of any of the above stated silver / water embodiments with DiSodium ED7A, at a level of 0.5 to 10 ppm in the final product.

[93] A further class of embodiments is the combination of any of the above-stated embodiments of silver / water with about 50 to 75٥ / by volume of substitution of 10٥ / ο of iodine of povidone replacing about 25 to 50٥ / ο of the silver / water mixture in the final product.

[94] A further class of embodiments is the combination of any of the above stated silver / water embodiments with various commercially available antibiotics (either in liquid form or in powder form) to result in synergistically effective combination therapies.

[95] Another class of embodiments are the methods for using all of the above compositions against human or animal pathogens, either: (1) internal, (2) external or (3) both internal and external.

[96] A further class of embodiments includes the use of AgEDTA for human and / or animal health and welfare.

[97] Another additional class of embodiments includes the use of other silver agents such as, for example, silver EDDS, silver curcuminate, silver berberim, and silver tetracycline.

[98] Another additional class of embodiments includes the use of other metals such as zinc, copper, copper alloys, titanium, platinum and their alloys and mixtures, interchangeably with silver to both in the preparation and in the methods of applying the treatment disclosed herein. For the sake of brevity, money is mostly referred to herein.

ΜΑ 29428Β1 however, it will be understood that the other materials disclosed herein may also be

[99] In another preferred embodiment of the invention, the additional inorganic silver products can be at least partially, or in some cases, substantially, completely substituted by the silver compositions of the present invention. . Specifically, silver (eg silver ions, Ag +, silver metal) can be controllably attached or fixed, eg between layers of clays and / or in cages in zeolites. . These attachments can take place by controlling the load, eg, the silicate layer, the load of the zeolite cage, as well as the distances between the layers or the size of the zeolite cage. In this regard,! ؛ Money may be tied or tied closely or relatively loosely, depending on the particular application of health or welfare and the point of interaction between money and the biological organism (for example, at the surface of the biological part, or in an internal part, or in combination of the internal parts, etc.). Accordingly, the resulting products can include products which are quite fluid and thus are drinkable or sprayable; as well as products which are gel-like or paste-like and are spreadable to surfaces such as gels or pastes. Any of the metals discussed herein may reside in a crystalline or amorphous clathrate of at least one atomic layer of oxygen-containing or oxygen-containing molecules. Some metal / clathrate structures have been shown to have unexpected efficiency. Furthermore, in addition to the silver being incorporated in or on a structure of an oxide layer (for example clays) and networks (for example zeolites) silicates, phosphates, and oxides such as hydrotalcytes can also be used. Still further, desirable families of mica or clays which are capable of being used with the present invention (and which are capable of having different charge areas and / or different distances between layers) include, for example, illites, montmorillonites, chlorites, and vermiculites.

[100] Clays or micas, as well as zeolites, are very desirable as a carrier of metal ions for several reasons including many of which are naturally occurring or easily drifted, the particles can be maintained within a desirable colloidal size range. that make them, for example, suspended in liquids (eg, water) and are typically very biologically familiar (eg, little or no side effects). In this regard, once the silver is placed, for example, on or in a clay or a zeolite, the molecules are then heated to moderate temperatures (eg, 100 to 2OO٥C) to fix the silver to or in the clathrate, all these materials can be made in a wide range of viscosities from very fluid to very viscous.

[101] Still still, the silver metal or silver ions incorporated into a silica gel by diffusion and drying are also desirable mechanisms for the delivery of the metal ions of the present invention.

[102] In another preferred embodiment of the combinations of the invention of the above-indicated particles, the organic and / or inorganic structures can be used to positively affect the health and welfare of humans and animals. Specifically, the particles of the metals, according to the invention, can be used alone, as used above. Additionally, the metal particles can be combined, for example, with the organic compounds discussed above (for example, AgEDTA). Still further, the metal ions according to the present invention can be combined with any of the inorganic compounds (eg, clays or zeolites). Still further, the metal ions of the present invention can be combined with both organic molecules (eg, AgEDTA) and inorganic molecules (eg, clays or zeolites). This combination of silver metal or silver ion delivery systems can be constructed so that, for example, internal consumption of any of the systems. ' delivery of money above may result in money being delivered to different parties, for example, to an organization. Particularly, for example, in humans, some silver can be absorbed by the loop, through the intestine, as well as through the colon and / or the small intestine, etc. Additionally, depending on, for example, the amount of clays or zeolites relative to water (as well as various gelling compounds disclosed herein) the resulting products can be very liquid (low viscosity) to very viscous (high viscosity). . In this regard, in general, the more clay or zeolite provided relative to the water (as well as the gelling agent) the more viscous the end product.

[103] EXAMPLES

[104] COMPOSITION TRAINING

[105] Silver / water compositions can be made according to the methods set forth in United States Patent No. 6,214,299, the subject matter of which is specifically incorporated herein by reference.

[106] A preferred method for producing a composition comprising silver according to this invention uses an electrochemical cell comprising electrodes and comprises the steps of:

ΜΑ 29428Β1

[107] (a) placing at least two silver electrodes in contact with a quantity of high purity water.

108 ؛] (b) transmitting an electric current through the silver electrodes to thereby separate the silver particles from said silver electrode in a sufficient manner to cause the production of the clay particles suspended in it! ' water; and

[109] (c) agitating the water during said production of the suspended silver particles thereby to disperse the silver particles in at least a uniform concentration in said water such as a high amount and a substantially uniform distribution of the particles silver suspension can be produced in batches.

[110] Another preferred method for producing a composition comprising silver / water compositions uses an electrochemical cell and comprises the steps of:

[111] (a) establishing an electrical circuit including a current source, and a first conductor electrically connects to said current source and a second conductor electrically connects to said current source, where said first conductor is disposed within the space of the second conductor, and where at least one of the conductors is made of elemental silver, or alternatively, zinc, copper, copper alloys, titanium, platinum and their alloys or mixtures:

[112] (b) close the circuit by placing the first conductor and the second conductor in communication with a fluid resistance;

[113] (c) operating the current source to supply the alternating current simultaneously to the first conductor and the second conductor so that the voltage is increased and decreased in the first and second conductors in an alternating tandem to thereby drive the particles of silver (or other metal) to separate from the first electrode and enter the fluid resistor and become suspended in said fluid resistor; and

[114] (d) select the adjustment of the electrodes by their removal towards the fluid resistance to compensate for the decrease in the length of the electrode due to the gradual separation of the silver particles to thereby avoid the formation of arcing. place between the electrodes and said resistive fluidic and to maintain a desirable current density at the tip of the electrodes.

[115] Each water chamber or tank which produces silver compositions has a power supply consisting of eight transformers (an acceptable transformer for use in the present invention is Fran'ceformer, Part No. 48765) rated for 120. VAC consumption and 10,500 VAC maximum output at 3Omilliamps. Each transformer is preferably equipped with a 45-microfarad capacitor (such as Aerovox. Part No. Μ24Ρ3745ΜΡ2) branch in parallel through a transformer output lug.

[116] The combination of transformer and capacitor can be beneficial in some cases and very desirable in others. In particular, the transformer helps in bringing in voltage and sine waves of the AC supply current in phase with each other. The degree to which voltage and currents have in phase with one another is known as factor power. The shutdown of the factor power is at 1.0, the closing of the phases reasserts in volts and amps and the most energy is delivered to the electrodes (for example, the energy is typically determined by multiplying the volts times the amps ).

[117] Each reservoir is fitted with a transparent cover made, for example, of a suitable polymer, and is constructed to receive eight sets of electrodes. Each series of electrodes consists of a fixed electrode, made, for example, of 18-gauge silver plate, flanked by two consumable electrodes, made of, for example, 18-gauge silver wire (.9999 purity) . The electrodes are preferably bent in the middle and the ends twisted together in a double helix to obtain a desirable voltage and a combination of energy density. Each series of electrodes is operated by a transformer.

[118] Since each tank is filled for production, the electrodes are adjusted so that the attached electrodes are in good contact with the water (eg, at least 1/3 to ٨A of the plates are submerged), and the consumable electrodes are above the surface of the water. Once the power supply is energized, the water rises and forms a cone-like structure around each consumable electrode. This cone-like structure is known in the literature as a Taylor cone. Initially, water is very pure, and thus has a high electrical resistance. As a result, once using, for example, a fixed current, a 10,000 volt transformer, the voltage applied across the electrodes may initially be very high, eg, about 6500 to 8500 volts, and the consumable electrodes may be 5 to 10 mm above the water surface, thereby achieving a desirable voltage and desirable current density at the consumable electrodes. This results in a relatively large Taylor cone due to the low conductivity of the water.

ΜΑ 29428Β1 relative to the high conductivity of the electrode (for example, a wide field is created). Elm silver nanoparticle product as silver particles are removed from the consumable electrodes at the air / water / silver electrode interface. As water takes up more and more silver particles, the electrical resistance of the water / silver mixture drops. In a fixed current or current limiting arrangement, the applied voltage will then drop or decrease as a function of time (see, eg, Figure 31). As a result, the consumable electrodes are typically lowered to be closer to the surface of the water, for example, may be only 102mm above the surface. Under simple conditions, the Taylor cones will also be small because of a small difference in the conductivity between the electrodes and the water (for example, a small field is present). In general, the consumable electrodes and / or the water level must be properly adjusted during the production process in order to maintain the initial geometry. Although the Taylor cones become small during this process (thus representing, for example, metal particles entering the solution) the small Taylor cones will remain present at the end of the treatment, the water in each tank is agitated air. throughout the process to maintain homogeneity.

[119] Once a desired or target ppm or silver in the silver / water solution is reached, the product can then be pumped, if desired or required, through a filter of 1 into one of several tanks carrying very high capacities. large, for example, 2,300 to 6,500 gallons, and analysis before being bottled for shipment, ، analysis is done by a digestion process using heat and nitric acid, and the analysis takes place using a Perkin-Elmer Analyst 300 Atomic Absorption spectrophotometer. The produced silver / water composition can be subsequently combined with other ingredients to give a hydrogel. a sheet material, or may be bottled as such, or may be combined (eg, either as a liquid or dried and added as a powder) with other additives, as discussed elsewhere herein.

[120] With further reference to Figure 31, what is shown is the real time voltage drop and silver concentration versus time data corresponding to a series of formation of the inventive silver / water compositions. Clearly, as the series progresses, the voltage decreases as the silver concentration decreases. A corresponding decrease in the size of the Taylor cones on each consumable electrode is also noted. The silver concentration data on this diagram will not be taken as quantitative, however, but representative, due to sampling and mixing results (e.g. the silver / water mixture may not be completely homogeneous at any time of sampling).

[121] Referring now to Figure 32. it is shown two traces of silver concentration for this same batch, as well as several additional concentration data points, gray line with squares indicates instantaneous silver concentration (as determined by atomic absorption spectroscopy) based on a 60 ml sample obtained by pipetting approximately halfway down the reservoir and about halfway between the center and the reservoir wall. The black line with diamonds indicates the instantaneous concentration of silver as abruptly approached by a previously calibrated device measuring the electrical resistance of the above 60 ml aliquot of liquid. In terms of raw resistivity data, water initially (eg, time = zero) and electrical resistivity of about 175 kilo-ohm centimeters. In contrast, at the 31 hour point in the series, the water / silver mixture had a resistivity of about 62.7 kilo-ohm centimeters.

[122] Immediately below the concentration / resistivity data point at the 32 hour point is a single data point presented as a meter. This data point shows the silver concentration as determined by atomic absorption spectroscopy after high voltage excitation, but leaving the mixer / bubbler operation continued for another 20 hours to homogenize the mixture.

[123] One conclusion that can be drawn from Figure 32 is that silver cannot be initially distributed homogeneously in the reservoir since silver is formed, despite the presence of the mixer / bubbler operation during the process. during the series. Rather, there may be some time lag after completing the silver additions to the bath before the mixer / bubbler can catch up, and distribute the silver evenly through the water.

[124] Figure 33 is another diagram of instantaneous voltage and silver concentration as a function of time during the series of silver / water production. This diagram also shows the instantaneous power factor of the power supply transformer. Thus, the power factor starts at about 0.8, increases to a maximum of about 0.97 around 6 hours, and decreases to a low degree of about 0.6 after about 30 hours. In addition, the voltage / time data was mathematically fitted to the equation y = -2.1333 ، n (x) + 8.7057, where y is voltage and X is time. The ppm of silver in water is represented by '' squares and starts around 1 ppm and reaches a maximum of around (for example, due to water not being completely pure after filtration) 11 ppm after about 30 hours.

[125] PHYSICAL CHARACTERIZATION

ΜΑ 29428Β1

[126] Analysis of the silver content in the silver compositions of this invention can be performed by flame-atomic absorption spectroscopy (acetylene) (FAAS), plasma inductively coupled plasma (ICP), emission spectroscopy. atomization (AES) or other techniques known to those skilled in the art to be sensitive to silver in an appropriate concentration range. If the particles of the silver composition are small and uniform in size (eg, 0.01 micrometers or less), a reasonably accurate test can be obtained by directly atomic absorption colloid flow or ICPAES. This is because sample preparation for atomic absorption spectroscopy essentially ionizes all of the agent allowing its easy detection.

[127] If the compositions have particles as large as 0.2 microns, it is preferred to use a digestion procedure. The digestion procedure is not essentially ideal for silver compositions which can be made or stored in contact with halides or other anionic species which can react with finely distributed silver, or combined with protein or other material. gelatinous. One embodiment of the digestion procedure is as follows:

[128] 1. Take 10 ml aliquot of a shaken or completely mixed silver composition to be analyzed, and place it in a clean polycarbonate bottle or other suitable material container (usually the bottle) with a lid. suitable greenhouse. It is preferred a size of 30 to 100 ml.

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

[130] 3. With the lid of the bottle tightly in place, heat the silver composition to a temperature of at least about 8O٥C, and preferably about 9O٥C to 10OC with gentle stirring for sufficient time to dissolve the silver. - dissolution is essentially instantaneous.

[131] 4. Place the resulting mixture at room temperature with the lid in place. Shake the bottle completely. This digestion procedure also dissolves any surface layer of silver oxide that may be present on the silver particles.

[132] 5. Use atomic absorption spectroscopy. ICP / AES, or equivalent means for analyzing the silver content of the silver mixture. Preferably, a newly prepared titre or titles will be used, preferably prepared according to the instructions of the manufacturer of the equipment, with an appropriate dilution if necessary.

[133] 6. In reporting the results, account should be taken of all dilutions during preparation, including the 1% dilution caused by the addition of nitric acid.

[134] The silver concentration of the silver / water compositions of the present invention corresponding to the data in Figures 31, 32, 33, etc., was determined using the Perkin Elmer atomic absorption spectrometer (aa) AAnalyst 300. Samples of the inventive silver / water compositions were digested according to the procedure described above.

[135] Principle

[136] The Perkin Elmer AAnalyst 300 system consists of a high efficiency burner system with a Universal GemT p nebulizer and atomic absorption spectrometer.

the burner system provides the thermal energy necessary to dissociate the chemical compounds, providing the atoms of the free analyte so that atomic absorption occurs, the spectrometer measures the amount of light absorbed at a length of d specific wave using a hollow cathode lamina as the first light source, a monochromator and a detector. A deuterium arc lamp corrects the absorbance background caused by non-atomic species in the atom cloud.

<td>[137] ANALYSIS OF PHYSICAL / CHEMICAL FORMS OF SILVER and SILVER / WATER</td><td>COMPOSITIONS</td>

[138] A. Introduction

[139] A sample of a composition, nominally containing 22 ppm silver in!] Water, was analyzed using a time-of-flight secondary ion emission mass spectrometer (TOF-SIMS) to determine the form of silver in the composition, the conclusion is that the volume of silver exists as silver (0) (which

<img file="MA29428B1_D0002.tif" />

ΜΑ 29428Β1 esl, a metallic silver) and that there is a surface coating which is like average of the composition, for example, silver (II) oxide (AgO). As mentioned above silver (II) oxide is usually a stoichiometric combination of silver (I) and silver (III).

[140] B. Experimental Procedure

[141] A few drops of the 22 ppm inventive silver composition were dry-evaporated on a silicone substrate at room temperature, the residue was analyzed by TOF-SIMS, and it is denoted as a sample. A reference silver (II) oxide (AgO) material was analyzed by placing few particles of the reference powder as received from the vendor on a silicone substrate, and it is denoted as

[142] The time-of-flight secondary ion emission mass spectrometer (TOF-SIMS) technique is based on the principle of bombardment of a solid sample with a pulsed, finely focused beam of primary ions, and then the analysis secondary ions produced from the sample surface via a time-of-flight mass spectrograph. this analytical technique is sensitive to the surface, deriving its information from a layer that extends approximately 20 to 40 A (one Angstrom = 1χ10-4 microns) below the surface, the TOF-SIMS technique is used normally as a study instrument to identify the composition of unknown samples. It is capable of quantification if the appropriate microanalytical standards are available for calibration. This analysis was performed using standard high mass resolution conditions.

[143] C. Resultas

[144] Negative ion masses were obtained for the Ag (؛ 1) O reference material and product samples, respectively, the mass spectral region for the two spectra showed the presence of more than one. species of silver oxide that was most likely present as at least a partial coating on the silver particles, the data suggested that silver (II) is the average oxidation state of silver present on the surface of the sample particles. The silver oxide signals (eg, AgO) show a significantly high intensity in the reference sample relative to the product sample which is likely because metallic silver is dominant in the sample. . It will be appreciated that since the average particle size in the sample is decreased, the ratio of silver to silver oxide will also decrease since more silver oxide will be present.

[145] ORPHOIOGY / COMPOSTON SIZE ANALYSIS

[146] It is likely that the unusual effectiveness of the silver / water preparations described herein is due to the ratio of the surface properties / internal properties (eg, oxide / metal) of the particles and / or the size distribution of silver nanoparticles and / or the morphology of silver nanoparticles. The smaller the average particle size, the larger the surface area and the greater the contribution of the particular surface chemistry. However, if the particles are excessively small it may have a loss in stability and / or other interactions which may adversely affect the product. The silver / water compositions of the present invention are remarkable because they are stable in essentially pure water without surfactants, etc. (eg, many of the (colloidal) silvers of the previous art require proteins to keep the silver particles in suspension). Also, silver / water compositions are essentially colorless while other colloidal silver preparations (especially with larger particle sizes) usually show colors. These properties are a result of the manufacturing conditions, as discussed above herein.

[147] Numerical analysis of the composition showed that there is an average particle diameter of 0.0106 micrometers with a range of 0.006 micrometers to 0.0851 micrometers. However, size distribution analysis shows that over 95٥ / of particles were between about 0.005 micrometers and about 0.015 microns in diameter.

[148] Further particle analysis was performed by SEM, EDS (EDAX) and TEM. In particular, the silver / water compositions were dried and placed on an EM grid and examined in a SEM (eg, scanning electron microscope) and two different TEMs (eg, transmission electron microscope). These analytical instruments resulted in the determination of particle size distribution in the range of 10 to 30 nm. However, some estimation of particle size was necessary in some of the photomicrographs generated because the particles tended to collect or agglomerate together in the drying, the size of the dried agglomerates was between 50 to 100nm. Figures 1-6 show various TEM photomicrographs of the dried silver particles of the silver / water compositions of the present invention. Figures 7a to 7d show various TEM photomicrographs of silver particles made according to the present invention, or these photomicrographs were generated by a different technique. In

<img file="MA29428B1_D0003.tif" />

ΜΑ 29428Β1 in particular the silver / water compositions of the present invention were placed on a film c is examined by c exempley0-TEM (eg, a different ٢EM from the TEM used to generate Figures 1 to 6), at a temperature of about 100 c. the silver / water composition of the present invention was therefore frozen substantially immediately. The cryo-ΤΕΜ was operated at about -1OO٥C and an energy level of about 100kV, and the generated photomicrographs are shown in Figures 7a, 7b, and 7c. these Figures 7a to 7c clearly show that the average particle size is less than 20 nanometers. In addition, Figure 7d shows the TEM analysis in the SAD mode. In general, these TEM photomicrographs (Figures 7a to 7c) show maximum particle sizes of ungrouped silver particles of 15 nanometers or less, and some smaller particles in the range of 3.5 to 5 nanometers, the Diffraction analysis shown in Figure 7d indicates that the particles are predominantly metallic silver, are twinned, and are substantially pure. There is a suggestion that these photomicrographs of a possible covering or coating, Figure 7e shows an EDAX spectrum (e.g., an energy scattering spectrum or EDS) of the silver particles taken from the silver / water compositions. Of the present invention, Figure 7e never shows any metal contaminants (eg. Au. Pt, etc.) in the silver, the copper present is of a microscope equipment requires. There is evidence of a significant amount of the oxygen present, which may be present in the copper, as well as being present as a coating (s) in at least part of the silver particles.

[149] Figure 8 shows an electron diffraction pattern taken from a silver particle of the present invention. These data suggest the presence of at least one species of silver oxide. This data is subject to some interpretation, however, as Fig. 9 shows, for example, electron beam damage occurring to silver particles during the data collection process. This damage to the electron beam was not so evident when examining colloidal silver produced by other manufacturers (discussed hereinafter). Thus, the collection of data using the SEM and TEM techniques is quite difficult because the energy from the electron beams is capable of damaging any surface compositions of interest. Thus, great care has been taken in generating and analyzing these results.

[150] Figure 42 shows the results of a further characterization instrument. In these cases, x-ray powder diffraction techniques have been used in an attempt to demonstrate the existence of the oxide phases. In particular, Figure 42 shows four X-ray diffraction patterns taken from four different locations on a 200 ppm dried silver / water composition made in accordance with the present invention. In addition, superimposed on four X-ray diffraction patterns are four reference diffraction patterns of species other than pure silver metal. In particular, a 32ppm silver / water composition made according to the present invention was concentrated to about 200ppm by a reverse osmosis water filtration process. In particular, the inventive silver / water composition was held through a reverse osmosis filtration system where the wastewater from the reverse osmosis filtration system contained more than one concentrated silver component. Once a 200ppm solution was obtained, this solution was dried in a floating nitrogen environment in order to produce a powder which could be subjected to X-ray diffraction. Specifically, the silver / water mixture was placed in a basin, the basin was covered with a plastic sheet, and nitrogen was introduced into one end of the plastic sheet / basin assembly: and Nitrogen depleted from the opposite end of the plastic sheet / basin assembly. the temperature of the apparatus does not exceed about 75 to 8O٥C in order to maintain the integrity of all components in the silver / water mixture. A sufficient amount of the dry powder (eg, made from a 200ppm solution) was then available for X-ray diffraction analysis.

[151] The generated X-ray diffraction patterns clearly show the presence of at least four separate species. In this regard, it is clear that a series of silver carbonate peaks occurs around 18 to 22 degrees. These peaks are probably due to the drying procedure. In this regard, co₂ in air was more likely to be present although attempts were made to create a fertile nitrogen layer over the 200 ppm solution during the drying procedure. Additionally, a series of peaks occurs around 33 degrees. However, each of these peaks could be attributed to silver oxide (AgO), silver carbonate (Ag₂CC&gt; 3), and / or silver oxide (AgO). Thus, the species present is not completely clear.

In addition, a strong peak of silver metal occurs at about 38 degrees. This strong peak can be seen in each of the X-ray diffraction patterns. It is noted, however, that a small amount of silver oxide (Ag₂0) the peak also occurs around 38 degrees. Even more, a strong peak of silver oxide (AgO) occurs around 37 degrees, in combination with a relatively strong peak of silver carbonate (Ag₂C٥3) also. It is further noted that the silver oxide (AgO) peak corresponds to one of the tetragon phases of silver oxide. What is clear from examining the generated x-ray diffraction data and comparing it to the existing database file is that at least one silver oxide phase is present in the inventive silver / water compositions according to the present invention. It is possible that a combination of the oxides is present due to the new processing techniques according to the present invention. It is noted that X-ray diffraction patterns are available for Ag₄04 to compared to the X-ray diffraction patterns of the present invention.

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[152] _ However, Ag₄O₄ does not exist on the market. In this regard, a sample of Ag O has been obtained in the market and an analysis of TGA and DTA of such powders has been performed. In particular, Fig. 43 corresponds to TGA analysis and DTA analysis, respectively. It is clear from the DTA curve in Figure 43 that an endotherm for Ag₄O₄ exists around I81٥c. This endotherm also corresponds to a loss of weight shown in the TGA curve of figure 43. These experimental measurements correspond to Ag₄O₄decomposing into AgiO. a second very strong endotherm is shown at about 4O3٥C, as well as a corresponding second weight loss. These two experimental points correspond to AgO decomposing into Ag metal.

[153] Figure 10 shows an SEM photomicrograph of novel silver electrodes prior to being used in the process according to the present invention. EDS elemental analysis was performed on the parts of the electrode marked with 1, 2 and 3. These three separate analyzes appear in Figures 11, 12 and 13, respectively. These analyzes showed essentially pure silver being present.

[154] Figure 14 shows an SEM photomicrograph of the tip of a silver electrode used after it was used in the process according to the present invention. And the elemental analysis of EDS was performed on parts of the used electrode marked with 1 and 2. These two separate analyzes appear in Figures 15 and 16, respectively. Figure 17 shows an SEM photomicrograph of the tip of the used electrode at high magnification (approximately 3500Χ). Parts 4 and 5 were examined by EDS elemental analysis and were also found to be substantially pure silver.

[155] Comparison of Silver Particles of Colloidal Argents Available on the Market

[156] In an effort to understand the differences in performance (eg, biological efficacy) of the silver / water compositions of the present invention, compared to colloidal silvers, the differences in physical properties were examined. , Figures 18a and 18b are TEM photomicrographs of the silver particles which correspond to the silver particles in a first colloidal silver obtained from General Nutrition Center in 2004 and known in the market by GNC Liquid Colloidal Silver Dietary Supplement (25 ppm) ( GNC). Figures 19a and 19b are TEM photomicrographs of the silver particles which correspond to a second colloidal silver known in the market by Silverado. Figures 20a and 20b are TEM photomicrographs of the silver particles which correspond to a third colloidal silver known in the market by Vitamin World Bioorganic Advanced Colloidal Minerals (3 ppm) (Bioorganic). Figure 21 is an overall TEM photomicrograph comparison of silver particles from two silver / water compositions (labeled ASAP 20 and ASAP 10) and from three colloidal silvers known in the market by GNC, Silverado and Bioorganic, discussed above, the clear differences in particle sizes and shapes are evident from these photomicrographs, thus showing that they are physical differences, structural and chemical potentials between different colloidal silvers, which may help by partially explaining the differences in biological efficacy between different products of similar general chemistry.

[157] CHARACTERIZATION OF SPECTROSCOPY

[158] RAMAN SPECTROSCOPY

[159] Additional analyzes of silver / water mixtures were performed by Raman spectroscopy. A number of analytical approaches on three different Raman spectrometers were performed. The reason for the use of Raman and Raman Resonance spectroscopy has been the belief that different modes (and / or amplitudes) of vibration might be apparent in the different colloidal silvers when compared to the silver / water compositions herein. invention, as well of comparison with pure or demineralized water. In addition, these different observed modes of vibration in water molecules can help to better define colloidal systems and to explain the difference in biological efficacy in different silver products.

[160] In a first series of Raman spectroscopy measurements, a Confocal Raman microscope from Vitech (Ulm, Germany) was used, the model number is CRM200. Spectra were obtained using a Nikon 6Οχ immersion objective (NA = 1) with an integration time per spectrum of 15 seconds (for example, three acquisition times separated by 5 seconds), the CCD was centered around 1,799 wave numbers. a droplet of a solution was placed in a small wall in a petri dish and the immersion objective was decreased herein, the Raman laser source was 532nm with about 10mW. The confocal detection system was used with the confocal volume being approximately 0.3 X 0.3 X 0.75 micrometers (approximately 7χ10 Ε-8 picoliters).

[161] Figures 22a and 22b show the results plotted data collected for 7 samples. Two of the samples are the same, although marked differently (10PR and 10 PSU) and

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ΜΑ 29428Β1 correspond to `` ASAP 10 mentioned above (e.g. 10 ppm silver of the inventive silver / water composition). HPIC corresponds to water of high purity (HPLC degree Ultrapure) obtained from Alfa Aesar. DI corresponds to demineralized water. GNC corresponded to the dietary supplement of GNC cash (25 ppm). G -32 ”corresponded to the inventive silver / water composition of 32ppm. W corresponds to Vitamin World Bioorganic Advanced Colloidal Minerals (3 ppm) (previously referred to as Bioorganic). Clear differences are shown between different samples. For example, the first mode of extension (eg, wave numbers around 3400 to 3500 1 / cm) in these various water and water-based solutions shows great differences. In addition, the vibration / rotational behavior below 500 1 / cm also shows clear differences between samples. Some differences can be seen also in the binding modes around 1600 1 / cm. Without wishing to be bound by any particular theory or explanation, it appears as if the different behaviors of the silver / water compositions of the present invention can influence, for example, or at least help to explain, the effectiveness of such compositions. compared to the other samples studied.

[162] A second set of Raman data was generated from a different spectrograph system. As long as the numbers generated between the two datasets are different (which strongly suggest that the Raman spectroscopy data for water is dependent on the analytical device used) the data in this dataset also shows remarkable differences between the compositions. of the present invention when compared to other colloidal silvers or other waters. In this set of Raman spectroscopy measurements. a Raman reflection microscope was used. Spectra were obtained using an Olympus 20χ objective (ΝΑ = 0.4). the CCD detector was centered at four different wave numbers, namely, 1600, 2500, 3400 and 4400 1 / cm. the laser source for the Raman was 514.5nm with approximately 11.5mW. additional information regarding the spectrum can be found in any of Figures 23a, 23b and 23c. the marking of samples in these Figures consists of the following text. All of the Raman spectroscopy data sets strongly suggest that different molecular motion exists in these different samples which may contribute (or at least demonstrate) the biological efficiency of the silver / water compositions according to the present invention.

