OA17317A - Antimicrobial composition including at least one or more aggregation(s) silver particles. - Google Patents

Antimicrobial composition including at least one or more aggregation(s) silver particles. Download PDF

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Publication number
OA17317A
OA17317A OA1201300384 OA17317A OA 17317 A OA17317 A OA 17317A OA 1201300384 OA1201300384 OA 1201300384 OA 17317 A OA17317 A OA 17317A
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OAPI
Prior art keywords
silver
antimicrobial
aggregations
antimicrobial composition
infections
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OA1201300384
Inventor
Robert Hutchings
Original Assignee
OOSTERLAAK Nei
Robert Hutchings
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Publication of OA17317A publication Critical patent/OA17317A/en

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Abstract

The present invention relates to an antimicrobial composition having antimicrobial properties and activity towards, inter alia, tuberculosis (TB), malaria and cancer. More particularly, but not exclusively, the present invention relates to an antimicrobial composition, having antimicrobial activity, useful in the treatment and prevention of infections, diseases and/or disorders. The present invention further relates to processes for preparing the antimicrobial composition and the use of the said composition in the manufacture of antimicrobial preparations for the treatment and/or prevention of infections, diseases and/or disorders.

Description

Antimicrobial Composition
Technical Field
The présent invention relates to an antimicrobial composition having antimicrobial properties and activity towards, inter alia, tuberculosîs (TB), malaria and cancer. More particularly, but not exdusively, the présent invention relates to an antimicrobial composition useful in the treatment and prévention of infections, diseases and/or disorders. The présent invention further relates to processes for preparing the antimicrobial composition and the use of the 10 said composition in the manufacture of antimicrobial préparations for the treatment and/or prévention of infections, diseases and/or disorders.
Background to the Invention
Throughout history, silver has been regarded by many cultures as a versatile healing tool. Records showed that Hippocrates recognized the rôle of silver in the prévention of disease and that the Romans stored wine in silver vessefs to prevent spoilage.
It is reported that silver exhibîts a broad spectrum of antimicrobial activity in vitro. A possible 20 mechanism responsable for such antimicrobial activity involves the binding of silver to microbial DNA thereby preventing bacterial réplication, and the binding of silver to the sulfhydryi groups of the metabolicenzymes ofthe bacterial électron transport chain, thereby causing their inactivation. It is also considered a possible mechanism by the Inventor that silver forms a ligand attachment with the microbial cell surface, thereby blockîng cell 25 transport mechanisms causing inactivation of the microbe.
With the development of mass produced antibiotics, interest in silver as an antimicrobial médiane dedined. The widespread use and frequent over-prescribing of antibiotics has led to an increasing ina'dence of microbes acquiring drug-resistance to current antibiotics. The 30 emergence and spread of antibiotic résistance is an alarming concem in clinical practice.
In addition to silver having broad antimicrobial activities, silver has few, if any, known résistant microbial, in particular, bacterial strains.
The art teaches of a wide range of commercially and historically available silver-based antimicrobial products, certain of which are discussed herein below.
When used in ionic form, preferred silver salts include but are not limited to silver nitrate, silver acetate, silver citrate, silver oxide, and/or silver carbonate. Dilute solutions of silver nitrate are known in the art to hâve antiseptie properties. One of the earliest uses of silver nitrate was in the préparation of infant eye drops in 1881. The use of silver nitrate in this application was highly instrumental for the prophylaxis of gonococcal ophthalmia in newboms. Whilst eye infections and blindness of newboms was reduced by this method, incorrect dosage could cause damage to the eye in extreme cases.
Thus, despite the bénéficiai use of silver nitrate, several notable disadvantages are assodated therewith. The principal disadvantage of silver nitrate is its toxic and corrosive properties. While short term exposure to the chemical at low concentrations will not produce immédiate or even any side effects other than staining of the skin, long-term exposure, at high concentrations, can cause permanent blue-grey staining of the eyes, mouth, throat and skin, (argyria) and may cause eye damage whilst being extremely toxic to the human body.
A further silver-based product that is known and used in the art is silver sulfadiazine. Silver sulfadiazine is routinely used as an adjunct in the prévention and treatment of infection in bum victims (see U.S. Patent Number 3,761,590 to Fox