[163] A third set of Raman data was generated using a Renishaw Confocal Raman Micro-spectrograph multiple line laser. This system has been configured to allow measurements from both the above and immersed in the sample. The facility was designed to examine a sample volume greater than 100x to 100٥χ than that described in the first set of measurements. The Leica DL DM microscope reflection micro-spectrography was adjusted with either a 20x (NA = 0.5) water immersion or a 5x (NA = .12) dry objective. The rear aperture of each lens has been rated to be the same or to exceed the laser beam diameter. Two laser frequencies were used, these being 50mW multi-line argon laser at ٨A power plant for 514.5nrn and a 20mW HeNe laser at 633nm. The high resolution grids were fitted in the monochrometer optical channel which allowed continuous scans of ode numbers from 50 to 4000 (1 / cm). It uses integration times of ten to 20 seconds. The fluid sample was placed below the objective in a 50ml beaker. All lasers were used to examine resonance bands, while the first laser was used first to obtain a Raman spectrum. The sample size was approximately 25ml. measurements made with a 5x dry objective were made with the objective placed approximately 5mm below the fluid to interrogate a volume of approximately 7mm below the water menisci, the immersion measurements were made with) With a 20x immersion objective placed approximately 4mm into the sample allowing examination of spatial volume, the CCD detector acquisition areas were individually adjusted for each objective to maximize signal strength and signal-to-noise ratios. A representative spectrum for the silver / water compositions of the present invention is shown in Figure 24a. Figure 24b shows the Raman spectrum of three different metal / water solutions made according to the present invention, plot 1 corresponds to a 13ppm silver / water solution: plot 2 corresponds to a zinc / water solution 10ppm: and plot 3 corresponds to an 11 ppm copper / water solution.

[164] While the numbers produced between the three data sets are somewhat different (which strongly suggest that the Raman spectroscopy data for water be dependent on the analytical instrument and set-up of that instrument), the data in this dataset demonstrates remarkable differences between the silver / water compositions of the present invention when compared to other colloidal silver or other waters. All of the Raman spectroscopy data sets strongly suggest that different movement and molecular bonds exist in these different samples which may contribute (or at least demonstrate) the effectiveness of silver / water compositions according to the present invention. In addition, the differences in the Raman patterns for the three different metal / water solutions shown in Figure 24b also suggest a different possible efficiency.

[163) UV-VIS SPECTROSCOPY

[164] Further analysis of the silver / water mixtures was performed by UV-Vis spectroscopy. Uv-Vis spectroscopy has been used in addition to Raman spectroscopy to search for additional distinguishing vibration modes and / or amplitudes in a different part of the spectrum. A simple spectrometer / V

ΜΑ 29428Β1

UV-٧is was used to collect the data. In this regard, energy absorption spectra were obtained using a UV-Vis microspectrophotometer. This information was acquired using dual beam scanning monochrometer systems capable of scanning the wavelength range from about 190 nm to about 00 nm. The UV-Vis spectrometer that was used to collect absorption spectra was Jasco MSV350. the instrument was set up to support the measurement of low concentration liquid samples using a 10mm X 10mm fused quartz cuvette. Data was acquired over the above wavelength range using both a photo multiplier tube (PMT) and a photo diode detector with the following operational parameters: a collection of width band of 2nm, a resolution of 0.5nm: and a baseline background subtracted from the produced spectra. In this regard, the UV-Vis signature for pure water has been subtracted from the spectra produced in order to show more representative spectral signatures for the silver / water mixture.

[165] Both tungsten halogen and hydrogen D2 energy sources were used as primary energy sources for the MSV350. The optical path of the spectrometer was placed to allow the energy beam to pass through the ectiantillons with the focus towards the center of the control cuvettes. The sample preparation was limited to filling and covering and placing them physically on the cuvette holder in the fully enclosed compartment. The data output was measured and displayed as absorbance units (by Beer-Lambert law) against wavelength and frequency. The primary difference between the samples corresponding to the two spectra shown in each of Figures 48a and 48b was the concentration of silver in the silver in each of the samples. Specifically, the higher amplitude curve in each of Figures 48a and 48b corresponds to a 32ppm silver / water solution: and the low amplitude curve corresponds to a 10ppm silver / water solution. the wavelength or frequency positions of the peaks (i.e., the locations of peaks and tilts) are quite similar.

[166] As discussed above herein, silver (eg, silver ions, silver metals, Ag +, etc.) can be attached or fixed in a controllable manner, eg, between and / or on clay layers and / or in zeolite cages. One method of effecting the displacement of, for example, silver ions in or over clays, mica, or zeolites, is to provide ionic silver species in a soluble state and to present said species in a composition or mixture. clay or zeolite. The concept of exchanging, for example, one silver ion for another positively charged ion is sometimes referred to as BEC or CCE (these are both shorthand nomenclatures to relate to the cation exchange capacity of d 'a system). In this regard, most kaolin materials are known to have cation exchange capacities which are in the range of 2 to 5 (i.e., 2 to 5 meq / 100 grams). Montmorillonite clays, for example, have cation exchange capacities around 100 meq / 100 grams. Whereas, zeolites can have cation exchange capacities of several hundred meqs / 100 grams. For example, a zeolite well known as India 4Α zeolite is 400 to 500 meq / 100 grams for its BEC or CCE number. In general, the higher the number of BECs or CCEs, the greater the capacity for the material to receive cations.

[167] Experimental procedures to determine whether kaolins or zeolites are capable of becoming delivery / carrier systems of silver (or other metal cations) have been conducted. In particular, the following steps were used to prepare and therefore analyze silver-clay samples, as well as silver-zeolite samples.

[168] Generally speaking, the kaolin and Zeolite Linde 4Α materials were first washed three times with deionized water to remove possible chlorine contamination which could cause some silvers to leave the products (e.g. eg silver ions) to precipitate (or react undesirably) before silver, the starting materials could preferably attach in / on the kaolin and / or zeolite structures. These washed materials were then mixed with a solution of silver nitrate (AgcNo3) in appropriate concentrations corresponding to the expected or known CEC of each respective material. The resulting treated materials on the other side were washed with deionized water to remove any unused silver nitrate. The samples were dried overnight in an electric resistance drying oven at about 12O٠C. In particular, the washing process was as follows;

[169] About two grams of each sample of kaolin or zeolite were placed in a centrifuge tube, demineralized water was added next, the sample and the demineralized water mixture were then stirred in a device. wrist shaker for about 40 minutes, the mixture was then centrifuged for about 30 minutes at about 1000 r / min. the excess liquid was then decanted from the sample tube. The steps of adding deionized water, shaking, centrifuging, and decanting were repeated for a total of three washes.

[170] Once the initial 2 gram sample had been properly washed, in order to remove possible chlorine contamination, silver nitrate was present in the cleaned kaolin and zeolite materials. In particular, about 0.09 grams of silver nitrate was present in the kaolin mixture and about 4.25 grams of fAgNO3 was introduced into the Linde 4Α zeolite. In particular, the quantities

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ΜΑ 29428Β1 measured silver nitrate was added to each tube, deionized water was then added to fill the tube, the mixture was then stirred in a wrist shaker for about 40 minutes, and then, centrifuged for about 30 minutes at about 1000 r / min. the liquid was then decanted. This process of adding silver nitrate, adding deionized water, agitation on a hand shaker, centrifugation, and decantation was repeated for a total of three times. After the washing and silver nitrate introduction procedures were performed, the samples were removed from the centrifuge tube and placed in an aluminum crucible (ΑΙ2Ο3) and dried overnight at about 120 ° C in a Electric resistance heating furnace, the resulting kaolin / silver and zeolite / silver materials were then characterized by SEM photomicrographs and SEM EDS (EDAX) techniques. Figures 44a and 44b show SEM photomicrographs for the kaolin samples made according to techniques discussed above herein. It is clear from these micropliotographs that as a book or as a sheet of kaolin structures (for example, the SiO2 and ΑΙΟ2Ο3 layers identified as X and Y in Figure 44a) clearly show that the silver cations have been located around the edges of clay materials. There was clearly some type of attachment or exchange of money, as shown by the brighter "like page" parts of these photomicrographs (Note: the X and Y parts are representative of various other like book structures in the sample). Figures 45a and 45b show an EDS (EDAX) analysis of the samples shown in Figures 45a and 45b, respectively. These analyzes clearly show the presence of aluminum and silicon, as would be expected for kaolin, as well as some titanium (which suggests the presence of rutile). Very small peaks of silver can also be seen, which correspond to the BEC numbers for kaolin being relatively low at 2 to 5.

[171] Figure 46 shows an SEM photomicrograph corresponding to zeolites processed according to the procedures discussed above herein. Due to the higher zeolite CEC number (i.e., approximately 500) the cubic-like zeolite structures in Figure 46 appear to be glowing in the photomicrograph (see, for example, Part A in Figure 46) . This glow suggests that there was a substantial even distribution of silver in and throughout all of the zeolite structures. In this regard, if there were the bright spots of the silver metal by itself shining brightly, then the silver would not have been embedded in / on the zeolite. Figure 47 is an EDS (EDAX) analysis of the sample shown in Figure 46. Again, relatively high amplitude peaks of aluminum and silicon are present, but extremely high peaks of silver are present (i.e., compared to peaks of AG in kaolin depicted on figures 45a and 45b). These very high silver peaks correspond to the much greater ability of the zeolite to capture silver in its structure (i.e., high BEC) compared to the structure of kaolins (i.e. -d., a low BEC) shown in figures 44a and 44b

[172] PROOF OF THE EFFECTIVENESS OF THE COMPOSITION OF 22 PPM SILVER AGAINST BACILLUS SUBTIIIS

[173] A. Purpose of the Example

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

[175] B. Materials and Methods

[176] Test organism.

A test suspension containing endospores of Bacillus subtilis (Ane # 19659) was prepared from a culture grown on nutrient agar, to which additional sporulation enhancing ingredients were added. Dishes were harvested with sterile water and the endospores were purified by repeated centrifugations and re-suspensions in water. The final wash was in 70% ethanol for 30 minutes, to ensure destruction of all vegetative bacteria. The spores were resuspended in water containing 0.1% Tween 80 (brand of polysorbate surfactant) to prevent aggregation.

Neutralizer. the neutralizing mixture is composed of 12.7٥ / ٠ of Tween® 80 (brand of polysorbate), 6.0% of Tamol® 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 the subsequent growth of bacteria.

[177] Killing procedure as a function of time;

[178] a) 9.9 ml of the aliquot of the disinfectant (inventive silver composition of 22 ppm, in water) was placed in a sterile 20 mm x 150 mm tube. The tube was equilibrated in a 2O٥C water bath.

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2٩٩٩ ملأ 2 Wk

[179] b) 9.9 ml of the aliquot of the disinfectant (inventive silver composition of 22 ppm, in water) was placed in a sterile 20 mm X 150 mm tube. The tube was equilibrated in a 2O٥C water bath.

[180] c) At 30 mins., 1 hr, and 4 hr, one ml of the organism / disinfectant suspension was removed from a tube containing nine ml of neutralizer. The tube was mixed perfectly.

neutralized suspension was diluted in series 1:10, in saline solution

[181] d) After two min, the physiological (PSS).

for 20 hr.

[182] e) The number of viable organisms in the selected dilution tubes was tested by membrane filtration. One ml of the aliquots were placed in duplicate, the membranes were washed with approximately 100 ml of sterile PSS and removed to the nutrient agar plates. The plates were incubated at 37 c

[183] ​​f) the number of colonies on each filter was counted and log reductions were calculated.

[184] Controls:

[185] a) The titers of the test suspensions were calculated by performing membrane filtration tests of the selected 1:10 dilutions of the test suspensions in PSS.

[186] b) A neutralizer control was performed by inoculating a mixture of 9 ml of neutralizer and 1 ml of disinfectant with 100 ml of a titer dilution containing 100 cfu. This produced about 10 cfu / ml in the tube, which was placed for 20 minutes before testing the membrane filtration using a duplicate of the 1 ml samples.

[187] C. Resuites

[188] Title of Bacillus subtilis;

Number of colonies

<td></td><td>Dilution</td><td></td>

<td>1: 1χ10®</td><td>1: 1x10</td><td> 1:1</td>

<td></td><td>Z</td><td>xio</td>

<td></td><td></td><td> 6</td>

<td>TNTC</td><td> 75</td><td> 7</td>

<td>TNTC</td><td> 58</td><td> 8</td>

[189] TNTC = too many to count

Dilution of B. subtilus spore / disinfectant suspension spore / disinfectant suspensions:

<td>time</td><td> 10١ 1: 1</td><td> 1 :1 10²</td><td> 1 :1 0</td><td> 1 :1 0</td><td> ؟10 1: 1</td><td> 1 :1 10®</td>

<td>30 min</td><td></td><td> ٠</td><td>TNTC</td><td>TNTC</td><td> 57</td><td> 10</td>

<td></td><td></td><td> »</td><td>TNTC</td><td>TNTC</td><td> 51</td><td> 7</td>

<td>1 hour</td><td></td><td> ٠</td><td>TNTC</td><td>TNTC</td><td> 28</td><td> 3</td>

<td></td><td></td><td> ٠</td><td>TNTC</td><td>TNTC</td><td> 55</td><td> 3</td>

<td>2 hours</td><td></td><td>TNTC</td><td>TNTC</td><td> 126</td><td> 23</td><td> -</td>

<td></td><td></td><td>TNTC</td><td>TNTC</td><td> 183</td><td> 17</td><td> -</td>

<td>4 hours</td><td>TNTC</td><td>TNTC</td><td> 88</td><td> 12</td><td> -</td><td> -</td>

<td></td><td>TNTC</td><td>TNTC</td><td> 69</td><td> 12</td><td> -</td><td> -</td>

[190] TNTC = too many to count

[191] Neutralization witness: 1: Ixio®

[192] D. Discussion

[193] titer results showed a viable concentration of s spore. subtilis of 6.65xio٥ per ml in the original Suspension. Inoculation of 9.9 ml of disinfectant with 100 ml of this suspension produced an initial concentration of 6.65x1 Os spores per ml in the analysis tube.

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[؛¹⁹د] The results of these procedures allow log reductions (IR) and percent kill (PK) values ​​to be calculated. They are listed in the table below. Values ​​were calculated using the formulas ؛ IR = - Log (S / So) and PK = (1- (S / So)) X 1٥0; where s = concentration of organisms at a specific time; and So = the initial concentration of organisms at time zero.

<td>Time</td><td>logarithmic reduction</td><td>Percentage of killings</td>

<td>30 min</td><td> 0,090</td><td> 18,8</td>

<td>1 hour</td><td> 0,205</td><td> 37,6</td>

<td>2 hours</td><td> 0,634</td><td> 76,8</td>

<td>4 hours</td><td> 1,928</td><td> 98,8</td>

[195] The data from the neutralization witness showed that the disinfectant was adequately neutralized, current counts correspond to those resulting from dilution without appreciable killing.

[196] The disinfectant preparation tested here showed good sporicidal activity against B. subtilis spores. B. subtilis is a common species used in the sporicidal test 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 substitute for Bacillus anthracis, anthrax bacterium. Accordingly, these results are applicable to anthrax. It is expected that longer exposure will result in additional killing.

[197] PROOF OF THE EFFECTIVENESS OF SILVER 10 PPM and COMPOSITION OF 1.0٠ / ٥ OF TO AND SILVER 14 PPM and A COMPOSITION 1.5٥ / ٥ OF Η2Ο2 AGAINST SUBTLE BACILLUS

[198] A. Purpose of the Example

[199] The aim of this example is to demonstrate the antimicrobial activity of two silver-based compositions of the present invention on the bacterial endospores of a test organism of bacillus sousilis. This was accomplished by performing standard kill versus time assays using a suspension of B. subtilis endospores. This example, visualized with respect to the previous example (using 22 ppm silver), establishes the promoting effect of hydrogen peroxide (Η2Ο2) on the antimicrobial properties of the silver compositions. Hydrogen peroxide is stable in the presence of the silver compositions of the present invention. While hydrogen peroxide itself has significant antimicrobial properties, it is frequently broken down by catalase or other microbial enzymes. However, hydrogen peroxide is able to weaken bacterial cell walls and increase the entry of silver particles before any enzymatic destruction of hydrogen peroxide can occur.

[200] B. Materials and Methods

120111. Test Organism. A test suspension containing endospores from Bacillus subtilis (ATCC # 19S59) was prepared from a culture of nutrient agar, to which additional sporulation enhancers were added, the plates were harvested and the The sterile water and endospores were purified by centrifugations and repeated re-suspensions in water, the final wash was in 70% ethanol for 30 min, to ensure the death of the vegetative bacteria. The spores were resuspended in water containing 0.1% Tween® 80 (brand of polysorbate) to prevent aggregation.

[202] 2. the neutralizing mixture composed of 12.7% Tween 80, 6.0٥ / ο Tamol® SN (؟ sodium salt ark of naphthalene-formaldehyde condensate), 1.7% lecithin. 1% Peptone, and 0.1% Cystine. This solution was intended to neutralize all chemicals so that they would not affect the subsequent growth of the bacteria.

[203] 3. Killing procedure as a function of time:

[204] a) A 9.9 ml aliquot of each of the disinfectants (inventive compositions of collol'dal silver: one containing 14 ppm of silver and 15٥ / ο H 202: the other containing 10 ppm of silver and 1.0٥ / ο Η2Ο2) was placed in a sterile 20 mm x 150 mm tube. The tubes were equilibrated in a water bath at 2O٥C.

[205] b) Each tube of disinfectant was inoculated with 100 ml of the test organism suspension at zero time.

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[206] 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 from a tube containing nine ml of neutralizer. The tube was mixed perfectly.

[207] d) After two minutes, the neutralized suspension was diluted in series 1:10 in physiological saline solution (PSS).

[208] e) The number of viable organisms in selected dilution tubes was evaluated by membrane filtration. One ml of the aliquots were plated in duplicate, the membranes were washed with approximately 100 ml of sterile PSS and removed from the Columbia Agar plates. the plates were incubated at

37٠c for 20 hr.

[209] f) the number of colonies in the filter was counted and the logarithmic reductions calculated.

[210] 4. Controls:

[211] a) Titers of test suspensions were calculated by performing membrane filtration assays of selected 1:10 dilutions of test suspensions in PSS.

[212] b) A neutralizer control was performed by inoculating a mixture of 9 ml of neutralizer and 1 ml of disinfectant with 100 ml of the 1:10 dilution of the titr. This produced approximately 2,000 cfu / ml in the tube, which was placed for 20 minutes before the 1:10 dilution. both tubes were evaluated by membrane filtration using duplicate 1 ml samples. All the results are shown in Tables 1a and 1b.

[213] C. Results

[214] Title of Bacillus subtiiis spores:

Number of colonies

<td></td><td>Dilution</td><td></td>

<td>1: 1χ10®</td><td>1: 1x10</td><td>1: 1x1</td>

<td> 6</td><td>Z</td><td> ٢</td>

<td>TNTC</td><td> 36</td><td> 5</td>

<td>NC</td><td> 2٦</td><td> 4</td>

[215] INT = too many to count.

Table the

Solution containing 14 ppm silver and 1.5% Η2Ο2:

Dilution of B. subtilis spore / disinfectant suspension

<td>Time</td><td> 10١ 1: 1</td><td> 1 :1 10²</td><td> 1 :1 0</td><td> 1 :1 0</td><td> 1 :1 10</td>

<td>10 minutes</td><td> -</td><td> -</td><td>TNTC</td><td>TNTC</td><td>“22Τ</td>

<td></td><td> -</td><td> -</td><td>TNTC</td><td>TNTC</td><td> 265</td>

<td>30 min</td><td></td><td> -</td><td>TNTC</td><td>TNTC</td><td> 258</td>

<td></td><td></td><td> -</td><td>TNTC</td><td>TNTC</td><td> 273</td>

<td>1 hour</td><td></td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 55</td>

<td></td><td></td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 33</td>

<td>2 hours</td><td></td><td>TNTC</td><td> 207</td><td> 29</td><td> -</td>

<td></td><td></td><td>TNTC</td><td> 237</td><td> 24</td><td> -</td>

<td>4 hours</td><td> 59</td><td> 3</td><td> 1</td><td></td><td></td>

<td></td><td> 57</td><td> 5</td><td> 1</td><td></td><td></td>

<td>6 hours</td><td> 0</td><td> 0</td><td> 0</td><td></td><td></td>

<td></td><td> 3</td><td> 0</td><td> 0</td><td></td><td></td>

<td>8 hours</td><td> 1</td><td> 0</td><td> 0</td><td></td><td></td>

<td></td><td> 1</td><td> 0</td><td> 0</td><td></td><td></td>

[216] TNTC = too many numbers to count.

[217] Neutralization witness:

م%

ΜΑ 29428Β1

<td>Not diluted</td><td>1: 1 X 10١</td>

<td>TNTC</td><td> 195</td>

<td>TNTC</td><td> 210</td>

[218] TNTC = Too many to count.

Table 1b

Solution containing 10 ppm silver and 1.0% Η2Ο2: Spore / disinfectant suspension of s. subtilis:

<td>Time</td><td> 1 :1 10</td><td> 1 :1 10²</td><td> 1 :1 10³</td><td> 1 :1 10</td><td> 1 :1 10</td>

<td>10 minutes</td><td></td><td> -</td><td>TNTC</td><td>TNTC</td><td> 230</td>

<td></td><td></td><td> -</td><td>TNTC</td><td>TNTC</td><td> 287</td>

<td>30 min</td><td></td><td> -</td><td>TNTC</td><td>TNTC</td><td> 254</td>

<td></td><td></td><td> -</td><td>TNTC</td><td>TNTC</td><td> 260</td>

<td>1 hour</td><td></td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 146</td>

<td></td><td></td><td>TNTC</td><td>TNTC</td><td>TNTC</td><td> 124</td>

<td>2 hours</td><td></td><td>TNTC</td><td>TNTC</td><td> 64</td><td> -</td>

<td></td><td> -</td><td>TNTC</td><td>TNTC</td><td> 71</td><td> -</td>

<td>4 hours</td><td>TNTC</td><td> 72</td><td> 5</td><td></td><td></td>

<td></td><td>TNTC</td><td> 77</td><td> 5</td><td></td><td></td>

<td>6 hours</td><td> 0</td><td> 0</td><td> 0</td><td></td><td></td>

<td></td><td> 2</td><td> 0</td><td> 0</td><td></td><td></td>

<td>8 hours</td><td> 1</td><td> 0</td><td> 0</td><td></td><td></td>

<td></td><td> 1</td><td> 0</td><td> 0</td><td></td><td></td>

5؛

[219] TNTC = Too many to count.

[220] Neutralization indicator:

Undiluted 1: 1x10

TNTC 184

[221] TNTC = Too many to count.

[222] D. Discussion

[223] the data showed a viable concentration of B. subtilis spore of 2.59 X 10٥ spores per ml in the original suspension, inoculation of 9.9 ml of disinfectant with 100 XI of this suspension produced a first concentration of 2.59 X 0 spores per ml in the analysis tube.

[224] The results of these procedures allowed log reductions (LR) and percent kill (PK) values. They are listed in the following table. The values ​​were calculated using the formulas: IR = - logSSo) and PK = (1 &lt;S / So »X 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 minutes, the minimum 10 data was used to estimate the So values. The 6 hour and 8 hour exposure times did not produce numbers high enough to be reliable. Therefore, these data were not used in linear regressions. Linear regressions were performed on the log reduction rates using the 'fitted line traces' control in the Minitab statistical package. the regression equations produced, and the times required to perform a six-log reduction are shown with the log reduction and percent kill values ​​in the following Table 2.

<td></td><td colspan="2">Silver 14 ppm + 1.5٥ / ο of Η2Ο2</td><td colspan="2">Silver 10 ppm + 1.0% of Η2Ο2</td>

<td>Time</td><td>Log reduction</td><td>Percent of kill</td><td>Log reduction</td><td>Percent of kill</td>

<td>30 min</td><td> -0,03</td><td>لآ7٠-</td><td> 0,003</td><td> 0,6</td>

<td>1 hour</td><td> 0,66</td><td> 78,0</td><td> 0,28</td><td> 47,8</td>

AT

٩ املأ 2 Mk

<td>2 hours</td><td> 2,05</td><td> 99,1</td><td> 1,58</td><td> 97,4</td>

<td>4 hours</td><td> 4,63</td><td> 99,998</td><td> 3,54</td><td> 99,97</td>

[225] Regression Analysis

[226]! The equation for the calculated line 14 ppm: Y = -0.66704 + 1.32936Χ. ! 'Equation for the calculated line 10 ppm: ٧ = -0.59690 + 1.03933Χ. These equations predict that the time for a 6-log reduction is 5.02 hours for the 14 ppm composition and 6.35 hours for the 10 ppm composition.

[227] The neutralization witness data showed that the disinfectant was adequately neutralized, the expected counts corresponded to those expected from the dilution.

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

ANTIMICROBIAL

[229] PROOF OF EFFECTIVENESS OF THE COMPOSITION OF 10 PPM SILVER AS A LARGE RANGE

[230] A. Methods

[231] MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) tests were performed using a standard broth microdilution method, MIC is defined as the lowest concentration of an antibiotic that will prevent growth (in vitro) 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 drug concentrations, which may vary depending on dose, route of administration, degree of protein binding, location of infection, patient age and weight, and other factors, MBC is defined as the lowest concentration of an antimicrobial agent required to kill 99.9% of the body's initial inoculum.

[232] The assay was performed by increasing the pure cultures of each of the test organisms in the liquid culture. Turbidometric measurements were used to monitor the concentration of the culture. Serial dilutions of each test antibiotic were made in nutrient broth, dilutions were calculated to cover the ranges which can be expected for each organism for each agent: A standard amount of the test culture was added to each tube and the tube is returned to an incubator (37 ± 2٥c) for growth, the tubes were checked turbidometrically to determine bacterial growth. Below the MIC concentration the 15 tubes showed an increase in optical density over time indicating bacterial growth, the lowest concentration of the antibiotic that showed no growth is MIC. the growth step tubes were then subcultured in fresh medium. The growth step tube with the lowest concentration of the antibiotic that showed no growth on in the subculture is the MBC. the results are shown in Table 3.

[233] B. Results:

<img file="MA29428B1_D0004.tif" />

ΜΑ 29428Β1

Table 3

<td colspan="7">Antimicrobial (ppm)</td>

<td>Organization</td><td>Tetracycline</td><td>Ofloxacin</td><td>Penicillin G</td><td>Cefaperazone</td><td>Erythrmycin</td><td>Money</td>

<td>s.pyogenes</td><td> 0,625/=-5</td><td> 1.25/2,5</td><td> &gt;5,0</td><td> 0,313/1,25</td><td> 0,003/0,019</td><td> 2,5/5.0</td>

<td>s. mutans</td><td> 0,625/=5</td><td> 2,5/=5,0</td><td> 0.521/=5</td><td> 1,25/=5</td><td> 0,009/0,019</td><td> 2,5/10,0</td>

<td>s. gordonii</td><td> 0,156/0,625</td><td> 2,5/5,0</td><td> 0,009/0,039</td><td> 1,25/1,25</td><td> 0,005/0,019</td><td> 2:5/10,0</td>

<td>s. pneumoniae</td><td> 0,078/0,625</td><td> 2.5/2,5</td><td> 0,019/0,019</td><td> 0,313/0,313</td><td> 0,002/0,004</td><td> 7,512,5</td>

<td>s. faecalis</td><td> 0,5151=5</td><td> 1,25/5,0</td><td> 5,0/=5,0</td><td> &gt;5,0</td><td> 0,009/1,25</td><td> 10,0/10,0</td>

<td>s. aureus</td><td> 0,313/=5</td><td> 0,417/0.625</td><td> 2,5/=5,0</td><td> 5,0/5,0</td><td> 0,039/=5.0</td><td> 5,0/5,0</td>

<td>s. aeruginosa</td><td> 0,078/5</td><td> 0.156/0,313</td><td> 0,13/=5,0</td><td> 2.5/5,0</td><td> 2,5/=5,0</td><td> 1,67/5</td>

<td>E. coli</td><td> 1,67/=5</td><td> 0,104/0.158</td><td> &gt;5.0</td><td> 0,625/=5,0</td><td> 5,0/=5,0</td><td> 2.5/2,5</td>

<td>E. aerogenes</td><td> &gt;5</td><td> 0,078/0,156</td><td> &gt;5,0</td><td> 2,92/=5,0</td><td> &gt;5,0</td><td> 2,5/2,5</td>

<td>E. cloacae</td><td> 1,67/=5</td><td> 0,156/0,156</td><td> &gt;5,0</td><td> &gt;5,0</td><td> &gt;5,0</td><td> 2,5/2,5</td>

<td>typhymurium</td><td> 1.25/=5</td><td> 0,078//0,156</td><td> &gt;5,0</td><td> 1,25/2.5</td><td> 5,0/=5,0</td><td> 2,5/5,0</td>

<td>s. Arizona</td><td> 0.625/=5</td><td> 0.078/0,078</td><td> =5.0</td><td> 0,833/=5,0</td><td> 4,17/=5,0</td><td> 2,5/5,0</td>

<td>s. boydii</td><td> 1,25/=5,0</td><td> 0,078/0,156</td><td> &gt;5,0</td><td> 0,625/0,625</td><td> 5,0/=5,0</td><td> 1,25/1.25</td>

<td>K. pneumoniae</td><td> 2,5/=5,0</td><td> 0,417/0,625</td><td> &gt;5.0</td><td> &gt;5,0</td><td> &gt;5,0</td><td> 2.5/2,5</td>

<td>K. oxyloca</td><td> 1.25=5,0</td><td> 10,104/0,156</td><td> &gt;5,0</td><td> 1,25=5,0</td><td> &gt;5,0</td><td> 1,25/1,25</td>

[234] data are presented as MIC / MBC (minimum inhibitory concentration / minimum bactericidal concentration) in parts per million (ppm)); &gt; indicates that the concentration required to obtain the MIC or MBC was higher than the test parameters measured for the test. For example, the highest concentration of tetracycline used for s. pyogene was 5 ppm. At this concentration there was growth which remains in the growth step tube subculture. Therefore, the MBC should be&gt; (greater than) 5 ppm.

[235] The MIC / MBC of E. coli strain ο 157.Η7, which was associated with obstructions from hemorrhagic diarrhea and colitis, was determined in the subsequent study, MIC was determined to be to be 2.5 ppm and the MBC was determined to be 5 ppm.

[236] C. Conclusion

[237] The 10 ppm silver composition of the present invention was tested and found to be 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 superior activity to all other preparations examined (data not shown), the most interesting observation was. been the great range, which the 10 ppm silver composition possesses. The antimicrobial activity that was observed was fairly constant regardless of the particular organism examined. With the exception of Streptococcus faecal is 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 gram organisms. -positive and gram-negative. 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 (all of which had MBC values ​​of 10 ppm). The data suggests that the 10 ppm silver embodiment of this invention shows a spectrum equal to or greater activity than any antibiotic examined, antibiotics generally limited the limiting antibacterial spectra to susceptible organisms, but as the data demonstrate. , the silver composition of the present invention is also effective against gram-positive and gram-negative organisms. The data suggest that with low toxicity related to silver, in general, and the broad spectrum of antimicrobial activity of this silver composition, this preparation can be used effectively as an alternative to antibiotics.

[238] D. Reference for the Previous Example

[239] 1. US EPA IRIS Report for Silver-CASRN 7440-22-4

[240] 2. Fox C1, Modak SM. Mechanism of Silver Sulphadiazine Action on Burn Wound. Infections. Antimicrobial Agents Chemother. 5: 582-588. 1974.

[241] 3. Furchner, JE, Richmond CR, and GA Drake. Comparative Metabolism of Radionuclides in Mammals. Ι٧. Retention ofSilver-IIOm in the Mouse, Rat, Monkey, and Dog. Health Phys. 15: 505-514.1968.

[242] 4. Grier, N. Silver and its Compounds in Disinfection, sterilization, and Preservation. (Seymour s. Block, ed.) 2d Edn, pp 395-407. 1977.

Λ /

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[243 ؛. Hinder, JA, and JH Jorgensen. Procedure in Antimicrobial Testing in Diagnostic Microbiology. (CR

Mahon and G Manuselis, eds.) Pp 63-91.1995.

[244] PROOF OF THE EFFECTIVENESS OF THE MONEY COMPGSITIGN 32 PPM CGNTRE PSEUDOMONAS AERUGINOSA, SALMONELLA CHOLERAESUIS AND STAPHYLOCOCCUS AUREUS

[245] A. Methods

[246] Pseudomonas aeruginosa ATTCC # 15442, Salmonella choleraesuis ATTCC # 10708 and Staphylococcus aureus ATCC # 6538 were tested using official AOAC methods (Association of Official Analytical Chemists AOC Methods, vol. 1, 15 'edition, 1990, AOAC Arlington , VA) 955.14, 95515 and 964.02. Broth nutrient tubes (NBAOAC) were inoculated from the current culture, and tubes incubated at 37 ± 2٠c. Transfers to fresh nutrient broth tubes were made for three successive days with the final transfer being incubated at 37 + 2Χ for 48 to 54 hours. The pseudomonas culture was decanted into a cool tube to remove dandruff. The other cultures were turbulent for 3-4 seconds and allowed to stand for 10 minutes at room temperature. Finally the cultures were diluted 1: 100 in peptone water (PEPW) to which equine serum was added to give a 5% yield of total organic challenges. Test carriers (10mm long polished 304 stainless steel cylinders with 8mm outside diameter and 6mm inside diameter) were soaked in the challenge solution for 15 minutes, removed, drained. and dried at 37 ± 2٠c for 40 + 2 minutes before use.

[247] Resistance of Phenol. Five to one ml aliquots of each dilution of the test phenol were placed in the sterile test tubes and equilibrated in a 20 ± 2٥c water bath. At 30 second intervals, 0.5 ml of each challenge culture was added to the appropriate dilutions of phenol, stirred, and replaced in the water bath: After the appropriate exposure times of 5.10. and 15 minutes, part of the suspension was removed from the analysis tubes and transferred to the broth tubes (LETH). the LETH tubes were incubated at 37 ± 2٠c for 2 days.

[248] Titration of the bearer. For carrier titration, 10 ml of Tween® Peptone Void Solution (polysorbate brand) (PEPT) was prepared. Two carriers were placed in individual tubes, representing the first 1:10 dilution. the tubes were shaken vigorously enough to obtain bacteria in the solution and serial dilutions were made in 9 ml voids of the LETH medium. The dilution voids were incubated at 37 ± 2٥c. The last tube with growth indicated an organizations logo title in the carrier. AOAC requires carriers to have minimum populations of 1 x 10⁴ cfu / pore r.

[249] The silver composition test. Using the sterile glass pipettes, 10 ml aliquots of the prepared disinfectants were placed in sterile test tubes and equilibrated in a refrigerated water bath at 20 ± 2٥c. Without touching the sides of the test tubes, a contaminated dry carrier was added at 30 second intervals to each tube of silver composition and placed back into the water bath. For each organism the disinfectant was tested against 60 dry contaminated carriers at 5 and 10 minute exposure intervals. After exposure, carriers were removed from the disinfectant and transferred to a tube of LETH. Culture tubes were incubated at 37 ± 2٥c for 2 days and stored as positive (+) or negative (0) for the growth of the challenge organism.

[250] Witnesses. For each organism, a dry contaminated carrier was added to a tube of IETH as a positive control, the tubes of uninoculated medium are served as negative controls. After incubation, all negative tubes were pointed with colony 1-100 forming units (cfu) of the corresponding organisms to demonstrate the effectiveness of neutralization. To demonstrate the growth promotion of the media, the negative control tubes were also inoculated with the same cfu 1 - 100 for each of the three organisms. Inoculation volumes were plated in triplicate on Soybean Casein Digest Agar (SCDA) to check inoculation titers, tubes and dishes were incubated at 37 ± 2 ° C until growth was seen in all tubes.

[251] Upon neutralization of P. aeruginosa, the initial titer of the inoculum was found to be&gt; 100 cfu which was as high for the protocol. Since all of the original tubes had been sharp, a mock test was performed with the same lot of the medium used in the test by placing the carriers in the disinfectant tubes of each of the three a good number of silver compositions for 10 minutes. The carriers were under transferred to LETH voids. These tubes were then pierced with 1-100 cfu from the organism, the tubes were incubated as before and stored for growth or no growth. New tubes of sterile medium from the same batch were also inoculated as a check for growth promotion.

[252] B. Results

ΜΑ 29428Β1

[253] Initial Test using s. aureus demonstrated run results for sample # 1 and # 2, but sample # 3 failed. Upon examination, it was decided that Sample # 3 may be damaged prior to delivery. A new bottle was obtained from the same lot as sample # 3, and the new bottle was marked as sample # 4. S challenge. aureus was repeated using sample # 4. AOAC guidelines state that at any time point and organism, only carrier 1 is grown for each

[254] Positive controls demonstrated growth and negative controls demonstrated for all lots, time points, and organisms.

[255] The carrier assay was conducted in duplicate for all garlic organisms. The title reports is an average for replications. For the three organisms, the mean titration found on carriers varies from 5.5 x 10 to 5.S x 10® cfu / carrier. AOAC requires carriers to have a minimum of 10 χ10⁴ cfu / carrier.

[256] For P. aeruginosa 3/180 the carriers showed growth at 5 min time point and carriers 2/180 showed growth at 10 min time point. For s. aureus 16/180 carriers showed growth at 5 min time point and 2/180 carriers showed growth at 10 min time point. For s. cholera, then 6/180 carriers showed growth at the 5 min time point and 1/180 carriers showed growth at the 10 min time point.

[257] Pseudomonas test culture showed growth following 5, 10 or 15 minute treatment with 1:90 phenol and showed growth following 5 or 10 minute treatment with 1:80 phenol but no growth following the 15 minute treatment with 1:80 phenol. Staphylococcus culture showed growth following 5, 10 or 15 minute treatment with 1:70 phenol and showed growth following 5 or 10 minute treatment with 1:60 phenol but no growth following treatment. 15 minutes with phenol 1:60. Salmonella culture a shows growth following a 5, 10 or 15 minute treatment with 1: 100 phenol but no growth following a 5, 10 or 15 minute treatment with

[258] PROOF OF THE EFFECTIVENESS OF 32, 22, AND 10 PPM OF SILVER AND 22 PPM OF SILVER and 1.5% Η2Ο2 AND 10 PPM OF SILVER and 10 ppm to, AGAINST SALOMENELLA AND ESCHERICHIA COLI IN CENTLY INOCULATED BCEUF R2 SAMPLES

[259] A. Purpose of the Example

[260] The purpose of this example is to demonstrate the antimicrobial activity of the silver based embodiments of the composition of the present invention on samples of beef flank steak inoculated on the outer surface with a cocktail of five strains. salmonella species, or Escherichia coli 0157: h7 at a high level of inoculum solution (1 x 10® cfu / cm²) and separately at a low level of inoculum solution (Ixio * cfu / cm ؛) (cfu = colony forming the unit).

[261] B. Materials and Methods

[262] Samples of Beef. Beef tissue samples were obtained from abattoir within 8 hours of evacuation. the rectus abdominus muscle was stopped from the carcasses hanging in the cold cooler by making an incision between the 11th and 12th ribs and then peeling the muscle out along the normal seam. The aseptically screened samples were placed in plastic bags and on ice packs and were transported the same day to the laboratory, where the samples were promptly packaged in a Multi-VCA (Α-360) and placed in a chiller. at 4٥c. the samples used to examine had a pH between 5.8 and 6.0 and were no more than 36 hours post-evisceration. From randomly selected rectus abdominus muscles, 13 x 8 cm samples were cut and processed. After treatment, 3.5 cm ؛ A flame sterilized stainless steel coring device and surgical scalpel were used to aseptically search for two meat cores per collection interval of each sample. Tissue cores were placed in a sterile stomach bag with 25 ml of 0.1% peptone and mixed for two minutes in a stomach (lab bender 400). Periodic dilutions were prepared and spirally plated at 0 minutes, 20 minutes, 1 hour, 4 hours, and 24 hours after treatment on selective and recovery media.

[263] bacterial cultures. Bacterial cultures were obtained from the Kansas state University (KSU) stock culture collection and were stored using the Protected Bead storage system, the following cultures were used for collection of Salmonella: s. lille (UGA), s. montevideo (UGA), s. typhimurium (UGA), s. agona (KSU 05 from CDC trigger isolate), and s. newport (KSU 06 CDC trigger isolate), the following cultures were used for the collection of Escherichia coli:

ΜΑ 29428Β1

E: coli 0157: Η7 (CDC 01, 03), E coli Ο157.Η7 (USDA-FSIS 011-82 Rif resistant lOOppm), E. co // 0157: Η7 (AFCC

43895 HUS type I associates and II Rif toxins. Res.) And E. coli ATCC # 23740 (Genotype Κ-12 lambda

[264! Stock cultures were grown by placing a soaked bead in a solution of 5 ml of Difco® Soybean Tryptic Broth (TSB) and incubating for 24 hours at 35٠c. Afterwards, a 0.05 ml ring of the respective culture was inoculated into a 5 ml solution of TSB and incubated for 24 hours at 35٥c to obtain a pure culture. After incubation, 1 ml of the respective culture was inoculated into 49 ml of TSB and incubated for 24 hours at 35٥c. After incubation, samples were centrifuged (15.3OOXg at 4 ° C), and the supernatant decanted and the pellet was re-suspended with 50 ml of 0.1 ٥/٥ peptone and centrifuged (15.3OOXg at 4٠C) at a final time, the peptone was decanted and the remaining granule was re-suspended with 10 ml of 0.1٥ / ٥ peptone. The five 10 ml bottles of respective culture were mixed together to produce a 50 ml cocktail containing 109 cfu / ml of the salmonella species, the cocktail was diluted to 106 cfu / ml or to 104 cfu / ml using 0, 1 ٥/٥ peptone. Cultures were confirmed by culturing in selective and differential media, and biochemical analysis of presumptive colonies using API 2ΟΕ kits.

[265] Method of inoculation. Samples of beef flank steak (rectus abdominus muscle) were cut to 13 X 8 centimeters (104 cm2) and were inoculated with a cocktail of five strains of the species of salmonella, or Escherichia coli 0157: h7 at a level high inoculum solution (106 log Otent cfu / cm2&gt; and separately at a low level of inoculum solution (104 log cfu / cm2). This inoculum was misted onto the tissue surface using a plastic spray bottle with samples contained in a sealed inoculum diameter. The actual concentration of salmonella species on the meat suiface was approximately 5.0 and 3.4 log cfu / cm2 for the high and low level inoculum solution, respectively. For E. coli Ο157.Η7, the respective meat surface inoculation levels were 4.2 and 3.9 log cfu / cm2.

[266] The beef samples were then hung vertically on stainless steel hooks attached to a motorized track which pulled the beef samples through a model spray chamber (Kansas State University, I Food Safety Laboratory) while spray treatments were applied. Treatments with either the silver compositions of this invention or deionized water were applied to the beef at 20 psi from a distance of 13 centimeters in the model pressure rinse enclosure for 20 seconds, the spout sprayer (BETE NF0580 303) provided approximately 20 ml of solution to the surface of the beef sample. The temperature of the solutions and the treatment application room was approximately I4٠c. After processing, 3.5 cm 2 duplicates of core core samples were randomly drawn from the side surface of the beef sample at 0, 20, 60 and 240 minutes. Samples were cultured and enumerated in selective differential and recovery media. [Log reductions were calculated by subtracting the logiO cfu / cm2 of the inoculated / treated samples {at the indicated sampling times (0, 20, 60, and 240 minutes) from the log- | 0 cfu / ٠m2 of the inoculated samples ا / untreated at 0 minutes. Sample processing included using 32 ppm silver, 22 ؛ silver ppm, and 10 ppm silver compositions according to the present invention. Separately, combinations of 22 ppm ؛

Ag with 1.5 wght٥ / ٥ hydrogen peroxide and 10 ppm Ag with 10 ppm peroxydisulfate (K2S2O8) were examined.

[267] C. Results with a silver composition of 32 ppm ؛

[268] The use of a 32 ppm silver composition according to the present invention produced a reduction in bacteria in the beef steak. In the following, this reduction is expressed as the log ™ of the rate of the number of bacteria in the control at time 0 to the amount of bacteria in the sample treated at the sampling time (i.e., the treatment ).

[269] For Salmonella, at a lower initial level of bacteria (104), the following log reductions were recorded: 0.78 at 0 minutes, 1.11 at 20 minutes, 1.08 at 60 minutes, and 1, 23 to 240 minutes. Thus, at 4 hours (240 minutes), the ratio of the initial count of bacteria in the control to bacteria in the 32 ppm silver treated sample is 101 23. For the upper initial level of bacteria (106), the following log reductions were recorded: 0.86 at 0 minutes. 0.95 to 20 minutes, 0.98 to 60 minutes and 1.17 to 240 minutes. The results indicate that the 32 ppm silver embodiment of this invention shows an effective bactericidal effect for salmonella on beef steak. It will be appreciated that disinfecting a meat surface is an extreme challenge for any disinfectant.

[270] For E. coli, the lower initial level of bacteria (104), the following log reductions were recorded: 1.03 at 0 minutes, 1.28 at 20 minutes, 1.42 at 60 minutes, and 1, 58 to 240 minutes. For the upper initial level of bacteria (106), the following log reductions were recorded: 0.65 at 0 minutes, 0.60 at 20 minutes, 0.83 at 60 minutes and 0.87 at 240 minutes, the results indicate that the 32 ppm silver embodiment of this invention shows an effective bactericidal effect for pathogenic E. coli on steak from

AT

ΜΑ 29428Β1

271 ؛] D. Results with a composition of silver 22ppm

[272] the results with 'Money in water. For Salmonella at a lower initial level of bacteria (104), the following log reductions were recorded: 0.41 at 0 minutes, 0.43 at 20 minutes, 0.48 at 60 minutes, and 0.68 at 240 minutes. For the upper initial level of bacteria (106), the following log reductions were recorded: 0.24 to 0 minutes, 0.24 to 20 minutes, 0.42 to 60 minutes and 0.61 to 240 minutes, the results indicate that the 22 ppm embodiment of this invention provides an effective bactericidal effect for Salmonella in beef steak.

[273] results with silver and water and 1.5 wght% hydrogen peroxide. For Salmonella, for the lower initial level of bacteria (104), the following log reductions were recorded: 0.34 to 0 minutes, 0.33 to 20 minutes, 0.36 to 60 minutes, and 0.62 to 240 minutes. For the higher initial level of bacteria (106). the following log reductions were recorded: 0.28 to 0 minutes, 0.14 to 20 minutes, 0.30 to 60 minutes and 0.69 to 240 minutes, the results indicate that silver 22 ppm with 1.5 wght٥ / ٥ of hydrogen peroxide of this invention provides an effective bactericidal effect for Salmonella in beef steak.

[274] E. Results with a composition of Silver 10 ppm

[275] Results with a composition of Silver in water. For Salmonella, for the lower initial level of bacteria (104), the following log reductions were recorded: 0.38 at 0 minutes, 0.41 at 20 minutes, 0.39 at 60 minutes, and 0.61 at 240 minutes. For the upper initial level of bacteria (106), the following log reductions were recorded: 0.24 (at 0 minutes, 0.21 to 20 minutes, 0.41 to 60 minutes and 0.54 to 240 minutes, the results indicate that the 10 ppm silver embodiment of this invention provides an effective bactericidal effect for Salmonella in beef steak.

[276] results with a composition of silver in water with 10 ppm K2S70a. For Salmonella, for the lower initial level of bacteria (104), the following log reductions were recorded: 0.26 to 0 minutes, 0.28 to 20 minutes, 0.35 to 60 minutes, and 0.58 to 240 minutes. For the upper initial level of bacteria (106), the following log reductions were recorded: 0.03 to 0 minutes, 0.16 to 20 minutes, 0.21 to 60 minutes and 0.36 to 240 minutes, the results indicate that l The 10 ppm silver with the 10 ppm potassium peroxydisulfate (K2S2G8) embodiment of this provides an effective bactericidal effect for Salmonella in beef steak.

[277] PROOF OF THE EFFICIENCY OF 10 PPM SILVER FOR FOOD PROCESSING

[278] A. Purpose of the Example

[279] The purpose of this example is to demonstrate the utility of the gent-based composition embodiments of the present invention for the processing of a variety of human foods, the studies in this section were performed in Ghana. , West Africa, Air Force Station Hospital under Dr. Kwabiah, Korie-Bu Teaching Hospital under Sr. Sackey, and Justab Clinic / Maternity Hospital Hospital under the direction of Dr. Abraham. In total, fifty eight (58) patients were treated using a silver / water composition of the present invention comprising silver IC ppm. the composition has been used both internally and externally as an alternative to traditional antibiotics, foods processed have included malaria, upper respiratory tract infections, urinary tract infections, sinusitis, infections. vaginal yeast, ear, nose, and eye infections, fungal skin infections, and sexually transmitted diseases, such as gonorrhea.

[280] B. Treatment methods and results

[281] Abdominal pain and Diarrhea, the method involves the step of administering approximately 5 to 25 ml of silver composition, one to five times a day daily until there is a response. . One patient has been treated with about 10 ml (about two teaspoons) of a composition of the present invention three times in a day, the patient has full recovery within a day.

[282] Bronchitis, the method comprises the step of administering ca. 2 to 25 ml of oral silver composition, one to five times a day until there is a response. Two patients were treated with about 5 ml (about one teaspoon) each of a composition of the present invention twice a day for three days. The patients have a full recovery in three days.

[283] Vaginal yeast (Candida). The method includes the step of administering ca. 5 to 25 ml of silver composition, one to four times a day as douclies vaginal vaginal douclies until there is a

٦ ك

ΜΑ 29428Β1 answer. Five patients were treated with about 10 ml (about two teaspoons) each of a composition of the present invention for twice a day. Patients showed full recovery in

[284] Conjunctivitis, the method comprises the step of administering ca. in several drops of a composition of silver, one to five times a day to the infected eye until there is a response. Two patients were treated with several drops of a composition of the present invention in each of the infected eyes twice a day, the patients recovering fully after one day.

[285] cuts and external infections (including infections of the skin with Staphylococcus, septic ulcers and infected abscesses), the method involves the step of administering a silver composition, one to five times. the day to the infected area until there is a response. Six patients were treated with about 5 ml (about one teaspoon) each of a composition of the present invention on the infected areas twice a day, the patients showed full recovery within three days.

[286] Otitis externa, the method involves the step of administering a silver composition one to five times a day to the infected ear until there is a response. Six patients were treated with approximately two drops of a composition of the present invention in infected ears three times a day. Patients showed full recovery after about four days.

[287] Antrite. the method involves the step of administering a silver composition one to five times a day to the infected ear until there is a response. One patient was treated with approximately two drops of a composition of the present invention comprising in the infected ear three times a day, the patient showed full recovery within four days.

[288] Fungal infection of the skin. The method involves the step of administering a silver composition topically one to five times a day to the infected area until there is a response. Two patients were treated with about ten ml (two teaspoons) each of a composition of the present invention three times a day. The patients showed a full recovery within eight days.

[289] Gonorrhea, the method involves the step of administering a silver composition to the infected area until there is a response. Two patients were each treated with about ten ml (two teaspoons) of a composition of the present invention three times a day. The patients showed no symptoms in six days.

[290] Malaria, the method involves the step of administering a silver composition one to five times a day orally to patients until there is a response. Eleven patients were treated in a first study with about ten ml (two teaspoons) each of a composition of the present invention three times a day. The patients showed resolution of symptoms within five days. More detailed protocols on Malaria have been discussed hereinafter.

[291] Halitosis and Gingivitis. The method involves the step of administering a silver composition one to five times a day as a mouthwash until there is a response. Two patients were each treated with the composition as a mouthwash. There was resolution of symptoms within three days (gingivitis) and within one day (fialitosis).

[292] Upper genital infection. The method involves the step of administering about 5 to 25 ml of a silver composition, one to five times a day as a douching until there is a response. A patient was treated with about 5 ml (approximately one teaspoon) of a composition of the present invention twice a day, the patient's symptoms resolved within five days.

[293] Pharyngitis. The method involves the step of administering a silver composition one to five times a day as a gargle until there is a response. Four patients were each treated with about ten ml (two teaspoons) of a composition of the present invention three times a day, patients showed full recovery within six days.

[294] Retrovirus HIV infection). The method involves the step of administering a silver composition, comprising 5 to 40 ppm of silver one to five times a day orally until there is a response. A patient showing HIV (human immunodeficiency virus) was treated with about 5 ml (approximately one teaspoon) of a composition of the present invention twice daily. The patient's symptoms are resolved within five days.

AT/

ΜΑ 29428Β1

295 ؛] Sinusitis and Rhinitis, the method involves the step of administering a composition of silver, one to five times a day to the nose until there is a response. Six patients with nasal infection (four for sinusitis and two for rhinitis) were each treated with approximately drops of a composition of the present invention having in their nasal passage three times daily. The patients showed a full recovery within four days.

[296] Angina. The method involves the step of administering a silver composition one to five times a day as a gargle until there is a response. A patient was treated with a composition of the present invention three times a day, the patient showed full recovery in seven days.

[297] Upper respiratory tract infection, the method involves the step of administering a silver composition, one to five times daily orally until there is a response. Two patients were treated with about 5 ml (approximately one teaspoon) of a composition of the present invention three times a day, the patients showed full recovery within six days.

[298] Urinary tract infection, the method involves the step of administering a silver composition, one to five times daily orally until there is a response. Three patients were treated cliacun with about ten ml (two teaspoons) of a composition of the present invention two to three times a day. The patients showed full recovery within six days.

[299] C. Discussion

[300] These results are consistent with the various in vitro tests reported herein. Essentially, the silver composition is extremely effective against a large number of microbes in vitro. However, tests indicate that this effectiveness still remains in the presence of a large amount of organic matter. Silver compositions are widely effective in vivo where the organic background is extremely high. Many other disinfectants are ineffective in the presence of a large amount of organic material and / or are also caustic or toxic for use in vivo.

[301] Additional study of Malaria in Ghana, Africa

[302] Another more dosage study was also conducted in Ghana, the aim of this study was to use a very specific protocol and only focuses on the healing properties of the 10 ppm silver / water composition of the present. invention in patients who contracted malaria.

[303] The purpose of this protocol was to set out a procedure where the 10 ppm silver / water solution is according to the net hereof can be tested for its possible curative properties in the treatment of patients who have contracted an infection of Malaria with any of four (4) species of Plasmodium. An overview of the protocol is as follows:

[304] The tests were performed in medical departments or in hospitals by doctors of nedicine (MD) who are very familiar with the disease and its health ramifications. There were a total of 16 patients examined by the doctor, and the patients were required to take the silver product twice a day for five days, their blood was also drawn one day before the start of the test. , and then each day until the blood test has shown that the parasite has been eliminated for at least two days, patients would only be paid if they fully adhered to the program to take the money and get daily blood tests

Protocol Details:

Number of Medicinal Doctors (MD) to involve in the test: 2 the number of patients to be tested by the doctor: 16 to 8 males and 8 females

[305] Total number of days for the test: 15

[306] A dose of 10 ppm silver / water composition to be given for the treatment of patients: a total daily dose of one ounce divided into two equal doses; half ounce (3 teaspoons) taken in the morning and half ounce (3 teaspoons) taken in the evening. Patients were treated with a silver / water solution for the first five days (5) of a 15-day total trial, or if the parasite was not completely gone on day five, treatment with ؛ ' silver / water was continued until the parasite left, or until day 15, which never occurred before.

ΜΑ 29428Β1

307 ؛] In the case of a patient whose parasites were gone in two or three days, the silver / water was continuous until day five, and a note was made in the records as when the parasite was been completely gone.

[308] In the case of a patient who still harbored the parasite after taking the silver / water for the 15 days, the trial would be terminated as usual, and this patient was recorded as treatment failure within registers.

[309] For the patient who was cured in less than five days, the date of complete parasite disappearance was recorded, the patient continued to receive silver / water until day five, and was continued in the trial. by day

Blood test:

[310] blood tests to be used: The presence (or absence) of the parasites in the blood of patients was determined either by the Acridin orange strain test or by the Giemsa Suctia test , in thin and thick blood smears from each of the patients, the patients' blood was tested on day zero (0) to ensure that they, in fact, had an active case of malaria. If the blood test has confirmed an active case of malaria, then the patient is selected for acceptance for the test. Selection included recording vital data such as name, age, patient reported onset of illness, informing patients that they are requested during the test, that they would be paid for full compliance, and the fact that failure to comply would result in the test being abandoned without pay. For those patients who agreed to join the test, they were given a set of written instructions indicating how to take the silver product each day, where to go each day for their blood tests, and emphasizing the need to l Complete adherence to the test protocol in order to be remunerated for each day the above protocol was strictly adhered to in the most recent study in Ghana, Africa. All patients received the same dose, and their blood was examined daily for the existence of Plasmodia parasites, the following table listed the parts of Lists 4 of the previous studies discussed above (Study 1 and Study 2). , as well as the new Study 3, which follows the protocol immediately stated hereinafter.

[311] Summary

Table 4

<td>Study</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td>

<td>Number of patients</td><td> 11</td><td> 16</td><td> 16</td><td> 13</td>

<td>Age range</td><td>8 to 75</td><td>2 to 90</td><td>3 to 61</td><td>15 to 57</td>

<td>Males / females</td><td>N / A</td><td>N / A</td><td>N / A</td><td>N / A</td>

<td>Average daily dose</td><td>10 ml</td><td>5 ml</td><td>15 ml</td><td>15 ml</td>

<td>Shortest recovery time +</td><td>3 days</td><td>3 days</td><td>2 days</td><td>3 days</td>

<td>Longest recovery time</td><td>7 days</td><td>10 days</td><td>8 days</td><td>6 days</td>

<td>Average recovery time</td><td>5.0 days</td><td>6.3 days</td><td>4.3 days</td><td>4.0 days</td>

<td># patients examined for plasmodia</td><td> 0</td><td> 7</td><td> 16</td><td> 13</td>

<td># patients w. plasmodia</td><td> 0</td><td> 0</td><td> 0٠</td><td> 0*</td>

<td># treatment failure</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td>

+ Recovery times are those taken by patients to be asymptomatic, as estimated by doctors.

* Each of these patients was tested daily for 14 days. After six days, none of their blood sample tested negative for Plasmodia.

[312] Clearly, the 10 ppm silver / water solution of the present invention has positive effects against the malaria parasite.

[313] PROOF OF 100 PPM MONEY EFFICIENCY AGAINST MALARIA (IN VITRO)

[314] INTRODUCTION

ΜΑ 29428Β1

[315] Globally, malaria has been and remains a major public health concern. The disease is caused by parasitic protozoa of the genus Plasmodium, the life cycle of this organism is complex, with the parasite alternating between sexual reproduction in an invertebrate host (mosquito) and asexual reproduction in a vertebrate host in addition to mammals in As long as vertebrate Helots, birds and reptiles also serve as Helots for malaria parasites. The part of the life cycle in the mosquito is the sporogonic phase, leading to the formation of the sporozoites which are injected by the vector into the vertebrate host in the period of feeding, the sporozoites cause schizogonic folding, with the proliferation of parasites in the erythrocytic and exoerythrocytic sites. The parasite is extracellular during its sporogonic phase, moving into an intracellular location during the schizogonic stages of development. The in vitro culture of the parasite requires conditions of stimulation in the mosquito vector for the sporogonic phase of the life cycle and, for the schizogonic phase, conditions favoring the growth of the exoerythrocytic and erythrocytic sites of the vertebrate hosts.

[316] Malaria is one of the most prevalent parasitic diseases in the world and accounts for as much as a third of the infectious diseases of importance in terms of mortality worldwide. The protozoan parasite that causes malaria is from the genus Plasmodium. Four species of Plasmodium protozoa cause malaria: Plasmodium falciparum. Plasmodium vivax. Plasmodium malrrae, and Plasmodium ovale. -Transmitted mainly by the Anopheles mosquito, malaria infections can also occur from contact with infected blood, such as blood transfusions.

[317] Classic symptoms of malaria include Classic symptoms of malaria include fever, headache, chills, vomiting, tremor, and convulsions. In some rare forms of falciparum malaria, cold and fever may be absent and the patient may present with dementia or coma, periods of remission may last from a few weeks to several months. Severe anemia is often the cause of death from malaria infection.

[318] Plasmodium falciparum:

This parasite has several important characteristics. These include the crescent shape of gametocytes, the rate of growth of the latter and the localization of the pigment around the nucleus (perinuclear distribution) which is absent in the gametocytes of other primate malaria parasites.

p. falciparum also differs from other human species in its greater virulence and lethal effects, while the schizogony of the erythrocytic stages is largely confined to the capillaries and sinusoids of the internal organ. The popular name for the disease caused by p. falciparum is third malignant malaria.

MATERIALS AND METHODS

Saline citrate:

Sodium chloride 9 gm

Sodium citrate 20 gm

Distilled water 1000 ml

The Giemsa strain:

Giemsa satin powder 75 gm

Absolute alcohol 75 ml.

Glycerol 25 ml.

The Field strain:

Field's solution # 1

1. Dissolve 1.6 g of methylene blue in 1 liter of distilled water.

ΜΑ 29428Β1

2. Dissolve 2.6g of Na2HP٥4 (anhydrous) in the solution from step 1.

3. Dissolve Azure 1 Ig in the solution from step 2.

4. Dissolve 2.6 g of ΚΗ2ΡΟ4 in the solution from step 3.

5. Place on medium heat with stirring or shaking for 45 minutes to 1 hour.

6. Leave on at room temperature for 24 hours.

Filtered.

Field Solution # 2

1. Dissolve 2g of Eosin Y in 1 liter of distilled water.

2. Dissolve 2.6g of Na2HP04 in the solution from step 1.

3. Dissolve 2.6g of ΚΗ2ΡΟ4 in the solution from step 2.

4. Filtered.

Wright strain.

Wright6 strain powder, Og

Giemsao strain powder, 6g

Methanol 1, 000ml

Shake overnight and filter before use.

Human blood type AB + serum / plasma

Human serum / plasma of blood group A +

Medium Incomplete RPMI -1640 (Personal Communication Dr. Sutar, Haffkine Institute, Parel)

Complete medium RPMI - ISO (Personal Communication Dr. Sutar, Haffkine Institute, Parel)

COLLECTION AND TREATMENT OF INFECTED BLOOD

[319] Parasitic erythrocytes were obtained by collecting 6 ml aliquots of blood in 1 ml of citrated saline by venipuncture from medically diagnosed cases of vivax Plasmodium and Plasmodium falciparum malaria from Kasturba Infectious Disease Hospital. Hospital, Bombay. Blood samples were collected in sterile 10 ml vials. the samples were examined by preparing thin smears and staining the smears with 10% Giemsa strain / Field strain / Wright strain, for identification and confirmation of the species of the malarial parasite. The percentage level of parasitaemia of the sample was recorded.

[320] Parasitized blood cells were washed twice with incomplete medium and once with complete medium and 6% of the cellular suspension was prepared in complete medium. Cultures were set up by distributing 0.5 ml of suspension in each petri dish. To this was added 1.5 ml of complete medium and the plates were incubated in an atmosphere of 5% CO2 and 14-17% O2. the medium was changed daily by aspirating the old medium with a sterile pasture pipette and adding 1.5 ml of medium

<img file="MA29428B1_D0005.tif" />

ΜΑ 29428Β1 full. Cultures were maintained by adding the fresh cells (of blood group Α + or sangu +: lava, and the cell suspension prepared in the same way) after a week, with washing twice a week, until the target parasite index has reached&gt; _to 1%. If the initial parasite index was more than 1 ٥/٠, then Blood Medium Mixture (BMM) was used directly for drug sensitivity (Thanh

2001) and (Tasanor, 2002)

PREPARATION OF FRESTS AND COLORING PROCEDURE

[321] Cultures were washed three times per week. For washing, cultures were removed from the plates and transferred to centrifuge tubes. About 5 ml of the incomplete medium was added to each centrifuge tube and mixed well, the tubes were centrifuged at about 1000 to 1500 rpm for about 10 minutes. After about 10 minutes, the tubes were removed from the centrifugation and the supernatant was discarded. Later, the cultures were subjected to at least two washes, one with incomplete RPMI-1640 medium and the other with complete RPMI-1640 medium. After three washes, the cultures were transferred to separate Petri dishes. A smear was prepared from each culture and stained with Giemsa / Field's / Wright's strain. About 1.5 ml of the medium was added to celiac plate and the smears were examined under a light microscope for parasitic index or ٥/٥ of parasitaemia for celiac culture was recorded, fresh erythrocytes were added to each plate any week (Pradhan, 1984)

[322] PREPARATION OF FROTTIS

[323] A drop of culture from the plate was taken in a micro coverslip. A mi'nce smear was made and air dried. This smear was fixed by dipping the coverslip into a coupling pot containing absolute alcohol. A 10% solution of the Giemsa strain was prepared and used for smear staining. The coverslips were kept submerged in 10% Giemsa strain solution for about 30-40 minutes and then washed in tap water.

[324] The Parasitic Index

[325] The Parasitic Index was calculated by counting the number of parasites per 100 erythrocytes in thin blood smears. A minimum of 100 fields or 10,000 RBCs were observed for this purpose.

[326] Culture Systems Plate cultures were prepared with a hematocrit of 5٥/٥ and approximately 1٥ / ο parasitaemia. The most diminished parasitaemia of the initial parasitaemia is the greater increase in the numbers of parasites which will take place during in-vitro growth.

[327] DRUG SENSITIVITY

[328] Sterial 16 mm flat bottom micro-well plates were used for drug sensitivity testing. One was used for a sample. First, the two wells were used for the control and received 50μΙ of BMM from the patients or the culture and 50μΙ RPMI from the complete medium and no drug. For the test of 50 μΙ of culture or BMM of the patients was mixed in the well containing 50 μΙ of the treated silver nanoparticles (ESNP) at various concentrations. Micro-well plates were covered and incubated at about 37C in a candle flask for about 48 hours, most parasites go into the schizont stage after 48 hours of incubation. After incubation using the micropipette, the supernatant medium was removed; everyone's blood was taken well to prepare for smears and observed for the development of schizont. the assay was assessed by counting the number of parasites in pre-incubation and post-incubation stained films and ESNP-related inhibition of schizont formation. For a valid test, the control well should show&gt; _10% schizont maturation. (Wemsdorfer and Wernsdorfer. 1995)

Results

[329] The in vitro test used as an indication of antimalarial efficacy shows conclusively that ESNP - 100 ppm is able to reduce parasite count in vitro. This is of importance since the parasites collected are from patients showing rigors with high temperatures and classic symptoms of malarial infections.

[330] PROOF OF THE EFFECTIVENESS OF 10 PPM MONEY AGAINST TUBERCULOSIS BACTERIA

[331] A. Goal

ΜΑ 29428Β1

[332] The purpose of this example is to demonstrate the efficacy of the silver composition of the present invention against bacteria which cause tuberculosis. This example describes the procedures for evaluating the present invention for tuberculocidal efficacy. The methodology is based on the Tuberculocidal Activity Test Method as accepted by the EPA December 11, 1985. [Submit to United States Environmental Protection Agency, 1986. Office of Pesticides and Toxic Substances. Data Call-in Notice, for Tubercuolocidal Effectiveness Data for All Antimicrobial Pesticides with Tuberculocidal Claims. (Received June 13, 1986).

[333] B. Material and Methods

[334] Subjects. The silver composition of the present invention comprises 10 ppm silver in water. The silver composition was evaluated using the liquid matrix against Mycobacterium bovis BCG (TMC 1028). the body causes tuberculosis in animals and can cause tuberculosis in humans. It is used as a dip for M. tuberculosis, the major cause of human tuberculosis, as tests have shown to have similar susceptibility to M. tuberculosis, the test organism was exposed to the silver composition in a four-fold duplicate exposure and quantified using a membrane of

[335] Procedure. A flask or culture of frozen stock was removed from storage and thawed. An equal volume of gelatin buffer (BUGE) was added to the cell suspension and homogenized with a Teflon® (brand of polytetrafluoroethylene) fabric grinder for 1 minute while maintaining 0 to 4 ° C in a cold bath. The homogenized cell suspension was diluted with Tween® 80 (polysorbate brand) saline (ST80) to approximately 107 µmL.

[336] Title of the challenge. Periodic ten-fold dilutions of the culture were prepared in control dilutions containing 9 ml of neutralizing broth (NEUB) per dilution 106. Three 1 ml aliquots of the appropriate dilutions were filtered through the membrane by first adding the 10-20 ml physiological saline solution (PHSS) in a filter housing and then adding a 1 ml aliquot of the dilution. appropriate, the filter was then rinsed with approximately 100 ml of PHSS.

The filters were aseptically removed from the filter housing and placed on 7Η11 agar plates. The plates were incubated in a humid chamber at 37 ± 2٥c for 21 days.

[337] Positive Witness. A tube containing 9 ml of S180 was prepared and equilibrated at 20 O.5OC. At time 0, 1 ml of test organism culture was added to the tube (1:10 dilution), the sample was held for 60 minutes. Ten-fold periodic delutions were prepared in control dilutions containing 9 ml NEUB through a 10.6 dilution. Three 1 ml aliquots of the appropriate dilutions were filtered through the membrane first by adding 10-20 ml of PHSS to the filter housing and then adding 1 ml of the appropriate dilution aliquot. The filter was rinsed with approximately 100 ml PHSS. The filters were aseptically removed from the filter housing and placed on 7Η11 agar plates. The plates were incubated in a humid room at 37 + 2 ° C for 21 days.

[338] the tests. Two 25 X 150 mm tubes containing 9 ml of the test sample were equilibrated at 20 ± o, s٥c in a water bath. To each tube containing the test disinfectant (i.e. a silver composition), 1 ml of the test organism culture was added, the tube was mixed by vortexing and placed back into the water bath. At 15, 30, 45, and 60 minutes, the 1.0 ml aliquots of the cell disinfectant suspension were transferred to 9 ml NEUB and mixed thoroughly. Periodic tenfold dilutions were prepared in control dilutions containing 9 ml of NEUB by a dilution of 10®. Three 1 ml aliquots of the appropriate dilutions were membrane filtered by first adding 10-20 ml of PHSS to the filter housing and then adding a 1 ml aliquot of the appropriate dilution. The filter was rinsed approximately with 100 ml PHSS. the filters were aseptically removed from the filter housing and placed on 7ΗΙΙ agar plates. the plates were incubated in a humidification chamber at 37 + 2OC for 21 days.

[339] Control of phenol. To demonstrate minimal culture viability and resistance, the culture was tested against 0.8% phenol solution. A 1 ml aliquot of the test organism culture was placed in 9 ml of the equilibrium phenol solution at 25 + 0.5 c and incubated for 20 minutes. After a period of exposure, 1 ml of the phenol / organism solution was removed and added to 9 ml of NEUB. Serial 10-fold dilutions were prepared in control diluions containing 9 ml NEUB through a 10® dilution. three 1 ml aliquots of the appropriate dilutions were membrane filtered by first adding 10 to 20 ml PHSS to the filter dish and then adding 1 ml of the appropriate dilution aliquot, the filter was rinse with approximately 100 ml PHSS. The filters were aseptically removed from the filter housing and placed on 7Η11 agar plates. The plates were incubated in a humid chamber at 37 ± 2٠c for 21 days.

<img file="MA29428B1_D0006.tif" />

ΜΑ 29428Β1

[340] Neutralization check. A 1 ml aliquot of the disinfectant was added to 8 ml of NEUB. the disinfectant / neutralizer broth was equilibrated at the same temperature as the test samples. One ml of the test organism culture was added to the mixture and mixed well, incubation continued for the approximate time it would take to filter a sample. In addition, a 1 ml aliquot of the test organism was added to 9 ml of NEUB and mixed thoroughly (1:10 dilution). Ten-fold serial dilutions of all tubes were prepared in control dilutions containing 9ml NEUB through 10® dilution. Three partial 1 ml aliquots of the appropriate dilutions were filtered through a membrane by first adding 10-20 ml of PHSS to the filter housing and then adding a 1 ml aliquot of the appropriate dilution, the filter was rinsed with approximately 100 ml PHSS. the filters were aseptically removed from the filter housing and placed in 7Η11 agar plates. The plates were incubated in a humid chamber at 37 ± 2 ° C for 21 days.

[341] C. Resultas

[342] The starting titration for the challenge culture was 4.7 X 10? cfu / ml. the positive control titer was 6.5 X 10 ؛ cfu / ml. The medium used in this study effectively demonstrated neutralization with a recovery of 95.2% in a disinfectant / neutralizer solution when compared to the control medium.

[343] For the assay plates, the predicted counts were underestimated and as a result, the reported counts showed to mark that the count is an estimate and that accurate counts are beyond the limit of detection for the plated dilutions.

[344] By calculating the log and percent reductions in disinfectant against M. bovis, estimated counts that have counts greater than â resulted in less than log and percent reductions (&lt;). The purpose of this is to demonstrate that the results are an estimate of and beyond the precise limit of detection for the plated dilutions. All reductions were calculated using the positive control as the organism's initial starting titer, the results for log and percent reductions are summarized below. As a measure of the resistance of the challenge culture, the phenol resistance of M. hovis showed a 1.81 log reduction with 20 min exposure to 0.13% phenol.

[345] Measure One:

<td>Exposure time</td><td>Logarithmic reduction</td><td>Percent reduction</td>

<td>15 minutes</td><td> 0,12؟</td><td> &lt; 12,3%</td>

<td>30 minutes</td><td> 0,22؟</td><td> &lt; 40,0%</td>

<td>45 minutes</td><td> &lt;1,57</td><td> &lt;97,2%</td>

<td>60 minutes</td><td> 1,56؟</td><td> &lt;97,0%</td>

[346] Measure two:

<td>Exposure time</td><td>Logarithmic reduction</td><td>Percent reduction</td>

<td>15 minutes</td><td> &lt;0,26</td><td> &lt;44,8%</td>

<td>30 minutes</td><td> &lt;0,20</td><td> &lt;36,9%</td>

<td>45 minutes</td><td> &lt;1,58 ,</td><td> &lt;97,3%</td>

<td>60 minutes</td><td> &lt;1,53 —</td><td> &lt;97,1%</td>

[347] D. Conclusions

[348] The use of the silver compositions of the present invention is effective against tuberculosis bacteria. A method comprising the step of administering the silver compositions of the present invention is effective against tuberculosis organisms.

[349] PROOF OF EFFECTIVENESS OF 10 PPM MONEY AGAINST CANDIDA

ALBICANS ATCC # 10231, TRICHOMONAS VAGINAIIS ATCC # 20235,

AND MRSA STAPHYLOCOCCUS AUREUS ATCC # ΒΑΑ-44

[350] A. Purpose of the Example

مر

ΜΑ 29428Β1

[351] The purpose of this example is to illustrate the efficacy of the silver compositions of the present invention against Candida albicans ATCC10231. Trichomonas vaginalis AT c 20235, and drug resistant Staphylococcus aureus ATCC ΒΑΑ-44.

[352] Candida albicans, a yeast, and Trichomonas vaginalisis, a protozoan, can cause several health problems including vaginal infections, diaper rash, and thrush, the results below show that the silver compositions of the present invention produced approximately 100% killing of all organisms, the results show the utility of the silver compositions of the present invention in a feminine hygiene product and in a diaper rash product.

[353] Staphylococcus aureus can cause severe blood poisoning when it enters a wound. It was easily treated in the past with penicillin, but the organism has now mutated to the point where it is completely resistant to penicillin, the next defense on the antibiotic scale has been methicillin, but resistant strains methicillin have become increasingly common, especially in hospitals. These strains are known as MRSA (Methicillin Resistant Staphylococcus aureus) and have been nicknamed superbug. people who contract MRSA can die within days. In the results reported in this example, a silver composition of the present invention was found to kill 91.6% MRSA in only 10 minutes, and 99.5٥ / ٠ in one content. the results show the utility of the silver compositions of the present invention in killing MRSA, a known infectious threat.

[354] B. Methods and Results

[355] Using the USP Preservative Rapid Challenge test with a composition of the present invention comprising 10 ppm silver in water, the following results were obtained. These results prove that the silver compositions of the present invention can be effective against yeast infections, protozoan infections, and infections by drug resistant bacteria.

[356] Candida albicans ATCC # 10231. the initial concentration of the yeast Candida albicans was 6.8 x 0 cfu / ml. After contact for either 10 minutes, 30 minutes, 1 hour, or a day with the silver composition, there are colonies detected.

[357] Trichomonas vaginalis ATCC # 30235. the initial concentration of the protozoan Trichomonas vaginalis was 6.0 X 0 cfu / ml. After contact with the silver composition for either 10 minutes, 30 minutes, 1 hour, or one day, there was 0% motility of 100 organisms. Thus, one hundred (100) parasites of Trichomonas vaginalis were analyzed by microscopy for flagellar motility. None of the hundred (100) parasites demonstrated motility after only ten (10) minutes of contact with the silver composition indicating inhibitory or lethal properties of the silver composition in the parasites, the outer membranes of twenty five (25) percent of the parasites were ruptured after one day contact (1).

[358] Staphylococcus aureus MRSA ATCC # BAA-44. The initial concentration of methicillin resistant Staphylococcus aureus (MRSA) was 6.0 X 10 ج cfu / ml. After contact with the silver composition, there were 500,000 cfu / ml detected after 10 minutes of contact (91.6% killed), 70,000 cfu / ml after 30 minutes of contact (98.8% killed), 30,000 cfu / ml after 1 hour of contact (99.5% killed), and less than 10 cfu / ml after one day of contact (virtually complete killing).

[359] EXPECTED EFFECTIVENESS AND LACK OF CYTOTOXICITY OF SILVER 10

PPM, SILVER 14 PPM + 1.5% Η2Ο2, and SILVER 22 PPM BY INHIBITING DNA POLYMERASE AND IE REVERSE TRANSCRIPTASE IN THE CONTEXT OF ΙΉΕΡΑΤΙΤΕ B

[360] A. Purpose of the Example

[361] The purpose of the example is to illustrate the efficacy of silver compositions of the present invention against hepatitis B. This example proves that the silver compositions of the present invention have antiviral properties. Any agent used in antiviral therapy should show little or no cytotoxicity thus the cytotoxicity of the silver compositions was analyzed.

[362] Hepatitis B is caused by a DNA virus from the hepadnaviridae family of viruses, hepatitis B virus (HBV) is a 3.2 KBS DNA virus, replicating almost exclusively in liver cells (hepatocytes). Replication involves two main enzymes: DNA polymerase and reverse transcriptase, the results of this example show that the silver compositions of the present invention interfere with replication involving DNA polymerase or transcriptase. inverse, the results

١/١

ΜΑ 29428Β1 of this example show that the silver compositions of the present invention have antiviral properties.

The results of this example show that the silver compositions of the present invention can be effective against hepatitis B.

[363] As a further detail, when hepatitis B enters the body of a new host, it infects the liver if it arrives after the host's immune system. In infection, the virus attaches itself to the membrane of a liver cell, and the nucleus particle of the virus enters the liver cell, the nucleus particle then discharges its content of DNA polymerase and DNA. DNA in the nucleus of liver cells. In the liver cell, virus replicates via reverse transcription and translational processes, which involve reverse transcriptase and DNA polymerase enzymes. DNA polymerase causes the liver cell to make copies of hepatitis B DNA. These copies of the virus are released from the membrane of liver cells into the bloodstream. From there, they can infect other liver cells and thus refold them efficiently, the incubation period of hepatitis B virus is approximately 6 to 25 weeks (i.e., the time before the examination and generally discernible histological or physical symptoms occur). However, there are several biochemical and histological changes that occur in the early stages after infection with the hepatitis B virus.

[364] B. Materials

[365] Solutions comprising silver compositions of 10 ppm, 14 ppm, 22 ppm and 32 ppm according to the present disclosure were used, the nucleotides dATP, dG٦P, dCTP. and [H] -dT7P were obtained from standard commercial sources, as were my compounds lamivudine (a synthetic antiretroviral agent) and zidovudine (AZT). The isolated hepatitis B virus was recently obtained from a person with hepatitis B infection and was taken by Haffine Institute, Mumbai INDIA (A WHO accredited testing laboratory), cell cultures of the test (Vero and Hep2) were developed as confluent monolayers by typical cell culture methods.

[366] C. Methods

[367] 1) Procedure for the DNA polymerase intestine test.

[368] Global approach. Hepatitis B virus extracts from human subjects were incubated with labeled nucleotides and an active inhibitor. The percent inhibition is calculated according to the amount of viral nucleic acid novo synthesized relative to lamivudine as positive control and phosphate buffered saline (PBS) as negative control.

[369] Specific procedure, the isolated hepatitis B virus was lysed to extract the free polymerase enzyme, which is free from contaminating enzymes. A virus extract (25 ml) was added to the reaction mixture comprising the nucleotides dATP, dGTP, dCTP and [³H] dTTP (25 mL). An active inhibitor (3 ml) was added to the mixture comprising virus extract and nucleotides, the resulting mixture was incubated at 37 ° C for 24 hours.

[370] A separate negative control experiment was performed in which phosphate buffered saline (PBS, 3ml) was used instead of inhibitor (3ml).

[371] A separate positive control experiment was performed in which the known DNA polymerase inhibitor (3 ml lamivudine at a concentration of 3 mg / ml) was used instead of the tested inhibitor (3 ml).

[372] the reaction was stopped by adding 25 ml EDTA and 25 ml TCA (trichloroacetic acid). the reaction mixture was then stained in ionic paper (DEAE paper). the paper was washed three times with TCA and then with ethyl alcohol. The filter paper was air dried and placed in a scintillation vial with a scintillation cocktail.

The radioactivity was measured by a liquid scintillation counter (Blue star). As a count control, a control silver composition was held through a complete procedure without vial loading, to look for any potential interference in the liquid scintillation counter.

[373] A reference for this method is ps Venkateswaran, I. Millman. and B. s. Blumberg. Effect of an extract from Phyllanthus niruri on hepatitis B and woodchuck hepatitis viruses: in vitro and in vivo studies, Proc. Natl. Acad. Sci. USA, 1987, 84, 274-278, which is incorporated herein by reference.

[374] 2) Procedure for assaying reverse transcriptase inhibition.

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ΜΑ 29428Β1

[375] A commercial viral enzyme preparation of murine leukemia virus reverse transcriptase

Moloney (MoMuLV) having Poly (A) d٢ (primer for RT) was used, 50ml of the MoMuLV preparation was combined with a mixture of dATP, dGTP, dCTP and HJdTTP nucleotides.

[376] This mixture was combined with 3 ml of the inhibitor to be tested, and the resulting mixture was incubated at 37 ° C for 24 hours.

[377] A negative control experiment was performed in which phosphate buffered saline (3ml PBSi) was used instead of the inhibitor.

[378] A positive control experiment was performed in which a reverse transcriptase inhibitor (3ml AZT at 0.625 microgram / ml concentration) was used instead of the tested inhibitor.

[379] the reaction was stopped by the addition of 25 ml EDTA and 25 ml TCA. the reaction mixture was stained on ionic paper (DEAE paper). the paper was washed three times with TCA and then with ethyl alcohol. The filter paper was air dried and placed in a scintillation vial with a scintillation cocktail, the radioactivity was measured by a liquid scintillation counter (Blue star).

[380] 3) Procedure for the Cytotoxicity test.

[381] Cells were prepared from Hep2 and Vero cell cultures. sanitary confluences which were maintained by the celiac passage 3 to 4 days. One day before the test cells are released from cultures using standard techniques and suspended in culture medium and dispensed into microtiter plate wells and placed in a 5% c٥2 incubator at 37 ± 2٠c. An aliquot (100 ml) of each test substance was placed in a well (in triplicate) with 100 ml of PBS as a control. Every 24 hrs the wells were examined under a high capacity of an inverted microscope for any cytopathic effect (CPE). All the results are shown in the following Table 5.

[382] D. Resultas

[383] Results for the reverse transcriptase intestine test:

Table 5a

<td>Sample</td><td>% inhibition</td>

<td>Negative control (PBS)</td><td> 0</td>

<td>Positive control (ZT)</td><td> 31,33</td>

<td>Arqent, lOppm</td><td> 89,52</td>

<td>Silver, 14 ppm with 1.5% Η2Ο2</td><td> 86.93</td>

[384] Results for the DNA polymerase inhibition test:

Table 5b

<td>Sample</td><td>٥/٠ inhibition</td>

<td>Negative control (PBS)</td><td> 0</td>

<td>Positive control (lamivudine)</td><td> 31,33</td>

<td>Money. 10 ppm</td><td> 71,73</td>

<td>Arqent, 14 ppm with 1.5% Η2Ο2</td><td> 65,6</td>

<td>Silver, 22 ppm</td><td> 60,89</td>

[385] Silver compositions of the present invention are highly effective in inhibiting DNA polymerase.

[386] Results for the reverse transcriptase inhibition test:

Table 5c

<td>Sample</td><td>٥/٠ inhibition</td>

<td>Negative control (PBS)</td><td> 0</td>

<td>Positive control (AZT)</td><td> 18,06</td>

f \ /

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<td>Silver, 10 ppm</td><td> 89,52</td>

<td>Silver, 14 ppm with 1.5% Η2Ο2</td><td> 86,93</td>

<td>Silver, 22 ppm</td><td> 84,46</td>

[387] Thus, the silver compositions of the present invention inhibit reverse transcriptase. The silver compositions of the present invention could be intended to be effective against human foods spread by viruses, such as hepatitis B.

[388] Results for the cytotoxicity test:

Table 5d

<td>Sample</td><td>Vero</td><td>Hep2</td>

<td>Control (PBS)</td><td>CPE No</td><td>CPE No</td>

<td>Silver, 10 ppm</td><td>CPE No</td><td>CPE No</td>

<td>Silver, 14 ppm with 1.5% Η2Ο2</td><td>Positive CPE</td><td>Positive CPE</td>

<td>Silver, 22 ppm</td><td>CPE No</td><td>CPE No</td>

[389] These results indicate that the composition of silver is essentially non-toxic. As expected, hydrogen peroxide, which is known to be cytotoxic, shows a cytotoxic effect. Thus, silver must be harmless to cells when used in vivo.

[390] 12. PROOF OF THE EFFECTIVENESS OF SILVER COMPOSITION AS A WATER DISINFECTANT

[391] A. Goal

[392] Tests were performed to demonstrate the effectiveness of the inventive composition in disinfecting drinking water.

[393] B. Methods

[394] A sample of pure river water was pierced with two whole rings of Klebsiella oxtyoca. 100 ml of aliquots of this pierced water solution were made up to 0.05 ppm, 0.1 ppm, 0.2 ppm, 0.5 ppm, or 1.0 ppm of the inventive silver composition. After an incubation of 5 to 60 minutes, the samples were filtered through the membrane. The filter was rinsed with approximately 100 ml of sterile water. Filters were aseptically removed from the filter housing and placed in coliform nutrient agar plates. the plates were incubated under growth conditions for 24 hours and counted.

[395] Table 6

<td>Sample</td><td>Silver (ppm)</td><td>Contact (min)</td><td>Total coliform (per ml)</td><td>Cfu / 100 ml</td>

<td>Pure water</td><td> —</td><td> ٠—</td><td> 36</td><td>TNTC</td>

<td>1 I</td><td> 1,00</td><td> 5,0</td><td> 0</td><td> 0</td>

<td> 2</td><td> 1,00</td><td> 10.0</td><td> 0</td><td> 0</td>

<td> 3</td><td> 1,00</td><td> 15,00</td><td> 0</td><td> 0</td>

<td> 4</td><td> 1,00</td><td> 30,00</td><td> 0</td><td> 0</td>

<td> 5</td><td> 0.50</td><td> 10,0</td><td> 0</td><td> 0</td>

<td> 6</td><td> 0,50</td><td> 30,0</td><td> 0</td><td> 0</td>

<td> ٦</td><td> 0,50</td><td> 60,0</td><td> 0</td><td> 0</td>

<td> 8</td><td> 0,20</td><td> 5,00</td><td> 0</td><td> 0</td>

<td> 9</td><td> 0,20</td><td> 10,0</td><td> 0</td><td> 0</td>

<td> 10</td><td> 0,20</td><td> 30,0</td><td> 0</td><td> 0</td>

<td> 11</td><td> 0,20</td><td> 60,0</td><td> 0</td><td> 0</td>

<td> 12</td><td> 0,10</td><td> 10,0</td><td> 0</td><td> 0</td>

<td> 13</td><td> 0,05</td><td> 20,0</td><td> 0</td><td> 0</td>

TNTC = Too many to count.

[396] The composition of silver has been found to be surprisingly effective. Even at the shortest time (20 minutes) taken for the incubation of the lowest concentration tested (0.05 ppm) there was complete killing of the bacteria.

ΜΑ 29428Β1 bacteria. At 0.20 ppm and higher there was complete kill at 5 minutes. It seems clear that a complete kill takes less than 5 minutes.

13971 PROOF OF EFFICIENCY OF SILVER 32 PPM AS SURFACE

DISINFECTANT

[398] The Environmental Protection Agency (EPA) has approved a 32 ppm silver composition of the present invention as a broad spectrum surface disinfectant for use in hospitals, medical environments, home-residences, etc. commercial buildings, and businesses. It has been approved for use against some of the most deadly pathogens including: Gram-positive bacteria, such as Staphylococcus aureus (currently considered to be the deadliest bacteria in hospitals in the United States of America), Gram-negative bacteria, such as Salmonella choleraesuis (responsible for food poisoning), and nosocomial pathogens, such as Pseudomonas aeruginosa (often found in burns and cuts).

[399] The silver compositions of the present invention can be sprayed in or around areas occupied in endangering the health or welfare of humans or animals. It is possible to disinfect the selected surfaces of the group consisting of walls, tables, chairs, light fixtures, bathrooms, glasses, porcelain, metal, glazed, enamelled and painted ceramics by spraying means or by wiping means. with a silver composition of the present invention. A preferred method of disinfection comprises at least one step of cleaning the surface to be disinfected, by applying, by means of spraying, wiping, sponging, or crumpling, a composition of the present invention, by completely wetting the area to be disinfected, allowing the surface to remain moist for at least 10 minutes at a temperature of at least 2O٥C (the temperature-temperature relationship may be adjusted via the Arrhenius equation or by other means known to those skilled in the art), and wipe the surface with a clean paper or tissue towel. Compositions for disinfecting surfaces include those comprising 5 to 40 ppm silver. A preferred composition of the present invention for disinfecting surfaces comprises (32 + 3) ppm silver. Another preferred composition of the present invention for disinfecting surfaces comprises (10 + 2) ppm silver. Another preferred composition of the present invention for disinfecting surfaces comprises silver (22 + 2) ppm.

[400] EVIDENCE OF THE EFFECTIVENESS OF SILVER COMPOSITION AS A SUPER DISINFECTANT

[401] A. Purpose of the Example

[402] The purpose of this example is to show the antimicrobial activity of a silver composition of the present invention (here silver 10 ppm, silver 14 ppm with 1.5 wght% hydrogen peroxide , and silver 32 ppm) against the test organism Yersinia pestis, the causative agent of bubonic plague. By performing a standard kill versus time test using the Y suspension. pestis, it is demonstrated that the silver compositions of the present invention are still effective against the bacterium of bubonic plague.

[403] B. Materials and Methods

[404] the strain of Y. Pestis, D27. was grown in a Columbia agar plate for about 24 hours at 30 ° C in a 5% 0 incubator. The growth of the plate was scraped in suspension, using 3 ml of sterile HPLC water. The suspension was transferred to a 50 ml conical centrifuge tube. The plate was then rinsed using an additional 2 ml of HPLC water. This rinse was added to the centrifuge tube. The tube was centrifuged at 3,500 X g for 5 minutes, the supernatant was discarded and the pellet was resuspended in 1 ml of HPLC water. to give a final concentration of approximately 10١٥ cells per

[405] method a involves the following steps:

[406] 1. A 9.9 ml aliquot of the silver composition to be tested was placed in a sterile 20 mm x 150 mm tube. the tube was equilibrated in a 2 ° C. water bath.

[407] 2. the tube of the silver composition was inoculated with 100 μΙ of the suspension of the test organism at time zero from a mixture, the tube was vortexed immediately and returned to the bath. 'water.

[408] 3. At 2 min. 3 min, 4 min, and 5 min for silver 10 ppm or 32 ppm or 2 min, 4 min, 6 min and 8 min for silver 14 ppm with 1.5٥ / ο v / v HQ1, ؛ ml of an organism / silver mixture was removed to 99 ml of a neutralizer in a 250 ml conical flask. the vial has been mixed perfectly.

ΜΑ 29428Β1

14091 4. The neutralized suspension was immediately diluted to physiological series 1 (PSS).

: 10 in saline solution

[410] 5. The number of viable organisms in selected dilution tubes and vials was assessed by membrane filtration. A few ml of the aliquots were placed in duplicate. The membranes were washed with approximately 150 ml (or 250 ml if the sample was taken from the neutralizer vial) of sterile phosphate buffered saline and removed to Columbia agar plates. The remaining whole contents (98 ml) of the he 4 and 5 min neutralizer vials were plated, the plates were incubated at 30 ° C in a 5% CO ؛ incubator for 72 hours.

[411] 6. The number of colonies on each filter was counted and log reductions were calculated.

[412] C. Resultas

[413] The results for 10 ppm silver are shown in Table 7.

Table 7

<td>Time</td><td>Logarithmic reduction</td><td>Percentage of kill</td>

<td>2 min</td><td> 2,63</td><td> 99,77</td>

<td>4 min</td><td> 3,20</td><td> 99,94</td>

<td>6 min</td><td> 3,46</td><td> 99,97</td>

<td>8 min</td><td> 3,68</td><td> 99,98</td>

[414] The calculated regression equation for these data is Y = 2.3965 + 0.1696 X. this indicates that the time for 6-log reduction is 21.2 minutes.

[415] The results for 32 ppm silver are shown in Table 8.

Table 8

<td>Time</td><td>Logarithmic reduction</td><td>Percentage of kill</td>

<td>2 min</td><td> &gt;7,61</td><td> 99,999998</td>

<td>4 min</td><td> &gt;7,61</td><td> 99,999998</td>

<td>6 min</td><td> &gt;7,61</td><td> 99,999998</td>

<td>8 min</td><td> &gt;7,61</td><td> 99,999998</td>

[416] The results for 14 ppm silver with 1.5% v / v HO ؛ are shown in Table 9.

Table 9

<td>Time</td><td>Loqarithmic reduction</td><td>Percentage of kill</td>

<td>2 min</td><td> 3,27</td><td> 99,95</td>

<td>3 min</td><td> 4,72</td><td> 99,998</td>

<td>4 min</td><td> 5,36</td><td> 99,9996</td>

<td>5 minutes</td><td> 6,47 )</td><td> 99,99997</td>

[417] The regression equation calculated for these data is Y = 1.371 ٠ 1.024 X. This indicates that the time for a 6-log reduction is 4.52 minutes.

[418] The silver composition of the present invention showed significant bactericidal activity against Y. pestis, the etiological agent of bubonic plague. The 32 ppm composition gave a greater than 7 log reduction (essentially total kill) in less than 2 min. data shows that 10 ppm silver takes some 20 min to achieve a 6 log kill. silver and hydrogen peroxide show significant synergism with a calculated kill of 6 log in 5 min. This is better than 10 ppm silver alone, the 14 ppm silver level was chosen because data from other experiments suggested that this silver level combined with hydrogen peroxide would achieve results approaching those of the product of !'money

[419] SUMMARY OF DATA

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ΜΑ 29428Β1

[420] The following Table A contains a summary of the above results in terms of the effects of the inventive silver composition on a wide variety of microbes and human diseases. In some cases, the data shown in the table is not repeated above. However, results were obtained using procedures explained above so that a person skilled in the art can easily replicate.

[421] Human diseases cured and Pathogens killed by the composition of Inventive silver

Table A

<td>Disease</td><td>Pathogenic</td><td>Effective concentration</td>

<td>Abscesses</td><td>Staphylococcus aureus</td><td>Killed @ 5 ppm</td>

<td>Osteomyelitis</td><td>Staphylococcus aureus</td><td>Killed @ 5 ppm</td>

<td>Bacillary dysentery</td><td>Shigella boydii</td><td>Killed @ 2.5 ppm</td>

<td>Burn infections</td><td>Pseudomonas aeruginosa</td><td>Killed @ 5 ppm</td>

<td>Plaque</td><td>Streptococcus mutans</td><td>Killed @ 5 ppm</td>

<td>Diarrhea (bloody)</td><td>Shigella boydii</td><td>Killed @ 2.5 ppm</td>

<td>Diarrhea</td><td>Escherichia coli</td><td>Killed @ 2.5 ppm</td>

<td>Ear infection</td><td>Haemophilus influenzae</td><td>Killed @ 1.25 ppm</td>

<td>Ear infection</td><td>Streptococcus pneumonia</td><td>Killed @ 2.5 ppm</td>

<td>Enteric fever</td><td>Salmonella tyhimurium</td><td>Killed @ 2.5 ppm</td>

<td>Epiglottitis (in children)</td><td>Haemophilus influenzae</td><td>Killed @ 1.25 ppm</td>

<td>Eye infections</td><td>Staphylococcus aureus</td><td>Killed @ 5 ppm</td>

<td>Ulccere - Corneal keratitis</td><td>Pseudomonas aeruginosa</td><td>Killed @ 5 ppm</td>

<td>Food poisoning</td><td>Salmonella Arizona</td><td>Killed @ 5 ppm</td>

<td>Food poisoning</td><td>Salmonella tyhimurium</td><td>Killed @ 2.5 ppm</td>

<td>Food poisoning</td><td>Escherichia coli</td><td>Killed @ 2.5 ppm</td>

<td>Endocarditis</td><td>Streptococcus faecalis</td><td>Killed @ 2.5 ppm</td>

<td>Endocarditis</td><td>Streptococcus gordonii</td><td>Killed @ 5 ppm</td>

<td>Meningitis</td><td>Haemophilus influenzae</td><td>Killed @ 1.25 ppm</td>

<td>Meninqitis</td><td>Enterobacter aerogenes</td><td>Killed @ 2.5 ppm</td>

<td>Meningitis</td><td>Pseudomonas aeruginosa</td><td>Killed @ 5 ppm</td>

<td>Meningitis</td><td>Streptococcus pneumonia</td><td>Killed @ 2.5 ppm</td>

<td>Nosocomial infection</td><td>Klebsiella pneumoniae</td><td>Killed @ 2.5 ppm</td>

<td>Nosocomial infection</td><td>Pseudomonas aeruginosa</td><td>Killed @ 5 ppm</td>

<td>Nosocomial infection (from the hospital)</td><td>Streptococcus pyogenes</td><td>Killed @ 1.25 ppm</td>

<td>Pneumonia</td><td>Haemophilus influenzae</td><td>Killed @ 1.25 ppm</td>

<td>Pneumonia</td><td>Pseudomonas aeruginosa</td><td>Killed @ 5 ppm</td>

<td>Pneumonia</td><td>Streptococcus pneumonia</td><td>Killed @ 2.5 ppm</td>

<td>Respiratory tract infections</td><td>Streptococcus pyogenes</td><td>Killed @ 1.25 ppm</td>

<td>Respiratory tract infections</td><td>E.:s٥</td><td>Killed @ 2.5 ppm</td>

<td>Respiratory tract infections</td><td>Klebsiella pneumoniae</td><td>Killed @ 2.5 ppm</td>

<td>Scarlet fever</td><td>Streptococcus pyogenes</td><td>Killed @ 1.25 ppm</td>

<td>Septicemia</td><td>Enterobacter aerpyogenes</td><td>Killed @ 2.5 ppm</td>

<td>Sinus infections</td><td>Haemophilus influenzae</td><td>Killed @ 1.25 ppm</td>

<td>Sinusitis</td><td>Streptococcus pneumonia</td><td>Killed @ 2.5 ppm</td>

<td>The gourmet</td><td>Staphylococcus aureus</td><td>Killed @ 1.25 ppm</td>

<td>skin infections</td><td>Staphylococcus aureus</td><td>Killed @ 5 ppm</td>

<td>skin infections</td><td>Streptococcus pyogenes</td><td>Killed @ 1.25 ppm</td>

<td>Streptococcal pharyngitis</td><td>Streptococcus pyogenes</td><td>Killed @ 1.25 ppm</td>

<td>Purulent arthritis</td><td>Haemophilus influenzae</td><td>Killed @ 1.25 ppm</td>

<td>Throat infections</td><td>Haemophilus influenzae</td><td>Killed @ 1.25 ppm</td>

<td>Tooth decay</td><td>Streptococcus mutans</td><td>Killed @ 5 ppm</td>

<td>Ureteritis (in men)</td><td>Trichomonas vaginalis</td><td>Killed @ 10 ppm</td>

<td>Urinary tract infections</td><td>E. coli</td><td>Killed @ 2.5 ppm</td>

<td>Urinary tract infections</td><td>Klebsiella pneumoniae</td><td>Killed @ 2.5 ppm</td>

<td>Urinary tract infections</td><td>Pseudomonas aeruginosa</td><td>Killed @ 5 ppm</td>

<td>Urinary tract infections</td><td>Streptococcus faecalis</td><td>Killed @ 2.5 ppm</td>

<td>Urinary tract infections</td><td>Enterobacter aerpyogenes</td><td>Killed @ 2.5 ppm</td>

<td>Vaqinite (in women)</td><td>Trichomonas vaginalis</td><td>Killed @ 10 ppm</td>

<td>Wound infections</td><td>Escherichia coli</td><td>Killed @ 2.5 ppm</td>

<td>Wound infections</td><td>Enterobacter aerpyogenes</td><td>Killed @ 2.5 ppm</td>

<td>Wound infections</td><td>Klebsiella pneumoniae</td><td>Killed @ 2.5 ppm</td>

Λ

ΜΑ 29428Β1

<td>Wound infections</td><td>Pseudomonas aeruginosa</td><td>Killed @ 5 ppm</td>

<td>Wound infections</td><td>Streptococcus faecalis</td><td>٦ué@2.5ppm</td>

<td>yeast infections</td><td>Candida albicans</td><td>Killed @ 10 ppm</td>

[422] EFFECTIVENESS OF SILVER COLLOID FORMULATED AS A HYDROGEL

[423] Modem wound care has come to recognize that for optimal healing a wound should be kept sterile and protected against dehydration and environmental contaminants, traditional dressings are effective as they provide protection against injury. environmental contaminants but are largely ineffective at preventing dehydration. Dressings can be made antimicrobial by the addition of a variety of disinfectant substances, but these substances are often harsh and killing cells or the body as well as microbes. In recent times wound care has been revolutionized by hydrogel materials which are available as a semi-solid (amorphous material) or as a soft sheet, the hydrogel is hydrophilic and therefore prevents dehydration of the skin. wound, the material like sheet is effective at excluding environmental contaminants and due to its hydrophilic nature the hydrogel can actually absorb the excess fluid exuding from the wound

[424] Hydrogels are formed by combining a hydrophilic polymer with other ingredients in a solute. The polymer forms a gel following a change in pH, temperature, or other triggering event. In a gel, a fine molecular network of the polymer surrounds regions of the solute. Although the composition can be a semi-solid amorphous material or as a firm sheet, the vast majority of the volume tends to be occupied by the solute unlike the hydrophilic polymer, hydrophilic polymers which are suitable for the production of hydrogels include gelatin, carboxymethyl cellulose (and other cellulose derivatives), other carbohydrate polymers from plants or from algae such as alginate, carrageenan, xanthan gum, grasshopper bean gum, gum traganth, guar gum, arabic gum and other plant gums, acrylic acid copolymers (such as Carbopol), and combinations thereof and hydrophilic polymers similar.

<img file="MA29428B1_D0007.tif" />

ΜΑ 29428Β1

[425] The aqueous component preferably contains the various additive substances which increase the physical characteristics of the hydrogel and / or increase wound healing. These include various vitamins, amino acids, and additional growth factors to increase healing or to reduce scar formation to decrease scarring. Common anesthetics such as novocaine, lidocaine, and derivatives can also be incorporated as additives to increase comfort. Since maintenance of the sterile wound is an important dressing goal, various antimicrobials or disinfectants are advantageously included. These include organic acids such as citric acid, dilute acetic acid, benzoic acid, proprionic acid, and lactic acid. Alcohols such as isopropanol or ethanol are useful, as are organic disinfectants comprising chlorinated phenolic compounds such as trichlorophenol CP (2,4,6), biguanides, chlorhexidine (once mixed with cetrimide), chlorliexidine gluconate, and chlorhexidine acetate, disinfectant surfactants including surfactants and amphotheric aldehydes such as formaldehyde and glutaraldehyde can be included, halogen disinfectants including iodine, iodopliores, and polyvidone-iodine are effective as are peroxides and other oxygenators such as hydrogen peroxide. Other beneficial ingredients include aluminum-zinc agents, furfuran derivatives, and quinoline astringent derivatives such as clioquinol. As beneficial as all of these antimicrobial agents may be, they all tend to suffer from the lack that they can be injurious to tissue and / or microbes can easily develop resistance.

[426] As amply demonstrated above, the inventive silver coat is highly effective antimicrobial, is very mild to tiuman tissue, and is effective against resistant microbes, the amorphous gel and hydrogel sheet are. both promote effective levels of colloidal silver in the healing moist environment. On the one hand the amorphous hydrogel slowly releases the colloidal silver as it slowly softens into a tissue exudate and gradually begins to dissolve. On the other hand the amplified hydrogel gives moisture to the tissue and simultaneously makes the colloidal silver available at the site. In addition, some current colloidal silver in the dressing has the advantage of being molecular silver, the gradual reduction of which over a prolonged period will release silver ions which have excellent oligodynamic activity.

[427] After the initial experiments Carbopol was selected as an effective hydrogel in forming an agent for use with the inventive colloidal silver. A basic formulation was developed which generally included the following ingredients as shown in Table 9a.

Table 9a

<td>Ingredient</td><td>Function</td><td>Provider</td>

<td>Colloidal silver solution (22 ppm or 32 ppm)</td><td>Active, anti-microbial and thinner</td><td>American Biotech Labs</td>

<td>Carbopol ERD2026</td><td>Rheology modifier</td><td>Noveon</td>

<td>Triethanolamine</td><td>Neutralizer, penetrating agent</td><td>E. Merck</td>

<td>Propylene glycol</td><td>Moistening</td><td>E. Merck</td>

[428] All raw materials were analyzed first for

1. Anti-bacterial acitvitis

2. Physical and chemical properties:

1. Appearance

2. Odour

3. pH

4. Sensation

5. Density 6. Foaming property

7. Flowability.

[429] Colloidal silver solution (22 ppm or 32 ppm):

ΜΑ 29428Β1

In this formulation of silver solution it is used as an active component (anti-microbial agent). It is also the only diluent in this specific formulation.

[430] A. Anti-bacterial activity:

<td>Culture</td><td colspan="2">Diameter of the inhibition zone</td>

<td></td><td>22 ppm</td><td>32 ppm</td>

<td>ISK</td><td>17 mm</td><td>18 mm</td>

<td>E. coli</td><td>14 mm</td><td>N / A</td>

<td>Ps. Aeruginosa</td><td>21 mm</td><td>22 mm</td>

[431] B. Physical and chemical properties:

1. Appearance

2. Odour

3. pH

4. Sensation

5. Density

6. Foaming properties

7. Flowability

Clear colorless liquid

Scentless

5,0

Not applicable

1,00

Not applicable

Not applicable

[432] Carbopol

[433] Carbopol is chemically known as carboxypolymethylene or carboxyvinyl polymer. It is a copolymer of acrylic acid and it is highly ionic (ie, hydrophilic) and slightly an acidic compound. Carbopol polymers must be neutralized in order to achieve maximum viscosity. It is used in pharmaceutical, cosmetic and fabric printing fields as a protective colloid, suspending agent, dispersing agent and emulsifier. In this formulation Carbopol is used as a gelling agent or protective colloid.

[434] A. Anti-bacterial activity

Not applicable

[435] B. Physical and chemical properties:

1. Appearance

2. Odour

3. pH

4. Sensation

5. Density

6. Foaming properties

7. Flowability

Dry, white powder Odorless

Not applicable

Not applicable

Not applicable

Not applicable

Not applicable

[436] Triethanolamine (TEA) CeH Ο3 (ΜοΙ. Wt .: 149.19)

In this formulation, Triethanolamine, an alkalizing agent neutralizes Carbopol to increase viscosity. It also increases the penetrating power of the active agent.

[437] A. Anti-bacterial activity (Not Applicable)

[438] B. Physical and Chemical Properties

<td> 1.</td><td>Appearance</td><td>Colorless viscous liquid</td>

<td> 2.</td><td>Odour</td><td>light ammonia</td>

<td> 3.</td><td>pH</td><td>Not applicable</td>

<td> 4.</td><td>Sensation</td><td>Not applicable</td>

<td> 5.</td><td>Density</td><td>1.1242 g / cc</td>

<td> 6.</td><td>Foaming properties</td><td>Not applicable</td>

AT/

ΜΑ 29428Β1

7. Flowability Not applicable

4391 ؛ Propylenealvcol

CsHsO ؛ Mol. wt .: 76.09

[440] Propyleneglycol is chemically known as 1: 2 propanediol. It is used as a moisturizer and sensation modifier in this formulation.

[441] A. Anti-bacterial activity Not Applicable

[442] B. Physical and Chemical Properties:

1. Appearance

2. Odour

3. pH

4. Sensation

5. Density

6. Foaming properties

7. Flowability

Colorless viscous liquid

Scentless

Not applicable

Not applicable

1.036 gm / cc

Not applicable

Not applicable

[443] Once the standard formula was developed, a number of manufacturing lots were manufactured to explore the possible range of formulations. From the 19 experiments performed, the following observations were accessed.

1. The increase in pH increases the gel viscosity.

2. increasing the amount of Carbopol increases the gel viscosity.

3. The higher the percentage of Carbopol, the higher the viscosity.

It can be concluded from the above experiments that a difference must be reached between the amount of Carbopol and the A used and the final pH obtained which must not be greater than 8.5. therefore formulation No. 18 has been kept as a standard and the manufacturing batch is weighed up to 10 Kg.

[444] INTRODUCTION OF PRODUCT DEVELOPMENT STUDIES:

[445] Carbopol based gel formulations should be standardized with respect to pH, feel, viscosity and consistency. To this end, various thoughts of work batches in the lab have been taken using water as the aqueous phase to achieve a product of suitable quality and feel before taking the main work lot.

[446] Production batch formulation No. SG 001:

<td>Part A: Distilled Water</td><td>83.50 g</td>

<td>Carbopol</td><td>00.62 g</td>

<td>NaOH 18%</td><td>00.60 g</td>

<td>Part B: Distilled Water</td><td>1.00g</td>

<td>Propylene glycol</td><td>5.00 g</td>

<td>NaOH 18%</td><td>1.50 g</td>

[447] Procedure: Weigh the given quantity of distilled water from part A and keep it in a water bath at 7O٥C. Add the Carbopol to the distilled water with constant agitation to avoid masses. Add to this 18% NaGH at 70 ° C after 20 minutes. Weigh all the ingredients of part B and keep in the water bath at 70 ° C for 15 to 20 minutes. Add part B to part A and stir for 10 to 15 minutes, cool to room temperature and analyze.

[448] Results:

Ι.ρΗ 10.8

2. Flowability 9O٥C =&gt; 5 min.

&lt;=45٠0

3. Viscosity Very sticky&gt; 5 min.

Γ

29428Β1

[449] Formulation of batch No. SG / 002: Part A: Distilled water 83.50 g

MY

<td>Part A:</td><td colspan="2">Distilled water 83.50</td>

<td></td><td>Carbopol</td><td>00.62 g</td>

<td></td><td>TEA</td><td>O1.2Og</td>

<td>Part B:</td><td>Distilled water 1.0</td><td></td>

<td></td><td>Propylene glycol</td><td>5.0 g</td>

<td></td><td>TEA</td><td>1.5 g</td>

[450] Procedure: Weigh the given amount of distilled water from part A and keep in the water bath at 7O٥C. Add Carbopol to distilled water with constant agitation to avoid lumps. Add to the latter TEA at 70 ° C after 20 minutes. Weigh all the ingredients in Part B and keep in the 7O٥C water bath for 15 to 20 minutes. Add part B to part A and stir for 10 to 15 minutes, cool to room temperature and analyze.

[451] Results: Ι.ρΗ 7.9 (SOP-08)

2. Flowability 9O٥C =&gt;&gt; 5 min

C =&gt;&gt; 5 min

3. Very sticky viscosity

Batch formulation No. SG / 003:

Part A: Distilled water 86.00 g

Carbopol 00.62 g

TEA 01.20 g

Part B: Distilled water 2.00 g

Propyleneglycol 5.00 g

TEA

1.50 g

[452] Procedure: Weigh the given amount of distilled water from part A and keep in the water bath at 70 ° C. Add Carbopol to distilled water with constant agitation to avoid masses. Add to this last TEA at 7O٥C after 20 minutes. Weigh all the ingredients in Part B and keep in the 7O٥C water bath for 15 to 20 minutes. Add part B to part A and shake it for 10 to 15 minutes. Cool it to room temperature and analyze.

[453] Results: 1. pH 8.62

2. Flowability 9O٠C = &gt;&gt; 5 min.

3. Viscosity Very sticky.

[454] Formulation of production batch No. SG / 004:

Part A: Distilled water 86.00 g

Carbopol 00.62 g

TEA 01.00 g

Part B: Distilled water 2.00 g

Propyleneglycol 5.00 g TEA 1.50 g

[455] Procedure: Weigh the given amount of distilled water from part A and keep in the water bath at 7O٥C. Add the Carbopol to the distilled water with constant agitation to avoid masses. Add to this THE at 70 ° C after 20 minutes. Weigh all the ingredients in Part B and keep in the 7O٥C water bath for 15 to 20 minutes. Add part B to part A and shake it for 10 to 15 minutes. Cool it to room temperature and analyze.

ΜΑ 29428Β1

[456] Results: 1. pH

8,5

2. Flowability 9O ° C = &gt;&gt; 5 min

45٠c =&gt;&gt; 5 min.

3. Very sticky viscosity

[457] Formulation of production batch No. SG / 005:

Part A:

Distilled water

86.0 g

Carbopol

0,62

TEA

1.2Og

Part B: Distilled water 2.00 g

Propylene glycol

7, OOg

TEA

1.50 g

[458] Procedure: Weigh the given quantity of distilled water from part A and keep in the water bath at 7O٠C. Add the Carbopol to the distilled water with constant agitation to avoid masses. Add to this THE at 7O٥C after 20 minutes. Weigh all the ingredients at 70 ° C for 15 to 20 min. add part B to part A and shake it for 10 to 15 min. cool it to room temperature and analyze.

[459] Results:

Ι.ρΗ

8,7

2. Pourability 90C = &gt;&gt; 5min.

45٠C = &gt;&gt; 5min.

3. Very sticky viscosity

[460] Formulation of production batch No. SG / 006:

Part A: Distilled water 85.00 g

Carbopol 00.62 g

TEA 01.00g

Part B: Distilled water 1.00 g

Propylene glycol 5.00 g

TEA 1.00 g

[461] Procedure: Weigh the given amount of Distilled Water from Part A and keep in a water bath at 7O٥C. Add Carbopol to Distilled Water with constant agitation to avoid masses. Add TEA to this at 70 C after 20 minutes. Weigh all the ingredients of Part B and keep it in a 7O٠C water bath for 15 to 20 min. add part B to part A and stir for 10-15 min. cool it to room temperature and analyze.

[462] Results:

Ι.ρΗ

8,4

2. Flowability 9O٠C =&gt;&gt; 5 min

ΜΑ 29428Β1

45٠c = &gt;&gt; 5 min

3. Very sticky viscosity

[463] Formulation of production batch No. SG / 007:

Part A: Distilled water 172 g

Carb0p0l1,24g

TEA 2.40 g

Part B: Distilled water 6.0 g

Propyleneglycol 10g

TEA 2.80 g

[464] Procedure. ' Weigh the given amount of Distilled Water from Part A and keep it in a water bath at 7O٥C. Add Carbopol to Distilled Water with constant agitation to avoid masses. Add TEA to this at 7O٥C after 20 minutes. Weigh all the ingredients of part B and keep in a 70٠0 water bath for 15 to 20 min. Add part B to part A and shake it for 10 to 15 min. cool it to room temperature and analyze.

[465] Results: Ι.ρΗ 8.28

2. Flowability 9O٠C =&gt;&gt; 5 min.

45'C =&gt;&gt; 5 min.

3. Very sticky viscosity

[466] Formulation of production batch No. SG / 008:

<td>Part A:</td><td>Silver solution (32 ppm)</td><td>86 g</td>

<td></td><td>Carbopol</td><td>0.62 g</td>

<td></td><td>TEA</td><td>1.20 g</td>

<td>Part B</td><td>Silver solution (32 ppm)</td><td>2.0 g</td>

<td></td><td>Propylene glycol</td><td> 5,0</td>

<td></td><td>TEA</td><td>1.5 g</td>

[467] Procedure: Weigh the given quantity of the Silver Solution of part A and keep in a water bath at 7O٠C. Add Carbopol to the silver solution with constant agitation to prevent lumps. Open TEA to it at 70 ° C after 20 minutes. Weigh all the ingredients of part B and keep in a water bath at 7O٥C for 15 to 20 min. Cut Part B to Part A and shake for 10 to 15 min. cool it to room temperature and analyze.

[468] Results: 1. pH 8.65

2. Flowability 9O٠C = &gt;&gt; 5 min.

45٠c = &gt;&gt; 5min.

3. Viscosity Very sticky

[469] Formulation of production batch No. SG / 009:

f® /

MY

29428Β1

Part A: Silver solution (32 ppm) 172 g

Distilled water 12 g

Carbopol 1.24 g

TEA 2.40 g

Part B: Silver solution (32 ppm) 6.00 g

Propyleneglycol 1O, Og

TEA 2.80 g

[470] Procedure: Weigh the given amount of the given solution of part A and keep it in a 7 خ O ° C water bath. Add Carbopol to the silver solution with constant agitation to prevent lumps. Add TEA to this at 7O٠C after 20 minutes. Weigh all the ingredients from part B and keep in a 70٥C water bath for 15 to 20 min. Add part B to part A and shake it for 10 to 15 min. cool it to room temperature and analyze.

<td>[471] Results: Ι.ρΗ</td><td> 8,54</td>

<td>2. Flowability:</td><td>9O٥C =&gt;&gt; 5 min.</td>

<td></td><td>45٥c =&gt;&gt; 5 min</td>

3. Very sticky viscosity

[472] Formulation of production batch No. SGI010:

Part A: Silver solution (32 ppm) I72g

Distilled water 24 g

Carbopol 1.39 g

TEA 2.40 g

Part B: Silver solution (32 ppm) 6.00 g

Propyleneglycol 5.00 g

TEA 2.80 g

[473] Procedure: Weigh the given quantity of the Silver Solution of part A and keep in a water bath at OC. Add Carbopol to the Silver Solution with constant agitation to prevent lumps. Add

TEA to this at 70 ° C after 20 minutes. Weigh all the ingredients of part B and keep in a water bath at 7O٥C for 15 to 20 min. Add part B to part A and shake it for 10 to 15 min. cool it to room temperature and analyze.

[474] Results: 1. pH 8.43

2. Flowability 9O٥C =&gt;&gt; 5 min.

45٠c =&gt;&gt; 5 min.

3. Viscosity: Very sticky

م

ΜΑ 29428Β1

[475] Formulation of production batch No. SG / 011:

<td>Part A: Distilled water</td><td>98 g</td>

<td>Carbopol</td><td>0.76 g</td>

<td>TEA</td><td>0.56 g</td>

<td>Part B: Distilled water</td><td>3.0 g</td>

<td>Propylene glycol</td><td>5.0 g</td>

<td>TEA</td><td>1.4 g</td>

<td colspan="2">[476] Procedure: Weigh the given quantity of Distilled Water from part A and keep in a water bath at 7O٥C. Add Carbopol to the Distilled Water solution with constant agitation to prevent lumps. Add TEA to this at 7O٥C after 20 minutes. Weigh all the ingredients of padie B and keep in a water bath at 7O٥C for 15 to 20 min. Add part B to part A and shake it for 10 to 15 min. Cool it to room temperature and analyze.</td>

<td>[477] Results: Ι.ρΗ</td><td> 8,05</td>

2. Flowability 9O٠C =&gt;&gt; 5 min.

45٠c = &gt;&gt; 5min.

3. Very sticky viscosity

[478] Formulation of production batch No. SG1012:

<td>Part a</td><td>Distilled water</td><td>98g</td>

<td></td><td>Carbopol</td><td>0.76 g</td>

<td></td><td>TEA</td><td>0.34 g</td>

<td>Part B</td><td>Distilled water</td><td>3.00 g</td>

<td></td><td>Propylene glycol</td><td>5.00 g</td>

<td></td><td>TEA</td><td>0.64 g</td>

[479] Procedure: Weigh the given quantity of Distilled Water from part A and keep in a water bath at 7O٥C. Add Carbopol to a solution of Distilled Water with constant agitation to prevent lumps. Add TEA to this at 70 ° C after 20 minutes. Weigh all the ingredients of part B and keep in a water bath at 7O٥C for 15 to 20 min. Add part B to part A and shake it for 10 to 15 min. Cool it to room temperature and analyze.

[480] Results Ι.ρΗ 6.35

2. Flowability 9O٠C =&gt;&gt; 5 min.

45٠c:&gt;&gt; 5 min.

3. Very sticky viscosity

[481] Formulation of production batch No. SG / 013:

at/

ΜΑ 29428Β1

<td>Part A: Silver solution (32 ppm)</td><td>86 g</td>

<td>Distilled water</td><td>12g</td>

<td>Carbopol</td><td>0.76 g</td>

<td>TEA</td><td>0.32 g</td>

<td>Part B: Silver solution (32 ppm)</td><td>3.00 g</td>

<td>Propylene glycol</td><td>5.00 g</td>

<td>TEA</td><td>0.64 g</td>

<td colspan="2">[482] Procedure: Weigh the given amount of silver solution and distilled water from part A and keep in a water bath at 7O٠C. Add Carbopol to Solution with constant agitation to prevent lumps. Add TEA to this at 7O٥C after 20 minutes. Weigh all the ingredients of part B and keep in a water bath at 7O٥C for 15 to 20 min. Add part B to part A and shake it for 10 to 15 min. cool it to room temperature and analyze.</td>

<td>[483] Results: 1. pH</td><td>β, τ</td>

<td>2. Flowability 9O٥C =&gt;&gt;</td><td>5 minutes.</td>

<td>45٠c =</td><td>&gt;&gt; 5 min.</td>

<td>3. Viscosity</td><td>very sticky</td>

<td>[484] Formulation of production batch No. SG / 014:</td><td></td>

<td>Part A: Silver solution (32 ppm)</td><td>86 g</td>

<td>Distilled water</td><td>12g</td>

<td>Carbopol</td><td>0.78 g</td>

<td>TEA</td><td>0.32 gm</td>

<td>Part B Silver solution (32 ppm):</td><td>3.00 g</td>

<td>Propylene glycol</td><td>5.00 g</td>

<td>TEA</td><td>0.64 g</td>

<td colspan="2">[485] Procedure: Weigh the given amount of the silver solution and Distilled Water from part A and keep in a water bath at 70 ° C. Add Carbopol to a Solution with constant agitation to prevent lumps. Add TEA to this at 7O٥C after 20 minutes. Weigh all the ingredients of part B and keep in a water bath at 7O٥C for 15 to 20 min. Add part B to part A and stir for 10 to 15 min. cool it to room temperature and analyze.</td>

<td>[486] Results Ι.ρΗ 6.6</td><td></td>

2. Flowability 9O ° C =&gt;&gt; 5 min. 45٠c =&gt;&gt; 5 min.

3. Very sticky viscosity

29428Β1

MK

[487] Formulation of production batch No. SG / 015:

Part A Silver solution (32 ppm) 86 g

Distilled water 12 g

Carbopol 0.68 g

TEA0.40 g

Part B Silver solution (32 ppm): 5.0 g

Propyleneglycol 7.0 g

TEA 0.6 g

[488] Procedure: Weigh the given amount of the silver and distilled water solution of part A and keep in a water bath at 7O٥C. Add Carbopol to the solution with constant agitation to prevent lumps. Add TEA to this at 7O٠C after 20 minutes. Weigh all the ingredients of part B and keep in a water bath at 7O٥C for 15 to 20 min. Add part B to part A and shake it for 10 to 15 min. cool it to room temperature and analyze.

[489] Results Ι.ρΗ 6.72

2. Flowability 9O٥C =&gt;&gt; 5 min.

45٠c =&gt;&gt; 5 min

3.٧ Tescollante viscosity

[490] Formulation of production batch No. SG / 016:

Part A: Silver solution (32 ppm) 86 g

Distilled water 12 g

Carbopol 0.64 g

TEA 0.40 g

Part B: Silver solution (32 ppm) 5.0 g

Propyleneglycol 7.0 g

TEA 0.6 g

[491] Procedure: Weigh the given amount of the silver and distilled water solution of part A and keep in a water bath at 7O٠C. Add Solution with constant agitation to prevent lumps. Add TEA to this at 7O٥C after 20 mins. Weigh all the ingredients of part B and keep in a water bath at 7O٥C for 15 to 20 min. Add part B to part A and shake it for 10 to 15 min. Cool it to room temperature and analyze.

[492] Results: Ι.ρΗ 6.8-7

2. Flowability 9O٥C =&gt;&gt; 5 min.

45٠c =&gt;&gt; 5 min

3. Sticky viscosity

[493] Formulation of production batch No. SG / 017:

ΜΑ 29428Β1

Part A: Silver solution (32 ppm) 86 g

Distilled water 12 g

Carbopol 0.62 g

٢EA0.4g

Part B: Silver solution (32 ppm) 5.0 g

Propyleneglycol 7.0 g

TEA 0.6 g

[494] Procedure: Weigh the given amount of silver solution and distilled water from part A and keep in a water bath at 70 ° C. Add Carbopol to Solution with constant agitation to prevent lumps. Add TEA to this at 7O٥C after 20 minutes. Weigh all the ingredients of part B and keep in a water bath at 7O٥C for 15 to 20 min. Add part B to part A and ؛ 'stir for 10 to 15 min. cool it to room temperature and analyze.

<td>[495] Results: Ι.ρΗ</td><td> 7,05</td>

<td>2. Flowability</td><td>9O٠C =&gt;&gt; 5 min.</td>

<td></td><td>45 ° c =&gt;&gt; 5 min</td>

<td>3. Viscosity</td><td>Sticky</td>

[496] Formulation of production batch No. SG / 018:

Part A: Silver solution (32 ppm) 86 g

Distilled water 12 g

Carbopol 0.58 g

TEA 0.4 g

Part B: Silver solution (32 ppm) 5.0 g

Propyleneglycol 7.0 g

TEA 0.6 g

[497] Procedure: Weigh the given amount of silver solution and distilled water from part A and keep in a water bath at 7O٥C. Add Carbopol to the solution with constant agitation to prevent lumps. Add TEA to this at 7O٥C after 20 minutes. Weigh all the ingredients of part B and keep in a water bath at 7O٥C for 15 to 20 min. Add part B to part A and shake it for 10 to 15 min. cool it to room temperature and analyze.

[498] Results: Ι.ρΗ 7.40

2. Flowability 9O ° C =&gt;&gt; 5 min.

45٥C = &gt;&gt; 5 min

3. Viscosity

Sweet

Z

٩ هه 2 هلأ 2 Mk

[499] Formulation of production batch No. SG / 019:

Part A: Silver solution (32 ppm) 86 g

Distilled water 12 g

Carbopol 0.54 g

TEA 0.4 g

Part B: Silver solution (32 ppm) 5.0 g

Propyleneglycol 7.0 g

TEA 0.6 g

[500] Procedure: Weigh the given amount of silver solution and Distilled Water from part A and keep in a water bath at 7O٥C. Add Carbopol to the solution with constant agitation to prevent lumps. Add TEA to this at 7O٥C after 20 minutes. Weigh all the ingredients of part B and keep in a water bath at 7O٠C for 15 to 20 min. Add part B to part A and stir for 10 to 15 min. Cool it to room temperature and analyze.

<td>[501] Results: Ι.ρΗ</td><td> 7,65</td>

<td>2. Flowability</td><td>9O٥C =&gt; 1 min.</td>

45٠c =&gt; 2 min

<td>3. Viscosity</td><td>Sweet</td>

[502] Note: Although the gel feel has improved, the consistency is not appropriate.

[503] Based on the above results the following instructions for a one kilogram production batch have been developed.

Part A Silver solution 860 gm

Distilled water 100 gm

Carbopol 5.80 gm

TEA 4.00 gm

ASAP Solution

Part B 50.0 gm

Propylene glycol 70.0 gm

TEA 6.00 gm

Output 1.0 Kg. After adjustment for moisture loss.

[504] Procedure: Take the required quantity of Distilled Water and Silver Solution in a clean sterilized container. Raise the temperature of a solution to 7O٥C with continuous agitation. Begin the addition of Carbopol in minute amounts with continuous agitation / homogenization. After all of the Carbopol has been added, continue for 30 minutes. (Adjust the time according to the size of the production lot). Then add TEA to the solution in phase A.

ΜΑ 29428Β1

[505] In a separate container, mix all the ingredients of part B. Raise the temperature to 7O٥C and slowly add part B to part A. will cool it to room temperature upon complete homogenization.

[506] Precautions: The dispersion of Carbopol should be done using a good homogenizer. Take a small test batch using a new production batch of Carbopol. Reduce the heating time since long heating leads to more water loss.

<td>[507] Results 1. pH</td><td> 7,4</td>

<td>2. Flowability</td><td>&gt; 5 min</td>

<td>3. Viscosity</td><td>Sweet.</td>

[508] This formulation was easily measured up to 10 kgs. In a pilot installation. No issues were recorded during the upgrade. Air removal by vacuum application is recommended to remove trapped air and to ensure uniform filling.

[509] This formulation has the following physical and chemical characteristics as shown in the table

Table 10

<td></td><td>Test</td><td>Specification</td><td>Results</td>

<td> 1.</td><td>Appearance</td><td>Golden yellow translucent gel</td><td>Admitted</td>

<td> 2.</td><td>Odour</td><td>Scentless</td><td>Scentless</td>

<td> 3.</td><td>Specific gravity</td><td> 1,02</td><td> 1,01</td>

<td> 4.</td><td>Flowability</td><td>At 45 ° and 90 ° - more than 5 min. to cross 1 chip from the origin</td><td>At 45 ° and 90 ° - more than 5 min. to cross 1 chip from the origin</td>

<td> 5.</td><td>5. cap. Foaming</td><td>&lt;10 ml</td><td>&lt;10 ml</td>

<td> 6.</td><td>Feel / viscosity</td><td>-1 sweet</td><td>-1 sweet</td>

<td> .٦</td><td>Viscosity TA 30٥ 370</td><td> 32.000 ±5000 30.000 ±5000</td><td> 34.000 33.500</td>

<td> 8.</td><td>PH</td><td>6.5 to 8.0</td><td> 7,4</td>

<td> 9.</td><td>Freeze and thaw</td><td>To pass SOP 1-10</td><td>Compared to the original</td>

<td> 10.</td><td>Optimal wavelength (X Max)</td><td>22 ppm - 400 +/- 20 nm 32 ppm - 450 +/- 20 nm</td><td>400 nm. * ٠ 450 nm. **</td>

<td> 11.</td><td>Light exposure</td><td>No additional discoloration</td><td>Admitted</td>

<td> 12.</td><td>Compatibility</td><td>No further discoloration from reaction of product 1 with vessels</td><td>Ref. Table 3</td>

<td> 13.</td><td>Moisture donation</td><td></td><td> ٥/٠ 10,27</td>

<td> 14</td><td>Moisture uptake</td><td></td><td> ٥/٠ 80</td>

15101 Microbiological evaluation

[511] It is reasonable to assume that the colloidal silver hydrogel possesses similar microbiological properties to the original colloidal silver which was extensively examined as demonstrated above. However, the addition of the hydrophilic polymer to produce the gel could directly interfere with the microbial properties of the silver or could thus prevent the diffusion of the silver whose effectiveness is diminished. Therefore, microbiological tests similar to those performed on the colloidal silver solution were also performed on the colloidal silver hydrogel.

[512] Initially, the hydrogel was tested to determine if the composition was self-sterilizing. The following protocol was followed:

[513] 100 ml vials of sterile Thioglycollate fluid medium (Anaerobic bacteria), sterile soybean casein digestion medium (Aerobic bacteria), and potato dextrose broth (fungi) were obtained. Samples of about 100 mg of test gel were aseptically transferred to groups of vials. One group was incubated at 3c and another group was incubated at room temperature / \ /

ΜΑ 29428Β1 for one week. After this time, the vials were checked and showed no turbidity or signs of microbial growth. Because the gel sample was not made under sterile conditions, it could be concluded that the composition is self-sterilizing. The 100 mg of gel used for testing, this corresponds to 2.2pg in 100 ml of medium or 0.02214 or 0.032pg of silver per ml of medium. At this concentration, silver would have no antimicrobial activity and therefore false negative results can be ruled out.

[514] A variety of test organisms were thus employed to compare the zone of inhibition obtained by either a 22ppm silver solution or 32ppm silver gel made as described above and as shown in Table 11. 0.1 ml aliquots or actively growing 18-hr cultures of the microorganism - (approximately 10® CFU / ml) were discarded from the sterile nutrient agar plates. A 10 millimeter diameter hole was punched into each plate inoculated with a cork borer. A test amount of (0/2 to 0.3 g) of the product was placed in each hole, and the plate was incubated for 24 hours. After this time the plaques were checked and the following areas of inhibition (total diameter of each area) were measured.

[515] Table 11

<td rowspan="2">Culture</td><td colspan="2">Silver solution</td><td colspan="2">Silver gel</td>

<td>22 ppm</td><td>32 ppm</td><td>22 ppm</td><td>32 ppm</td>

<td>E. coli</td><td>14 mm</td><td>14 mm</td><td>12 mm</td><td>13 mm</td>

<td>Ps. Aeruginosa</td><td>21 mm</td><td>22 mm</td><td>21 mm</td><td>20 mm</td>

<td>B. subtilis</td><td>15 mm</td><td>16 mm</td><td>14 mm</td><td>14 mm</td>

<td>MRSAI</td><td>17 mm</td><td>18 mm</td><td>16 mm</td><td>17 mm</td>

<td>MRSA2</td><td>16 mm</td><td>17 mm</td><td>16 mm</td><td>17 mm</td>

<td>S. aureus ATCC 6538 P</td><td>14 mm</td><td>14.5 mm</td><td>15 mm</td><td>15 mm</td>

<td>S. pyroqenes</td><td>16 mm</td><td>18 mm</td><td>16 mm</td><td>18 mm</td>

<td>S. typhi</td><td>17 mm</td><td>16 mm</td><td>16 mm</td><td>16 mm</td>

<td>Sh. Flexneri</td><td>20 mm</td><td>21 mm</td><td>20 mm</td><td>21 mm</td>

<td>K. penumoniae</td><td>17 mm</td><td>18 mm</td><td>18 mm</td><td>18 mm</td>

<td>C. diphteriae</td><td>16 mm</td><td>18 mm</td><td>16 mm</td><td>17 mm</td>

<td>C. albicans</td><td>39 mm</td><td>40 mm</td><td>39 mm</td><td>40 mm</td>

[516] These results show that the inhibitory effects of the gel are essentially equivalent to those of the colloid silver solution: this demonstrates that the gelling polymer does not negatively affect the antimicrobial powers of the silver colloid. Some cultures (S. pyogenes, C. diphtheriae and s. Aureus) were also grown on blood agar. The results suggested that the silver gel was also effective on a bloody, oozing wound.

[517] Similar tests were performed on the same bacterial strains using a variety of antibiotic agents. In some cases the antibiotics have been more effective than the silver compounds — in others they have been less effective. This demonstrates that the strains used were not weakened or discarded strains (see Tables 12a and 12b).

Gram Positive Bacteria (Table 12a)

<td>Antibiotic</td><td>Conc.</td><td>S. aureus</td><td>MRSAI</td><td>MRSA2</td><td>B. subtilis</td>

<td>Ampicillin</td><td>200 mcg</td><td>Claire</td><td>15 mm</td><td>18 mm</td><td>16 mm</td>

<td>Cefotaxime</td><td>30 mcg</td><td>26 mm</td><td>No inhibition</td><td>- Claire</td><td>12 mm</td>

<td>Cephalexin</td><td>30 mcq</td><td>Claire</td><td>1.0 mm</td><td>0.8 mm</td><td>Claire</td>

<td>Ciprofloxacin</td><td>5 mcg</td><td>28 mm</td><td>14 mm</td><td>14 mm</td><td>20 mm</td>

<td>Cloxacillin</td><td>1 mcq</td><td>Claire</td><td>Claire</td><td>13 mm</td><td>18 mm</td>

<td>Co-trimoxazole</td><td>25 mcg</td><td>Claire</td><td>No inhibition</td><td>No inhibition</td><td>15 mm</td>

<td>Gentamycin</td><td>10 mcq</td><td>Claire</td><td>No inhibition</td><td>11 mm</td><td>18 mm</td>

<td>Lincomycin</td><td>2 mcg</td><td>Claire</td><td>Claire</td><td>Claire</td><td>18 mm</td>

<td>Dfloxacin</td><td>5 mcg</td><td>Claire</td><td>15 mm</td><td>16 mm</td><td>22 mm</td>

<td>Peflofloxacin</td><td>10 mcg</td><td>30 mm</td><td>11 mm</td><td>13 mm</td><td>21 mm</td>

<td>Roxythromycin</td><td>15 mcg</td><td>Claire</td><td>1.0 mm</td><td>12 mm</td><td>20 mm</td>

<td>Tetracvcline</td><td>30 mcg</td><td>34 mm</td><td>No inhibition</td><td>0.7 mm</td><td>19 mm</td>

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Gram negative bacteria (Table 12b)

<td>Antibiotic</td><td>Conc.</td><td>E. coli</td><td>K. pneumoniae</td><td>S. typhi</td><td>Ps. Aeruginosa</td>

<td>Amikacin</td><td>30 mcg</td><td>Claire</td><td>18 mm</td><td>Claire</td><td>10 mm</td>

<td>Ampicilin</td><td>200 mcg</td><td>23 mm</td><td>18 mm</td><td>20 mm</td><td>13 mm</td>

<td>Cefotaxime</td><td>30 mcg</td><td>21 mm</td><td>20 mm</td><td>22 mm</td><td>19 mm</td>

<td>Ceftizoxime</td><td>30 mcg</td><td>18 mm</td><td>18 mm</td><td>15 mm</td><td>No inhibition</td>

<td>Chloramphenicol</td><td>30 mcg</td><td>22 mm</td><td>21 mm</td><td>23 mm</td><td>No inhibition</td>

<td>Ciprofloxacin</td><td>5 mcg</td><td>29 mm</td><td>22 mm</td><td>25 mm</td><td>15 mm</td>

<td>Cotrimoxazole</td><td>25 mcg</td><td>24 mm</td><td>19 mm</td><td>27 mm</td><td>Claire</td>

<td>Gentamycin</td><td>10 mcg</td><td>Claire</td><td>17 mm</td><td>Claire</td><td>No intlibition</td>

<td>Ofloxacin</td><td>5 mcg</td><td>Claire</td><td>29 mm</td><td>Claire</td><td>15 mm</td>

<td>Pefloxacin</td><td>10 mcg</td><td>Claire</td><td>25 mm</td><td>Claire</td><td>10 mm</td>

<td>Piperacillin</td><td>100 mcg</td><td>22 mm</td><td>15 mm</td><td>16 mm</td><td>10 mm</td>

<td>Tetracycline</td><td>30 mcg</td><td>19 mm</td><td>18 mm</td><td>16 mm</td><td>No inhibition</td>

[518] Manual cleaning test

[519] Since the hydrogel has the ability to increase the adhesion of silver to skin surfaces, the effectiveness of the gel as a hand cleanser was evaluated. For this test one square inch of volunteers was identified and then cleaned by approximately 1 g of gel. A control area was cleaned with sterile distilled water. The zones were buffered and the sample was streaked on nutrient agar, buffering was repeated hourly for four hours, streaked plates were incubated for 24 hours at 37 ° C and the results were evaluated.

[520] As shown in Table 13 below, the control samples increased so many bacteria that they are also numerous to count TTC). The areas treated with the silver gel remained essentially sterile for three hours and showed only slight growth at four hours. This should provide superior health care results for workers who require sterilization of the surface of their hands without the use of harsh components or sprinklers.

Table 13

<td>Time</td><td>Control</td><td>22 ppm</td><td>32 ppm</td>

<td>0 hour</td><td>TNTC</td><td>No growth</td><td>No growth</td>

<td>1 hour</td><td>TNTC</td><td>No growth</td><td>No growth</td>

<td>2 hours</td><td>TNTC</td><td>No growth</td><td>No growth</td>

<td>3 hours</td><td>TNTC</td><td>No growth</td><td>No growth</td>

<td>4 hours</td><td>TNTC</td><td>3 Cfu</td><td>2 Cfu</td>

<td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td>

Although hydrogels show exceptional wound healing properties, a disadvantage of the typical hydrogel is that microorganisms can often migrate through the matrix. Thus, if a wound is covered by the hydrogel and an area of ​​the wound becomes infected, infectious organisms may be able to travel through the hydrogel and infect other areas. This possibility was investigated by employing a hydrogel strip to bridge separate regions of a nutrient agar plate. Each plate was separated into two regions by removing a 2 cm strip of agar along one diameter of the plate. This bond was established by a 1.5 cm wide strip of hydrogel which coated over the agar by approximately 5 mm at either end. One side of the plate was then inoculated with about 0.5 m! culture and the plate was incubated to see if the microorganisms could cross the hydrogel bridge. the results in Table 14 prove that the silver hydrogel completely prevents migration.

Table 14

<td>Culture</td><td>Incubation area</td><td>Migration area</td>

<td>E coli</td><td>Hard growth</td><td>No growth</td>

<td>B. subtilis</td><td>Hard growth</td><td>No growth</td>

<td>MRSAI</td><td>Hard growth</td><td>No growth</td>

<td>Ps. Aeruginosa</td><td>Hard growth</td><td>No growth</td>

<td>Hydrogel control</td><td>Hard growth</td><td>Growth</td>

<img file="MA29428B1_D0008.tif" />

MY

29428Β1

From the results shown above, a prototype gel formula was chosen and the variations are suggested in the following examples.

[521] Example A

For a production batch of 1 Kg of the gel the components taken from Part A and Part B are given below

<td>Part a</td><td>Inventive silver colloid 32 ppm ppm</td><td>860 g</td>

<td></td><td>Distilled water</td><td>100 g</td>

<td></td><td>Carbapol</td><td>6.8 g</td>

<td></td><td>Triethanolamine</td><td>4.0 g</td>

<td>Part B</td><td>Inventive silver colloid 32 ppm</td><td>50 g</td>

<td></td><td>Propylene glycol</td><td>70 g</td>

<td></td><td>Triethanolamine</td><td>6.0 g</td>

[522] First take the required amount of distilled water and silver solution in a stirrer and start stirring. Slowly add in Carbapol (Noveon. USA), agitation should be vigorous enough to distribute the Carbopol and to prevent lumps. The temperature should be maintained between 60-7O٥C during agitation.

[523] Combine all the ingredients of part B in a beaker. Heat to 7O٠C and add to part A with vigorous stirring. Continue kneading and cool to room temperature. Find the yield of the production batch. It should be approx. 1000 gm. Triethanolamine causes Carbopol to gel.

Example B.

[524] Prepare all the ingredients as in Example A including the addition of 1% collagen. This will give a gel with both an antimicrobial effect as well as beneficial collagen which has a type of scaffolding to accelerate wound healing.

Example c:

[525] Prepare all the ingredients as in Example A but including the addition of aloe (powder or solution) in the range 1 to 5٥ / ο. This will impart additional wound healing properties.

Example D:

[526] Prepare all the ingredients as in Example A with the addition of 1 to 10% by weight of Maltodextrin. This will provide a gel formulation which stimulates wound granulation.

[527] Summary of the results of silver hvdroael

[528] It was possible to prepare the Carbopol-based gels using the inventive colloidal silver solutions of 22 ppm and 32 ppm. the gels thus prepared have many advantages over their solution counterparts in terms of their ability to stay in place while maintaining the properties of the parent silver solutions, the amorphous hydrogel nature of the drug confers the advantage of accelerating healing wounds in dampness and also limit the severity of burn wounds by limiting thermal shock. Moreover, the active agent, the colloidal silver solution were examined on a cell line in a previous study and were found to be non-cytotoxic.

[529] A full physicochemical evaluation of the gel was made with various batch manufacturing groups and detailed sets of methods were prepared to standardize and control the product and processes during manufacturing.

[530] Microbiological studies have been performed with intensity and prove that the gel maintains its bactericidal nature. Studies on silver migration have been simulated and show conclusively that freezing can deliver silver to the wound for a period of time. The formulation design will also not allow the migration of microbes in and out and vice versa.

مر

ΜΑ 29428Β1

31] These tests demonstrate that the hypothetical evaluation of the silver hydrogel on the basis of a publication (Journal of Wound Care Vol 12, No 8 SEPT 2003) where the alternative silver dressings were evaluated with the points given for:

1. Antimicrobial zone of inhibition:

2. Microbial challenge test:

3. Microbial transmission test; and

4. silver content of the dressings.

[532] In the first run, the silver hydrogel would be placed in group B and in each of the three remaining runs the silver hydrogel would score in group A giving it total points of 20, on par with Calgitrol AG and Acticot, commercial products that scored the highest in this review.

[533] The antibacterial and antiviral properties of the colloidal silver solution open up several significant uses for the silver hydrogel beyond dressing. As demonstrated above, hydrogel is an ideal antibacterial hand cleanser. Additionally, the non-irritant nature of the silver colloid and hydrogel make the combination an ideal personal lubricant for male or female sexual use with or without condoms or diaphragms where the combination would fight bacteria, mycetes (note effectiveness on Candida albicans) and dangerous viruses such as HIV and disinfects reusable barriers such as diaphragms. Since the hydrogel contains little of any oil, it has no harmful effects on condoms or diappliragmas, unlike some other personal lubricants.

[534] HYDROGEL DETERGENT FOR HANDS (Note: Hydrogel and SILGEl mean the same inventive product and are used interchangeably herein)

[535] Clean hands are reported to be the single most important factor in preventing the spread of dangerous germs and antibiotic resistance in healthcare facilities. As a result, it was decided to verify the effectiveness of the hydrogel known as SILGE1 as a hand hygiene product according to the MMWR guidelines dated October 25, 2002A / OL 51 / No RR-16.

the following standard operating procedure was used:

Material request (SOP):

A standard suspension of Serratia marcescens (10 ⁸ cfu / ml), tap water, sterile insulating gloves, sterile sampling solution, sterile tryptic soy agar, sterile pipettes, sterile test tubes.

Method:

1. 5 ml of suspension saturated with Serratia marcescens is applied to the hands and surfaces of the hands.

2. Distribute 3 ml of test material over the hands and lower 1/3 of the forearms.

3. Add 2 ml of tap water to the hands and soap them (refer to Fig. 1)

4. Rinse hands and forearms in tap water for 30 seconds at room temperature.

5. Repeat the procedure from step 2 to step 4.

6. After the 1st, 3rd, 7th and 10th washes, the sterile insulating gloves used for sampling were placed in the right and left hands.

7. 75 ml of sterile sampling solution is poured into the gloves.

8. All surfaces of the hands are massaged for one minute.

/

ΜΑ 29428Β1

Q- Samples are obtained aseptically for quantitative analysis by a viable counting method, using Tryseptic Soy Agar.

10. The step-plate technique is employed using, 10 ١,10'2 and 10 3 as dilutions.

The plates are incubated at 37٥ c for 24 hours.

Materials and Methods The procedures in the above designated SOP were used

Medium uses St. Trypsic Soy Agar.

Cultures used 16 hr aged Serratia marcescens culture (Approx. Density is 10® CFU / ml).

<td>Incubation temperature:</td><td>37٥c</td>

<td>Incubation time</td><td>24 shots.</td>

<td>Evaluated products</td><td>Silgel 22 and 32 ppm, Spitaderm, Clear Liquid, Sterillium</td>

: The results are present in tables 15a - 15th, 16a - 16th

Results

Left hand

Table 15 a: Spitaderm (Annex-11)

<td rowspan="2">No. of washes Dilution</td><td colspan="4">CFU / ml</td>

<td>Original</td><td> 10٠١</td><td> 10’2</td><td> 10’3</td>

<td>1st wash</td><td> 10</td><td>None</td><td>None</td><td>None</td>

<td>3rd lavaqe</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>5th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>10th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

Table 15b: Silgel 32 ppm

<td rowspan="2">No. of washes Dilution</td><td colspan="4">CFU / ml</td>

<td>Original</td><td> 10</td><td> 10’2</td><td> 10’3</td>

<td>1st lavaqe</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>3rd lavaqe</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>7th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>10th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

Table 15 c: Silgel 22 ppm

<td rowspan="2">No. of washes Dilution</td><td colspan="4">CFU / ml</td>

<td>Original</td><td> 10'-</td><td> 10’2</td><td> 10*</td>

<td>1st wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>3rd wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>7th lavaqe</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>10th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

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Table 15 d: sterillium (Annex- II)

<td rowspan="2">No. of washes Dilution</td><td colspan="4">CFU / ml</td>

<td>Original</td><td> 10”</td><td> 10*</td><td> ٥’10</td>

<td>1st wash</td><td> 30</td><td> 30</td><td>None</td><td>None</td>

<td>3rd wash</td><td>None</td><td> 10</td><td>None</td><td>None</td>

<td>7th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>10th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

Table 15 e: Clear liquid (Annex- II)

<td rowspan="2">No. of washes Dilution</td><td colspan="4">CFU / ml</td>

<td>Original</td><td> 10”</td><td> 10*</td><td> 10*</td>

<td>1st wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>3rd wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>7th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>Orne wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

Right hand

Table 16a: Spitaderm (Annex - II)

<td rowspan="2">No. of washes Dilution</td><td colspan="4">CFU / ml</td>

<td>Original</td><td> 10’’</td><td> 1'0’2</td><td>د'0ا</td>

<td>1st wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>3rd wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>5th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>10th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

Table 16 b: Silgel 32 ppm

<td rowspan="2">No. of washes Dilution</td><td colspan="4">CFU / ml</td>

<td>Original</td><td> 10’1</td><td> 10*</td><td>د'0ا</td>

<td>1st wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>3rd lavaqe</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>7th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>10th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

Table 16 c: Silgel 22 ppm

<td rowspan="2">No. of washes Dilution</td><td colspan="4">CFU / ml</td>

<td>Original</td><td> 10”</td><td> 10*</td><td> 10*</td>

<td>1st lavaqe</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>3rd wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>7th lavaqe</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>10th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

Table 16 d: sterillium (Annex - II)

<td rowspan="2">No washes Dilution</td><td colspan="4">CFU / ml</td>

<td>Original</td><td> 10٦</td><td> 10*</td><td> 10*</td>

<td>1st wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>3rd wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>7th lavaqe</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>10th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

ΜΑ 29428Β1

Table 16e: Clear liquid (Annex - II)

<td rowspan="2">No. of washes Dilution</td><td colspan="4">CFU / ml</td>

<td>Original</td><td> 10”</td><td> 10*</td><td> 10*</td>

<td>1st wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>3rd wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>7th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

<td>10th wash</td><td>None</td><td>None</td><td>None</td><td>None</td>

Conclusion: SIlGEl 32 ppm and SIlGEl 22 ppm meet the TFM (Tentative Final Monograph) criterion for efficacy as hand hygiene products which specifies efficacy as a 2 loglO reduction in the indicator organism on each hand after 1st use and reduction of 3 logiO of indicator organism on each hand within 5 minutes of the 10th use. In addition, SIIGEL was more effective as a hand wash compared to sterillium and Spitader. Finally being a 'cleaning tape', SIIGEL, as a hand detergent, would be well tolerated as it eliminates the need for a bin and also does not make the user's hand sect or tilted to irritation, but rather will tend to be Irydrate the area of ​​use.

[536] HYDROGEL AS A WOUND DRESSING MATERIAL

[537] Introduction

[538] Hydrogel dressings can be used as primary dressings (amorptic and gas soaked) or as primary or secondary dressing (Plates) to control the thickness of the wound partially and totally, deep lesions (amorphous, soaked with gas). gauzes), wounds with necrosis or molting, minor burning and tissue damage by radiation.

[539] Almost all hydrogels on the market today do not have an antimicrobial agent included. This is because antibiotics and antiseptics are potentially cytotoxic and often delay wound closure.

Since the inventive silver solution is non-cytotoxic, it was decided to prepare a hydrogel using the inventive silver nanoparticles processed.

Recently a specially prepared hydrogel has been introduced for the management of radiation induced dermatitis. These dressings have a high specific heat to provide a cooling effect and will absorb at least three times in water, serum or blood.

[540] The advantages

Are calming and reduce pain

Rehydrate the wound area

Facilitate autolytic debridement 'Replace zone space (amplified, gas soaked)

Provide minimal to moderate absorption

Easily applied and removed from the wound

Can be used when infection is present

Provide a view of the injury area

[541] Disadvantages

Not usually recommended for wounds with heavy exudate.

Some require secondary dressings

Dehydrate easily if left uncovered "Some can be difficult to protect

Some can cause maceration

[542] Procedure

Λ

ΜΑ 29428Β1

[543] The hydrogel of the present invention was prepared in sheet format as a candidate wound dressing, the sheet format of the hydrogel may be interchangeably referred to as SILDERM.

[544] RESUITAS

[545] SILDERM - LOSS OF HUMIDITY

[546] Goal:

Determine the moisture loss capacity of SIIDERM.

[547] Procedure:

[548] Equipment requests:

Analytical balance.

[549] Material requests:

* Plastic container

[550] Method:

Determine the weight of an empty bin.

Place the SIIDERM sheet on the tray.

Determine the weight of the container + the SILDERM sheet.

Note this reading as t = 0 shot.

* Take the reading every 1 hr.

* Take the reading at night.

* Then a trace of a VS time diagram. Moisture loss.

Determine the percentage of moisture loss.

Results: See Table 17 below and Figure 34

Table 17: SILDERM moisture loss capacity

<td>Time (hours) time (eures)</td><td>Weight</td>

<td></td><td>68 gm</td>

<td> 1</td><td>64 gm</td>

<td> 2</td><td>60 gm</td>

<td> 3</td><td>56 gm</td>

<td> 4</td><td>51 gm</td>

<td> 5</td><td>48 gm</td>

<td> 22</td><td>40gm</td>

Conclusion: We can conclude that the SILDERM sheet can lose 30 ٥/٥ of its weight in humidity.

[551] SIIDERM TAP -HUMIDITY

[552] Goal:

حم لاك

ΜΑ 29428Β1

Determine the moisture uptake capacity of dehydrated SIIDERM.

[553] Procedure:

[554] Equipment requests:

Analytical balance.

[555] Material requests:

Cup

[556] Method:

Determine the weight of the SIIDERM sheet in grams.

Note this reading as t = zero Hrs.

Fill the cup with water.

Place the SIIDERM sheet in the cup by submerging it completely.

0 at intervals of one hour remove the drop of dry leaf and determine its weight.

Take the reading at night.

Then a plot of time VS. Moisture uptake.

"Calculate the percentage of moisture uptake of the dehydrated gel.

Results (See Table 18 below and Figure 35)

<td></td><td>Weight</td>

<td> 0</td><td>45 gm</td>

<td> 1</td><td>48 gm</td>

<td> 2</td><td>51 gm</td>

<td> 3</td><td>53 gm</td>

<td> 4</td><td>54 gm</td>

<td> 5</td><td>56 gm</td>

<td> 6</td><td>57gm</td>

<td> 22</td><td>68 gm</td>

Conclusion: The dehydrated SIIDERM sheet can absorb up to 52٥/٥ of its weight in moisture.

[557] SIIDERM - RELEASE OF MONEY

[560] Goal:

Determine the sustained release of silver nanoparticles from SIIDERM

[561] Principle:

The hydrogel dressing sheets are placed over a wound normally for 48 to 72 hours. In this situation it would be desirable to determine the antimicrobial activity of the dressing in this time period relative to the silver release.

[562] Equipment requests:

UA

Incubator. Laminar current

[563] Material requests:

Sterile nutrient agar plates, sterile cotton holders. Micropipette (Capacity 100 pl - 1000 pl) 16 hr culture. From Pseudomonas aeruginosa (wild type)

[564] Method:

Cut a 4 cm X 3 cm piece of SILDERM.

Place the Silderm in a nutrient agar plate buffered with Ps. Aeruginosa (wild type).

'' Incubate at 37 ° C for approximately 18 hrs.

'Look for the inhibition zone vertically and horizontally.

Then place the same piece of SILDERM in a nutrient agar plate recently buffered with Ps. Aeruginosa.

Incubate as above.

Repeat this procedure for a minimum of 7 days.

Results: At printing time SIIDERM a shows inhibitory activity for 3 blots as given in Table 19 below.

Table 19: SIIDERM challenge test

<td>Transfer n</td><td>Vertical inhibition</td><td>Horizontal inhibition</td>

<td>Transfer 1</td><td>52 mm</td><td>35 mm</td>

<td>Transfer 2</td><td>53 mm</td><td>35 mm</td>

<td>Transfers</td><td>51 mm</td><td>34 mm</td>

Conclusion: SILDERM hydrogel was able to exert a sustained antimicrobial activity on 3 definitions of a new inoculum every 24 hours, the additional test is in progress.

[565] The composition of the medium for the embodiments discussed above was as follows:

Nutrient agar:

Peptone 1٥.٥gm

Sodium chloride 5.0 gm

Meat extract 3.0 gm

Distilled water 900 ml

Agar 2.5 gm pH 7.2 ± 0.2

[566] In addition, it will be possible to develop formulations of SIIDERM with the following:

Collagen Collagen, the most abundant protein in the body, is fibrous and insoluble and is produced by fibroblasts. Its fibers are found in connective tissues, including skin, bone, ligaments, and cartilage. During wound healing, collagen encourages the deposition and organization of

ΜΑ 29428Β1 collagen and newly formed granulation tissue in the injury area. It also stimulates new tissue development and wound debridement, creating an environment conducive to

Maltodextrin Maltodextrin is a wound healing promoter which accelerates healing by activation and attraction of macrophage, thereby reducing infection and increased granulation.

Platelet-Derived Growth Factor (PDGF) PDGF promotes chemotactic recruitment and proliferation of cells involved in wound repair and increasing granulation tissue formation. It is mainly used to treat diabetic lower extremity neuropathic ulcers.

[567] DiSodium EDTA as ADDITIVE

[568] Disodium EDTA has been known to increase the antibacterial effect of various compounds, both natural and synthetic, by a mechanism which has been presumed to result in increased permeability of the bacterial wall, thereby facilitating the entry of antibacterial compounds.

[569] The metal chelator and internal membrane permeabilization of bacteria, ethylene tetraacetic acid (EDTA), have been shown to be able to increase the activities of various anti-microbial agents against Pseudomonas aeruginosa, addition of an inhibitory concentration of EDTA markedly reduced the MICs of cefprozil against E. coli and Serratia marcescens.

[570] It has been reported that imipenem, ceftazidime and cefepime plus 150mcg of EDTA can increase the mean diameter of zone of inhibition for p. aeruginosa. One study reported that Ethylenediaminetetraacctic acid (EDTA) influenced the susceptibility of p. aeruginosa. EDTA when used in conjunction with AgO a significantly increases the antibacterial action of the latter, so that Klesbiella pneumoniae and Staphylococcus aureus souclia resistant to 70 micrograms / ml AgNo3 are observed to become susceptible to 10 microgram / ml of this compound.

A specific composition and set of tests were designed to determine whether the silver / water compositions of the present invention would work favorably with disodium EDTA. Specifically, disodium EDTA was obtained from West Coast Laboratories in Mumbai, India. Disodium EDTA is also denoted by NEDTA (Disodium Ethylenediaminetetra Acetic Acid) and has the formula: (CI-NCHCOOHCH COONah 2ΗΟ and has a molecular weight of 372.24.

[571] the medium used for this test was: nutrient agar: (HiMedia) 1000 ml; Β.Νο. 1G115 exp. Aug 2006; Digestive peptic of animal tissue 50.00g; yeast extract 1.50 g; beef extract 1.5Og; sodium chloride 5.00g: Agar Agar type-1 25g; pH 7.4 +/- 0.2.

MICROBIAL STRAINS the 32 ppm silver / water composition alone, the 22 ppm silver / water composition alone, and the 32 ppm silver / water composition, as well as the 22 ppm silver / water composition, were added to NazEDTA. and were each tested against a control group of microorganisms, including:

Escherichia coli (Strain resistant to several drugs) of the faecal sample; Pseudomonas aeurginosa (Strain resistant to several drugs) of sputum; and

Methicillin-resistant Staphylococcus aureus (Strain resistant to several drugs) from pus in the lumbar region.

the MDR strains indicated above were obtained from p. D. Hinduja Hospital (MUMBAI, India).

Shigella flexneri (strain of lab)

Salmonella typhi (lab souclia) /

ΜΑ 29428Β1 bacterial souctia were cultured for 24 hrs. at 37٥c in nutrient agar (pH 7.4).

[572 32ppm and 22ppm dilutions with N 2EDIA were made in sterile distilled water. Each microorganism was suspended in sterile saline and diluted to 10 ^ colony forming units (cfu / ml): They were buffered on the surface of nutrient agar (pH 7.4) using cotton buffers. n sterile, the wells (10 mm in diameter) were punched with agar and 0.1 ml of the respective dilutions were delivered there. After incubation for about 24 hours at 37 ° C, all plates were examined for any zones of growth inhibition and diameters were measured in mm using a zone reader (Hi Media). The results are given in Table 20.

Results and Discussion

Table 20 Money / water + NEDTA.

<td>System</td><td>E. coli</td><td>MRSA</td><td>vs. albicans</td>

<td>Silver / water 32 ppm- ctr.</td><td>21 mm</td><td>24 mm</td><td>27 mm</td>

<td>Silver / water 32 ppm + 0.5٥ / ο Na ؛ EDTA</td><td>22 mm</td><td>29 mm</td><td>40 mm</td>

<td>Silver / water 22 ppm +</td><td>20 mm</td><td>23 mm</td><td>29 mm</td>

<td>Silver / water 22 ppm + O, 5 ° / o Na2 EDTA</td><td>22 mm</td><td>31 mm</td><td>&gt; 40 mm</td>

[573] 0.5 ppm disodium EDTA certainly increases the potency of the silver / water compositions of the present invention at all 22 and 32 ppm concentration levels.

[574] EDTA silver as a carrier alone Antibacterial

[575] A specific composition and set of tests were designed to determine whether chelated silver like silver EDTA (or AgEDTA) possesses antibacterial qualities. Specifically, commercially available silver compositions were obtained from AKZQ Nobel and Alpha Chemicals.

[576] Equipment requests:

Incubator, laminar flow

[577] Material requests:

Sterile nutrient agar plate, sterile cotton holders. Micropipette ؛ Capacity 100 pi- 1000 Ijl) of 16 hour old culture of the following strains (density a ^ pr. Is في CFU / ml) ',' Escherichia coli (wild type), Escherichia coli (MDR), Pseudomonas aeruginosa (type wild). Pseudomonas aeruginosa (MDR), Staphylococcus aureus ATCC 6538Ρ, Methicillin resistant Staphylococcus aureus.

[578] Method:

. Dab the 16 hr old crop. of the test organisms are given in a sterile nutrient agar plate.

Allow the plates to rest for 15 minutes for absorption.

After 15 minutes aseptically pierce the wells on the agar surface using a 10 mm cork borer.

Distribute 100 µl of the appropriate sample dilution into the wells. Hold for 15 minutes for pre-diffusion.

Incubate the plates at approximately 37٥c for approximately 24 hrs. and observe the results.

Measure the zone of inhibition in mm using the HiMedia zone reading.

[579] Results: See Table 21 below and Figures 36 and 37.

<td rowspan="2">Organization</td><td rowspan="2">Concentration</td><td colspan="2">Inhibition zone</td>

<td>AKZO</td><td>ALPHA</td>

<td rowspan="3">E. coli (wild type)</td><td>28 ppm</td><td>20 mm</td><td>22 mm</td>

<td>57 ppm</td><td>22 mm</td><td>24 mm</td>

<td>114 ppm</td><td>22 mm</td><td>25 mm</td>

<td rowspan="3">E. coli (MDR)</td><td>28 ppm</td><td>20 mm</td><td>19 mm</td>

<td>57 ppm</td><td>21 mm</td><td>21 mm</td>

<td>114 ppm</td><td>23 mm</td><td>22 mm</td>

<td rowspan="3">Ps. Aeruginisa (wild type)</td><td>28 ppm</td><td>21 mm</td><td>20 mm</td>

<td>57 ppm</td><td>27 mm</td><td>24 mm</td>

<td>114 ppm</td><td>28 mm</td><td>27 mm</td>

<td rowspan="3">Ps. Aeruginisa (MDR)</td><td>28 ppm</td><td>15 mm</td><td>17 mm</td>

<td>57 ppm</td><td>21 mm</td><td>20 mm</td>

<td>114 ppm</td><td>25 mm</td><td>22 mm</td>

<td rowspan="3">S. aureus (wild type)</td><td>28 ppm</td><td>16 mm</td><td>15 mm</td>

<td>57 ppm</td><td>19 mm</td><td>18 mm</td>

<td>114 ppm</td><td>22 mm</td><td>21 mm</td>

<td rowspan="3">MRSA</td><td>28 ppm</td><td>19 mm</td><td>20 mm</td>

<td>57 ppm</td><td>21 mm</td><td>22 mm</td>

<td>114 ppm</td><td>26 mm</td><td>24 mm</td>

[582] Conclusion: Silver chelates such as silver EDTA possess antibacterial efficacy.

[583] COMBINATION OF ANTIBIOTIC THERAPY

[584] Once discovered, antibiotics were sold as a miracle cure and they literally are. The infections which were fatal before the century have been tamed to only disturbances during this century. But the drug has become almost fully bypasses. Overuse, over prescribing and / or overuse of antibiotics has allowed resistant strains of bacteria to grow and again strains of bacteria threaten health and life.

[585] Some of the other factors which contribute to the development of resistance by bacteria are the use of antibiotics for agricultural purposes and as food supplements in agriculture (eg, for poultry, cattle, pig, etc. .), over-prescribing of antibiotics is believed by many to be rampant in the agricultural industry in the United States and in many foreign countries. Therapy with antibiotics in agriculture is often started even before the culture specimen is sent to the laboratory. Avian influenza (eg Η5Ν1 or HPAI) has become very resistant to antibiotics due to the massive use of antibiotics by poultry farmers in Asia. Patients also have easy access to antibiotics. Inaccurate dosages and incomplete treatment times also contribute to the development of drug resistant strains. There are important clinical ramifications to the problem of resistance, resistance of pathogenic bacteria to antibiotics has had a severe effect on the treatment of infectious diseases. Many drugs, such as penicillin, which was thought to be a prodigy drug had great potential for effective control when it was first introduced, intended only for bacteria, adapted to them and greatly reduced their applicability.

[586] The problem of antibiotic resistance is today a global problem. It is expected that some common and highly pathogenic bacteria such as Staphylococcus aureus, particularly the souclia found in hospitals, are now known to be resistant to everything except vancomycin, and are early resistant to vancomycin as well. MRSA (Methicillin Resistant Staphylococcus Aureus) and VRE (Vancomycin Resistant Enterococci) are a cause of serious nosocomial infections and often hospital wards are firm, and even destroyed, once detected.

[587] With this problem it is imperative to find an alternative as either new antibiotics replace the old ones or make the use of existing antibiotics effective. Additionally, the growing threat of multi-drug resistant bacteria is a good reason to consider silver / water compositions according to the present invention.

[588] One of the ways to treat the ever increasing resistance of bacteria to antibiotics involves the use of combination therapy, which uses at least two different antibiotics with different modes of action. Various in vitro methods are available for measuring the synergistic effects of

ΜΑ 29428Β1 combinations of antibiotics, but the results may show abnormalities when different tests are used, also this does not totally eliminate the development of drug resistance.

[589] GOALS AND OBJECTIVES

[590] This study was conducted with the following goals and objectives:

1. To determine the Multi-Drug Resistance (MDR) model of clinical isolates.

2. To determine the sensitivity of clinical isolates to the silver solutions of the present invention.

3. Determine the antibiotic combination (synergy) by the approximation disk test.

4. Determine the minimum inhibitory concentration (MIC) of the antibiotics and silvery compositions of the present invention.

5. To study the synergistic action between the antibiotics and the silver solutions of the present invention by chessboard test.

[591] MATERIALS AND METHODS

Collection of clinical isolates

The following Multi-Drug Resistant clinical isolates were collected from PD Hinduja Hospital,

Cadell road, Mahim, Mumbai- 400016, India.

Esclierichia coli. (saddle insulator)

Pseudomonas aeruginosa, (isolated from sputum)

Methicillin resistant Staphylococcus Aureus. (MRSA-isolates pus from the lumbar region)

[592] Medium, Solutions and Antibiotic discs:

[593] Middle:

“Nutritious broth.

Nutrient agar.

Muller and Hinton agar.

[594] Solutions:

Antibiotic solution.

Silver / water solution (22ppm).

The composition of the medium and the solutions used for various experiments in the study are listed in Table 26 (hereinafter).

readily available antibiotic disc of the appropriate concentration were used, the disc content for each antibiotic is listed in Table 27 (hereinafter).

Preparation of inoculum:

٩ املأ 2 MK

A loopful of pure growth of the culture was inoculated into nutrient broth and incubated overnight at about 37 ° C.

500mcl of the overnight culture was transferred to 5ml of fresh nutrient broth and incubated for 4-6 hrs at approximately 37٠c. Culture density is set at around 10 ؛ at 10 ج cfu / ml.

[595] Antibiotic sensitivity test - Kirby Bauer method:

In this method, the antibiotic-soaked discs are placed in the agar plate, previously inoculated with the bacterial suspension. The antibiotic diffuses out of the surrounding environment. There is an alogarithmic reduction in antibiotic concentration as the distance from the disc increases. A clear area around the disc indicates the organism's susceptibility to the antibiotic. Bright areas are measured in mm and compared to a standard NCCLS diagram

[596] Method:

1. P of oors sterile cotton were immersed in the broth tubes above inoculum and using the extended surface in agar plates Μ.Η. to achieve confluent growth.

2. After allowing the inoculum to absorb into the medium, the antibiotic discs were placed in the extended surface plates using the sterile forceps.

3. the plates were incubated at about 37 ° C for about 24 hours.

4. LJne clarification around discs indicates sensitivity of the organism. The diameters of the zone are recorded and interpreted according to the graphs provided by NCCIS. (See Table 27) (Koneman 5 'ed.

[597] Determination of the sensitivity of isolates by the 10 ppm agar diffusion method: this was determined by the well test method in which the isolate is a mass seeded into the agar medium and a 10 ppm silver / water solution is added to the wells (10 mm) perforated in the inoculated solid medium. The size of the inhibition zone is then noted.

[598] Method:

1. At a molten end of Muller and Hinton agar 20 ml. 0.5ml of the inoculum is added and poured into a Petri dish and solidified.

2. the wells are perforated in the agar layer.

3. the different concentrations of the silver / water compositions are then added to one of the wells.

4. The plates were incubated at about 37 ° C for about 24 hours.

5. Note the size of the inhibition zones.

[599] Determination of the antibiotic combination by disk diffusion test.

This is a simple, qualitative test to examine the interaction between the isolate and the clinical combination of the antibiotic. In this test, the antibiotic discs are placed on the agar plate inoculated by the Kirby-Bauer technique. the discs should be separated by a distance that is equal to or slightly greater than the average of the diameters of inhibition produced by each single disc, the shape of the inhibitory zone obtained will indicate the kind of interaction between the isolate and the clinical combination of the antibiotic.

[600] Method:

1. Sterile cotton holders were soaked in the inoculum broth tubes and used on the extended surface of the Μ.Η agar plates. to achieve confluent growth.

2. After allowing the inoculum to absorb into the medium, two antibiotic discs (the combination to be studied) were placed on the extended surface using sterile forceps, at an equal or slightly greater distance from the total diameters. inhibition produced by each disc alone.

ΜΑ 29428Β1

3. The plates were incubated at about 37٥c for about 24 hours.

4. The shape of the inhibitory zones would indicate the type of interaction, i.e., synergy, antagonism or indifference.

Figure 25 is a diagram which shows potential interactions in a disk diffusion assay for bacterial synergy.

[601] Specifically, Part A demonstrates additive or indifference effects; each antibiotic produces a zone of inhibition which is not affected by the adjacent one; Part B demonstrates antagonistic effects in which the areas of antibiotic celiac release are diminished in the presence of another antibiotic; and part c demonstrates two possible manifestations of synergistic interactions. On the left side an enlarged inhibitory zone occurs where the two antibiotics meet. On the right, neither antibiotic is inhibitory in its own right, but bacterial growth is inhibited where the two antibiotics shed together.

[602] Determination of the minimum inhibitory concentration (MIC) of antimicrobial agents. This is a macrodilution broth susceptibility test. Periodic dilutions of the antimicrobial agent are prepared in broth to which the standard bacterial suspension is added. At the end of the incubation period. The tubes are visually observed for growth. The lowest concentration of the antimicrobial agent which prevents obvious growth is taken as MIC

Antibiotics used:

Amikacin: Mikacin inj. (250mg) Aristo labs. Mumbai India.

Manufacturing batch no.02D054, mfd Apr. 2004.

Cefoperazone: Magnamycin inj. (250mg) Pfizer ltd, Mumbai, India batch no. 32035153Α, mfd Mar 2003

Ciprofloxacin: Cifran (200mg / ml) Ranbaxy Labs, Jaipur, India

Manufacturing batch no. 9042601, mfd Mar 2004.

[603] Method:

1. An amount of the antimicrobial agent is serially diluted within an appropriate range.

2. A tube free of antimicrobial agent serves as a growth control.

3. Each of the tubes is then inoculated with a standardized bacterial suspension and incubated at approximately 37٠c for approximately 24 hours.

4. At the end of the incubation period, the tubes were visually examined for turbidity. Turbidity indicates that bacterial growth was not inhibited by the concentration of the antimicrobial agent in the medium.

5. MIC is the low concentration of the antimicrobial agent which inhibits visible growth.

[604] The study of the synergistic effect by chessboard analysis. Chessboard analysis is one method used, when multiple antibiotics and / or multiple dilutions are to be tested. Periodic two-fold dilutions are chosen so that concentrations of one sixteenth to at least twice MIC are included, drug A is serially diluted along the y-axis, while drug B is dilute serially along the x-axis. The resulting chessboard reports each combination of the two antibiotics, from a tube that contains the highest concentration of each from the opposite angle.

Protocol:

Λ /

ΜΑ 29428Β1

E

ة m

<img file="MA29428B1_D0009.tif" />

٠g

MIC

MIC

٠ س

MIC

ت

1:16

MIC

<td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>* go C</td><td></td><td></td><td></td><td></td><td></td><td></td>

1:16 1:8 1:4 1:2 2

MIC MIC MIC MIC MIC MIC

Drug A pg / ml

Arrangement of drug dilutions in the chessboard test.

The first row and column of tubes with only one drug served to confirm the individual MIC values ​​of the test isolates.

A tube without antibiotic is a positive control.

1. In a final volume of 5ml in each tube antimicrobial drugs diluted in broth should be added to the appropriate stock solutions.

2. Add 0.1 ml of the culture suspension.

3. Incubate at 37 ° C for 24 hrs.

4. The results are indicated by drawing an isobologram obtained by joining the dots which represent all the combinations with the same effect, including the equally effective concentration of the antibiotic used alone.

[605] Calculations:

Elion et al (1954) described a method for quantifying the MIC results obtained in terms of the Fractional Inhibitory Concentration Index (FIC), defined as the total of the FIC values ​​of two drugs in the combination.

FIC index = FIC of drug A ٠ FIC of drug B.

Drug A FIC = Drug A MIC in combination with Drug B.

MIC of drug A.

An index of less than 0.5 is considered evidence of synergism; an index greater than 2.0 is evidence of antagonism. (Koneman 5 'ed. 1997)

Fig 26 shows a checkerboard titration for antimicrobial synergy.

١ ك

Each box represents a tube. Increases in antibiotic A concentrations are distributed along the horizontal axis, and increases in antibiotic B along the vertical axis, the hatched boxes indicate bacterial growth. In a list A the antibiotics demonstrate the additive effect: the isobologram in the right is a straight line. List B represents the synergism where the isobologram is a concave curve, list c shows the antagonistic results with the convex curve.

Determination of the antibiotic sensitivity model by the Kirby-Bauer method.

1٩ ملأ 2 MN

Table: 22 Antibiograms of the isolates used for the study, (areas in mm.)

<td rowspan="2">Antibiotics</td><td colspan="3">Zone size (mm) of organisms</td><td rowspan="9">Solution: no inhibition Determination of the sensitivity of clinical isolates to the ASAP-agar fusion method</td>

<td>ئ</td><td>Ps.</td><td>MRSA</td>

<td>Amikacin</td><td> 20</td><td> 9</td><td> 22</td>

<td>Ciprofloxacin</td><td> -</td><td></td><td> 20</td>

<td>Kanamycin</td><td> 14</td><td></td><td> -</td>

<td>Gentamycin</td><td> 19</td><td></td><td> -</td>

<td>Tetracycline</td><td> -</td><td></td><td> 27</td>

<td>Nalidixic acid</td><td> -</td><td></td><td> -</td>

<td>Cefoperazone</td><td></td><td> 14</td><td> 23</td>

<td>Ceftazidime</td><td> 10</td><td> 16</td><td> -</td><td></td>

<td>Chloramphenicol</td><td> -</td><td></td><td> -</td><td></td>

<td rowspan="2">ASAP conc ppm</td><td colspan="3">Size of the area in mm of the organisms</td><td rowspan="7"></td>

<td>Ec</td><td>Pseudomonas</td><td>MRSA</td>

<td> 32</td><td> 16</td><td> 16</td><td> 13</td>

<td> 16</td><td> 15</td><td> 14</td><td> 11</td>

<td> 8</td><td> 11</td><td> 11</td><td></td>

<td> 4</td><td> -</td><td> -</td><td> ٠</td>

<td> 2</td><td> -</td><td> -</td><td> -</td>

Solution: - No inhibition

See figure 27 for photos

[607] Determination of the antibiotic combination by the approximation disk test.

Various antibiotic combinations controlled the synergistic or additive effect on the isolates, inhibitory areas indicative of possible synergy were observed only in case of MRSA for the combination of amikacin with Cefoperazone and amikacin with tetracycline. (See figure 28). Gn did not observe any inhibitory zones suggestive of the synergistic combination in the case of the other two isolates i.e. E.coli and pseudomonas, (see Figures 29 and 30).

[608] Determination of the minimum inhibitory concentration of antibiotics.

the MIC of the antibiotics which showed inhibitory areas suggestive of possible synergy was determined.

Table 23:

MIC of Amikacin

Stock: 125mcg / ml

Thinner: Nutrient Draft

MIC of Cefoperazone Stock: 100 mcg / ml Diluent: Nutrient draft Culture: MRSA

Solution: +: growth

No growth the MIC of amikacin for MRSA was found to be 0.8mca / ml.

<td>Tube n ”</td><td>Conc. Mcg / ml</td><td>Growth</td>

<td> 1</td><td> 0,2</td><td> +</td>

<td> 2</td><td> 0,4</td><td></td>

<td> 3</td><td> 0,6</td><td> +</td>

<td> 4</td><td> 0,8 1</td><td> -</td>

(\ /

٦ هه 2 مو 2 MA

<td> 5</td><td> 1</td><td></td>

<td> 6</td><td> 2</td><td></td>

<td> ٦</td><td> 3</td><td></td>

<td> 8</td><td> 4</td><td></td>

<td> 9</td><td> 5</td><td></td>

<td> 10</td><td>+ ve</td><td></td>

<td> 11</td><td>-ve</td><td> -</td>

Solution: ٠: growth

No growth

The MIC of Cefoperazone for MRSA was found to be 10mc٩ / ml.

MIC silver / water

Stock: 20 ppm silver / water solution

<td>Tube n</td><td></td><td>Growth</td>

<td> 1</td><td> 5</td><td> +</td>

<td> 2</td><td> 10</td><td></td>

<td> 3</td><td> 15</td><td></td>

<td> 4</td><td> 20</td><td></td>

<td> 5</td><td> 25</td><td></td>

<td> 6</td><td> 30</td><td></td>

<td> 7</td><td> 35</td><td></td>

<td> 8</td><td> 40</td><td></td>

<td> 9</td><td> 45</td><td></td>

<td> 10</td><td> 50</td><td></td>

<td> 11</td><td>+ ve</td><td> +</td>

<td> 12</td><td>-ve</td><td> -</td>

Thinner: Nutrient broth

Culture: MRSA

Table: 23a

<td>Tube n '</td><td></td><td>Growth</td>

<td> 1</td><td> 1</td><td></td>

<td> 2</td><td> 2</td><td> +</td>

<td> 3</td><td> 3</td><td> +</td>

<td> 4</td><td> 4</td><td></td>

<td> 5</td><td> 5</td><td> +</td>

<td> 6</td><td> 6</td><td> +</td>

<td> 7</td><td> 7</td><td> +</td>

<td> 8</td><td> 8</td><td> +</td>

<td> 9</td><td> 9</td><td> -</td>

<td> 10</td><td> 10</td><td> -</td>

<td> 11</td><td>e</td><td> +</td>

<td> 12</td><td>- ve</td><td></td>

Solution: +: growth.

No growth, the MIC of silver / water for MRSA was found to be 8ppm. MIC silver / water Stock: 20 ppm Diluent: nutrient broth

Culture: E. col ؛

رك

ΜΑ 29428Β1

Tahloan- 24

<td>Tube n</td><td></td><td>Growth</td>

<td> 1</td><td> 1</td><td> +</td>

<td> 2</td><td> 2</td><td> +</td>

<td> 3</td><td> 3</td><td></td>

<td> 4</td><td> 4</td><td> -</td>

<td> 5</td><td> 5</td><td></td>

<td> 6</td><td> 6</td><td></td>

<td> ٦</td><td> ٦</td><td></td>

<td> 8</td><td> 8</td><td></td>

<td> 9</td><td>Q</td><td></td>

<td> 10</td><td>٠ ve</td><td> +</td>

<td> 11</td><td>-ve</td><td> -</td>

Solution: +: growth. No growth. The silver / water MIC for E.coli was found to be 3ppm.

MIC silver / water

Stock: 20ppm silver / water

Thinner: Nutrient broth

Culture: Pseudomonas

Table: 25

<td>Tube n</td><td></td><td>Growth</td>

<td> 1</td><td> 1</td><td> ٠</td>

<td> 2</td><td> 2</td><td></td>

<td> 3</td><td> 3</td><td></td>

<td> 4</td><td> 4</td><td></td>

<td> 5</td><td> 5</td><td></td>

<td> 6</td><td> 6</td><td></td>

<td> ٦</td><td>T</td><td></td>

<td> 8</td><td> 8</td><td></td>

<td> 9</td><td> 9</td><td></td>

<td> 10</td><td>+ ve</td><td> +</td>

<td> 11</td><td>- ve</td><td> -</td>

Solution: +: growth. No growth. The silver / water MIC for E. coli was found to be 3ppm.

Study of the synergistic action by the chessboard test.

I. Combination of Amikacin and silver / water.

MIC of Amikacin = 0.8mcg / ml.

MIC of silver / water = 8ppm.

Culture: MRSA

كلم

ΜΑ 29428Β1

1.6

0.8

0.4

0.2

0.1

0.05

<td>I</td><td> -</td><td></td><td></td><td> -</td><td> ٠</td><td> -</td>

<td></td><td> -</td><td></td><td></td><td></td><td></td><td></td>

<td> +</td><td> -</td><td> ٠</td><td></td><td></td><td></td><td> -</td>

<td> ++</td><td> ٠</td><td></td><td></td><td></td><td> -</td><td></td>

<td> ++</td><td></td><td>ا</td><td> ٠'</td><td></td><td></td><td></td>

<td>باه</td><td> ++</td><td> ٠</td><td> ٠'</td><td> *</td><td> ٠</td><td></td>

<td>+ νβ c</td><td>هاب</td><td>٠ ه</td><td>حض ٠ ه</td><td> +</td><td></td><td></td>

0.5 I 2 4 8 12

<img file="MA29428B1_D0010.tif" />

Solution: +: growth. -: no growth.

The synergistic concentration for MRSA was found to be 0.05 mcg / ml of amikacin and Ippm of silver / water of the present invention.

Calculation of the FIC index:

Amikacin FIC = MIC of Amikacin in combination

MIC of Amikacin alone.

= 0,05/0.8 = 0,0625.

ASAP FIC = Spider / Water MIC in Combination

MIC silver / water only.

= 1/8 = 0,125.

FIC index = FIC of Amikacin + FIC of silver / water = 0.0625 + 0.125 = 0.1875.

The FIC index is indicative of synergy between Amikacin and silver / water

[609] II. Combination of Cefoperazone and silver / water.

MIC of Cefoperazone = 10mcg / ml.

MIC of silver / water = 8ppm.

Culture: MRSA

AT

٩ ^ 2 ملأ 2 MA

2.5

1.25

0.525

<td> ٠</td><td> ٠</td><td> ٠</td><td></td><td> ٠</td><td colspan="2">ا</td>

<td></td><td></td><td>ا</td><td></td><td></td><td colspan="2">-أ-</td>

<td> +</td><td> ٠</td><td> ٠</td><td></td><td></td><td></td><td></td>

<td> ++</td><td> -</td><td> ٠</td><td> ٠</td><td></td><td></td><td></td>

<td> *+</td><td> '٠</td><td> ٠</td><td></td><td></td><td></td><td></td>

<td>"ؤ -</td><td> ++</td><td> *</td><td> ٠</td><td></td><td></td><td></td>

<td>+ ٧e C</td><td> ++</td><td> ++</td><td> ++</td><td>ه</td><td></td><td></td>

12 8 4 2 1 0.5 ه

Solution: t: growth. no growth.

The synergistic concentration of MRSA was found to be 0.625mcg / ml of Cefoperazone and 1 ppm of silver / water

Calculation of the FIC index:

Cefoperazone FIC = Cefoperazone MIC in combination

MIC of Cefoperazone alone.

= 0,625/10 = 0,0625.

FIC of ASAP = MIC of silver / water in combination

MIC of silver / water alone.

= 1/8 = 0,125.

FIC index = FIC of Amikacin + FIC of silver / water = 0.0625 t 0.125 = 0.1875.

FIC index is indicative of synergy between Cefoperazone and silver / water.

[610] III. Combination of Cefoperazone and Amikacin.

MIC of Cefoperazone = 10mcg / ml.

MIC of Amikacin = 8ppm.

Culture: MRSA

ΜΑ 29428Β1

<img file="MA29428B1_D0011.tif" />

<td> ٠</td><td> -</td><td> -</td><td colspan="2"></td><td> *</td><td> .٠</td>

<td></td><td></td><td></td><td colspan="2"> ’1'</td><td> ٠</td><td> ٠'</td>

<td> +</td><td></td><td></td><td colspan="2">ا</td><td>ا</td><td> ٠</td>

<td>f +</td><td></td><td></td><td></td><td></td><td>ا</td><td> '٠</td>

<td>ه'ه</td><td></td><td>ه ٠</td><td></td><td> -</td><td> ٠</td><td> -</td>

<td>بب</td><td colspan="2">ه + ا به</td><td>٠ هه</td><td>I</td><td> ٠</td><td></td>

<td>+ ve C</td><td colspan="2">+ ♦ إ ب +</td><td> ++</td><td> ++</td><td> ٠</td><td> ٠</td>

0.06 0.1 0.2

<img file="MA29428B1_D0012.tif" />

Solution: -t-: growth. no growth.

The additive concentration of Cefoperazone was found to be 1.25 of Amikacin was found to be 0.4.

Calculation of the FIC index:

FIC of Cefoperazone = MIC of Cefoperazone in combination MIC of Cefoperazone alone. = 1.25 / 10 = 0.125

FIC of Amikacin = MIC of Amikacin in combination

MIC of Amikacin alone = 0.4 / 0.8 = 0.5 the FIC index = MIC of Amikacin ٠ FIC of Cefoperazone = 0.125 + 0.5 = 0.625 the FIC index is indicative of the addition between Cefoperazone and money / water.

[611] DISCUSSION

[612] In this example, the three clinical isolates collected from PD Hinduja Hospital, Mumbai, India, the gregative isolates showed resistance to older antibiotics such as ampicillin, tetracycline, kanamycin, and older quinolones. such as nalidixic acid as well as the third generation cephalosporins - ceftazidime and cefoperazone. the clinical isolates of pseudomonas used for the study were also resistant to recent ciprofloxacin and to the semi-synthetic aminoglycoside, amikacin. The gram-positive isolate of MRSA was also resistant to older antibiotics and also to third generation cephalosporins such as ceftazidime.

[613] The study of their sensitivity to the silvery compositions of the present invention proved that the gram-negative isolates were readily sensitive to approximately 3ppm silvery solutions and the MRSA isolate was found to be inhibited by the. 8ppm silver solutions as determined by the diffusion method and macrodilution broth.

[614] The interaction of the two antibiotics in combination with the isolates was determined by the disk diffusion method, which indicated synergistic results between cefopoerazone and amikacin against MRSA. A chess board analysis was performed to confirm this. No addition or synergy between antibiotics was observed for gram-negative isolates by the disk diffusion assay.

[615] The chessboard analysis was performed and the FIC index of the two antibiotics was found to be 0.625 thus indicating the addition and no synergy of the combination of amikacin and cefoperazone.

AT/

ΜΑ 29428Β1

[616] The chessboard analysis was also performed to study the combination of silver / water solutions with amikacin and also with cefoperazone. The results proved that in the presence of inventive silver / water compositions, the effective concentration of the antibiotic was reduced by about four times. The FIC index of these combinations was found to be 0.1875 in each case indicating the synergy for the combination of silver / water with amikacin and silver / water with cefoperazone.

[617] The results of the study indicate that in the above clinical isolates of MDR the antibiotic dose could be considerably reduced in the presence of silver / water, which was not observed to be the case in the antibiotic combination. .

[618] These results show that the inventive silver / water compositions will have an important role to play in antibiotic combination therapy, particularly against strains resistant to multiple drugs.

TABLE 26

1. Nutrient broth:

Peptone 10.0 gm

Sodium chloride 5.0 gm

Meat extract 3.0 gm

Dextrose 5.0 gm

Phenol red (indicator) 0.001٥ / ٥

Distilled water 900 ml

2. Nutrient agar:

Peptone lO.Ogm

Sodium chloride 5.0 gm

Meat extract 3.0 gm

Distilled water 900ml

Agar 2.0٥ / ٥pH 7.2

3. Muller and Hinton agar

Acid hydrolyzate of casein 29,0gm

Beef starch 10,0gm

Amodin potato 2,5gm

1.2٥ / ٠ agar

Distilled water 1000ml

PH 7.6

TABLE 27

ΜΑ 29428Β1

Interpretation of the diameter of the zone (Document NCCLS, 1988)

<td rowspan="2">Antibiotics</td><td>Disk</td><td colspan="3">Zone diameter in mm</td>

<td>Conc (mcg)</td><td>Resistant</td><td>Intermediate</td><td>Sensitive</td>

<td>Amikacin (AK)</td><td> 30</td><td>14 ن</td><td>15 to 16</td><td>17 ة</td>

<td>Ciprofloxacin (RC)</td><td> 5</td><td> ٤15</td><td>16 to 20</td><td> ٦١&lt;</td>

<td>Kanamycin</td><td> 30</td><td>13 ة</td><td>14 to 17</td><td>18 ح</td>

<td>Gentamycin (GM)</td><td> 10</td><td>12 كا</td><td>13 to 14</td><td> &gt;-1*</td>

<td>Tetracycline (TE)</td><td> 30</td><td>14 كا</td><td>15 to 18</td><td>19 ة</td>

<td>Nalidixic acid (NA)</td><td> 30</td><td>14 كا</td><td>14 to 18</td><td>19 ح</td>

<td>Cefoperazone (CP)</td><td> 75</td><td>15 كا</td><td>16 to 20</td><td> ٦١&lt;</td>

<td>Cettazidime (FG)</td><td> 30</td><td>12 كا</td><td>13 to 17</td><td>18 ج</td>

[619] COMBINATION OF GENTAMYCIN AND SILVER / WATER COMPOSITIONS AS A POWDER DUSTING YOUR PIAIES

[620] INTRODUCTION

[621] Wound dusting powders are formulations used for the prevention or treatment of external bacterial infections of wounds, burns, skin ulcers or abscesses after incision.

[622] Powders intended for wounds are normally broad spectrum antibiotic / antiseptic preparations, the use of such powders does not exclude, where appropriate, concomitant therapy with antibiotics.

[623] Most of the wound products available on the market today are based on Povidone - iodine. Povidone - iodine is strongly cytotoxic in open wounds and has been specifically contraindicated in diabetic wounds. In addition, the iodine sublimates and should be reapplied approximately every 6 to 8 hours.

[624] Another potential field of application is in the veterinary sector. Pets often incur cuts, abrasions, and wounds either due to scratching to eliminate parasites as well as encounter with other animals. A mild but broad antimicrobial spectrum would be useful in this application.

[625] It was decided to formulate a fingerprint wound powder composed of a slow release preparation combining Gentamycin and the treated silver nanoparticles of the present intention. A talc-based preparation containing approximately 200 silver nanoparticles and approximately 100 ppm of Gentamycin hereinafter referred to as SILDUST

[626] RESULTS

[627] SILDUST-SENSITIVITY

[628] Aim: To determine the sensitivity of SILDUST and its constituents against microorganisms.

[629] Procedure:

[630] Equipment requests:

Incubator, * Laminar flow

[631] Material requests:

Nutrient agar plates, sterile cotton holders. Micropipette (Capacity 100 pl - 1000 pl), 16 hr. Aged culture of the following strains (Appr. density is 0 CFU / ml) Escherichia coli (^ DR) i Pseudomonas aeruginosa (MDR), Methicillin resistant Staphylococcus aureus.

[632] Method:

Surface area of ​​0.1 ml culture on the nutrient agar surface using sterile cotton gates. Hold aside for 15 minutes.

After 15 minutes aseptically pierce the wells on the agar surface using a 10 mm cork borer.

Add 10 mg of SIIDUST (200 ppm silver talc t 100 ppm Gentamycin) into a well.

Add 100 µl of 100 ppm Gentamycin to another well. Also introduce 200 ppm of silver talc + 100 µl of distilled water. Both serving as witnesses.

Incubate the plates at about 37٥c for about 24 hours and observe.

Measure the inhibition zone in mm using the HiMedia zone reader.

[633] Results: As given in Table 28 below and Figure 38.

Table 28: Sensitivity of SIIDUST

<td rowspan="2">Culture</td><td></td>

<td>100 ppm gentamycin</td>

<td>Escherichia coli (MDR)</td><td>24 mm</td>

<td colspan="2">Inhibition zone</td>

<td>200 ppm ASAPTalc</td><td>SILDUST *</td>

<td>17 mm</td><td>26 mm</td>

SIIDUST * ٠ 200 ppm ASAP Talc + 100 ppm Gentamycin

[634] Conclusion: There is a synergistic activity observed for SILDUST (containing 200 ppm of silver talc and 100 ppm of Gentamycin).

[635] Solution:

SIIDUST 1 - 200 ppm silver talc Talc + 50 ppm Gentamycin SILDUST 2 - 200 ppm silver talc + 100 ppm Gentamycin

[636] SILDUST - ANTIBACTERIAL ACTIVITY

[637] Aim: To determine the kill time of SILDUST against microorganisms.

[638] Procedure:

[639] Required Equipment:

Incubator, laminar flow, weighing scale.

[640] Material requested:

Sterile Red Phenol Dextrose Broth. Micropipette, 16 hr old culture. of the following strains (approximate density is 10٥ CFU / ml) Escherichia coli (MDR), Pseudomonas aeruginosa (MDR), Methicillin resistant Staphylococcus aureus.

[641] Method:

Prepare 5 ml of aliquot containing 2g of SIIDUST in a sterile test tube.

/

MA 22٩٩٩ مو

Inoculate ٥.1 ml of culture in the above solution. Vortex perfectly.

At time intervals of 0, 5, 10 .... 50 minutes, inoculate the sample loopful in a test in 5 ml of sterile Phenol Red Dextrose Broth. Vortex perfectly.

Incubate at about 37 ٥c for about 24 hours.

Watch the growth.

For the negative control, loopful of uninoculated SIIDUST was suspended in 5 ml of sterile phenol red dextrose broth and incubated at about 37 ° C for about 24 hours.

For the positive control, loopful of the culture in a test was inoculated into 5 ml of sterile Phenol Red Dextrose Broth and incubated at about 37٥C for about 24 hours.

[642] Results See tables 29, 30, and 31

Table 29 Escherichia coli (MDR)

<td>Time interval (minutes)</td><td>100 ppm gentamycin</td><td>200 ppm ASAPTalc</td><td>SILDUST *</td><td>Wokadine *</td>

<td> 0</td><td></td><td> +</td><td> +</td><td></td>

<td> 5</td><td></td><td> +</td><td> +</td><td></td>

<td> 10</td><td> +</td><td> +</td><td> +</td><td></td>

<td> 15</td><td> +</td><td> +</td><td> +</td><td></td>

<td> 20</td><td> +</td><td> +</td><td></td><td></td>

<td> 25</td><td> +</td><td> +</td><td>ه</td><td></td>

<td> 30</td><td></td><td> +</td><td> +</td><td></td>

<td> 35</td><td> +</td><td> +</td><td> +</td><td></td>

<td> 40</td><td> +</td><td> +</td><td></td><td></td>

<td> 45</td><td> +</td><td> +</td><td> +</td><td></td>

<td> 50</td><td> +</td><td> +</td><td></td><td></td>

<td>Positive control</td><td> +</td><td> +</td><td></td><td></td>

<td>Negative control</td><td> -</td><td></td><td> -</td><td> -</td>

SILDUST * 200 ppm ASAP Talc + 100 ppm Gentamycin Wokadine *&gt; 200 ppm of available iodine Solution:

t Growth

- No growth

[643] Conclusion: The combination shows synergistic activity. The tube with WOKADINE (discussed below at the end of the example) turned brown within seconds on adding the powder to the medium due to the release of iodine. However WOKADINE shows faster killing, such that high cytotoxicity is undesirable for wound healing.

[644] Table 30 Pseudomonas aeruginosa (MDR)

<td>Time interval (minutes)</td><td>100 ppm gentamycin</td><td>200 ppm ASAPTalc</td><td>SILDUST *</td><td>Wokadine *</td>

<td> 0</td><td></td><td> +</td><td></td><td></td>

<td> 5</td><td> +</td><td> +</td><td> +</td><td></td>

<td> 10</td><td></td><td> +</td><td> ٠</td><td></td>

<td> 15</td><td> +</td><td> +</td><td>t</td><td></td>

<td> 20</td><td> +</td><td> +</td><td> +</td><td></td>

<td> 25</td><td> +</td><td> +</td><td> ٠</td><td></td>

<td> 30</td><td> +</td><td> +</td><td> +</td><td></td>

<td> 35</td><td> +</td><td> +</td><td> +</td><td></td>

<td> 40</td><td> ٠</td><td> +</td><td> +</td><td></td>

<td> 45</td><td></td><td> +</td><td> -</td><td></td>

<td> 50</td><td> -</td><td></td><td></td><td></td>

<td>Positive control</td><td> +</td><td> +</td><td> +</td><td> +</td>

<td>Negative control</td><td> -</td><td> -</td><td> -</td><td> -</td>

SILDUST * 200 ppm ASAP Talc ٠ 100 ppm Gentamycin Wokadine *&gt; 200 ppm of available iodine

Λ

ΜΑ 29428Β1

Solution:

+ Growth

- No growth

[645] Conclusion: the combination shows synergistic activity.

[646] Table 31 MRSA

<td>Time interval (minutes)</td><td>100 ppm gentamycin</td><td>200 ppm ASAPTalc</td><td>SILDUST *</td><td>Wokadine *</td>

<td> 0</td><td> +</td><td>t</td><td></td><td></td>

<td> 5</td><td> +</td><td> +</td><td> +</td><td></td>

<td> 10</td><td> +</td><td> +</td><td></td><td></td>

<td> 15</td><td></td><td> +</td><td></td><td></td>

<td> 20</td><td></td><td> +</td><td></td><td></td>

<td> 25</td><td></td><td></td><td></td><td></td>

<td> 30</td><td></td><td></td><td></td><td></td>

<td> 35</td><td></td><td></td><td></td><td></td>

<td> 40</td><td></td><td></td><td></td><td></td>

<td> 45</td><td></td><td></td><td></td><td></td>

<td> 50</td><td></td><td></td><td></td><td></td>

<td>Positive control</td><td> +</td><td> +</td><td></td><td>t</td>

<td>Negative control</td><td> -</td><td> -</td><td> -</td><td></td>

SIIDUST * 200 ppm ASAP Talc 100 ppm Gentamycin Wokadine *&gt; 200 ppm of available iodine

Solution:

+ Growth

- No growth

[647] Conclusion: The combination shows synergistic activity.

[648] SIIDUST- ANTIBACTERIAL ACTIVITY

[649] Aim: To determine the susceptibility of the host bacteriophage to SIIDUST.

[650] Principle; An appropriate dilution must be achieved to remove the false positive test due to the death of the host by SIIDUST.

[651] Procedure

[652] Principle:

Even the bacteriophage and the host Escherichia are used as a detection system.

the concentration of silver in SIIDUST must be neutralized by dilution so as not to kill the host. The experimental aliquots were prepared as follows;

1. Ballast - Phage + SIIDUST 2. Witness - Phage t Saline

[653] Equipment requests:

Weigh scale, laminar air flow unit. Incubator.

[654] Material Request:

Petri dishes, Marker, Spatula, Micropipette.

[655] Method:

Prepare 2.5 ml of aliquot containing approximately 1 gm of SILDUST (which does not show bactericidal action) and Saline in sterile test tubes.

ΜΚ

Add to each approximately 0.1 ml of phage lysate (approx. 10١٥ infectious phage particles per ml).

Mix well in a vortex blender and incubate at approximately 37٥c.

* At t = 0, 1 and hourly intervals, then remove 0.5 ml of the aliquots and dilute to the pilot dilution of SILDUST which does not show bactericidal action.

Stain this dilution on a newly prepared confluence of the host's tpis. This should be done for the test as well as for the witnesses.

Incubate the plate at approximately 37٠c for approximately 24 hours.

Mix 0.1 ml of this dilution with 0.5 ml of the exponentially growing host and incubate at about 37 ° C for about 15 minutes.

Add 7 ml of the molten soft agar to the dilution. Vortex completely and cover in a St. nutrient agar plate.

Incubate the plate at approximately 37 ° C for approximately 24 hours.

Check the plates on the taps and list the plate forming units on the cover.

[656] Results: See table 32

Table 32 - SILDUST Pilot

<td>Dilutions</td><td>Results</td>

<td>ï</td><td> ٠</td>

<td>î</td><td> -</td>

<td> 10’3</td><td> -</td>

<td> 0</td><td> -</td>

Solution:

+ ٠ Presence of active phage particles.

- ٠ Absence of phage particles.

[657] SILDUST-ACTIVITE ANTIVIRAI

[658]: To determine the antiviral activity of SILDUST using a bacteriophage detection system.

[659] Procedure: The same as SILDUST “ANTIBACTERIAL ACTIVITY, Part 2

[660] Results: As for tables 33 and 34

[661] Table 33 SIIDUSI kill time

<td>لللآ</td><td>Saline</td><td>SILDUST *</td>

<td> 0</td><td> ٠</td><td> +</td>

<td> 1</td><td> +</td><td> +</td>

<td> 2</td><td></td><td> -</td>

<td> 3</td><td> ٠</td><td> -</td>

SILDUST * * 200 ppm ASAP Talc + 100 ppm Gentamycin

Solution:

AT /

ΜΑ 29428Β1 + »Presence of active bending particles.

- »Absence of bending particles

[662] Table 34 Enumeration of phages

<td>Time intervals (hours)</td><td>Saline (pfu / ml)</td><td>SIIDUST * (pfu / ml)</td>

<td> 0</td><td>TNTC</td><td>1.15xio٥</td>

<td> 1</td><td>TNTC</td><td>1.0 X 10 *</td>

<td> 2</td><td>TNTC</td><td>3.0x10®</td>

<td> 3</td><td>TNTC</td><td>None</td>

SILDUST * ٠ 200 ppm ASAP Talc + 100 ppm Gentamycin

Solution:

TNTC quite a few to count.

pfu / ml - Titer of infectious folding particles

[663] Conclusion: SIIDUST was found to show bactericidal activity against the host culture at a dilution of 10 ”2. antiviral activity was verified at the same dilution of SILDUST and found to be effective. The plaque forming units were found to decrease from 0 to zero within 3 hrs. showing that SILDUST could probably have activity against animal viruses as well.

the following composition was used in the experiments immediately above herein

[664] Composition of the medium

Nutrient agar:

Peptone 10.0 gm sodium chloride 5.0 gm

Meat extract 3.0 gm

Distilled water 900 ml

Agar 2.5 gm pH 7.2 ± 02

Phenol Red Dextrose Draft:

Proteose peptone 10.00 g / lt

Beef extract 1.00 g / lt

Sodium chloride 5.0 g / lt

Dextrose 5.0 g / lt

Phenol red 0.018 g / lt pH 7.4 ± 0.2 soft agar:

agar 1.0 ٥/٥

Saline:

0.9% sodium chloride

WOKADINE

Mfg. Lie. No .: AD / 200-A

Manufacturing batch No .: WNR 5008

Date of: March 2005

Mk 2S42SB٩

Expiration date: March 2008

Active ingredients:

Iodine Povidone IP 5٥/٥ w / w

Mfgd. by:

Navketan Research and Lab. Ltd.

[665] ADDITIONS OF SILVER / WATER TO THE POVIDONE DIODE 10% SOLUTION

[666] Another example of an additive which works favorably with the silver / water compositions of the present invention is iodine Povidone. Iodine is a well-known prophylaxis in medicine for the treatment of a wide range of pathogenic microbes. Iodine is commercially available in various concentrations, but generally the concentration employed, and preferred, is 10%. In this preferred embodiment of the invention, a synergistic combination has about 25 to 50٥ / ο by volume substitution of the silver / water mixture by replacing the 10% iodine solution. While some reactions between the silver / water mixture and iodine are possible, it turns out from experimental results that the synergistic combination of silver / water with iodine povidone can work as a topical disinfectant (e.g. , ointment) and / or as a prophylaxis against infection in cuts, burns and / or scrapes, etc.

[667] Specifically, the synergistic activity of the 32 ppm silver / water compositions combined with varying percentages of iodine ovidone (PI) was studied against numerous bacteria. Test methods and results follow. It can be concluded from these results that by adding these two materials together, a synergistic relationship exists. This synergism can be used to result in an excellent topical disinfectant.

[668] The following claims are thus to be understood to include what is specifically illustrated and described above, what is conceptually equivalent, what can obviously be substituted and also what essentially incorporates the essential idea of ​​the invention. Those skilled in the art will appreciate that various adaptations and modifications of the preferred embodiment just described can be configured without departing from the scope of the invention. The illustrated embodiment has been determined for the purposes of the example only and this should not be taken as a limitation of the invention. Therefore, it should be understood that, within the scope of the appended claims, the invention may be practiced other than as specifically described above.

ΜΑ 29428Β1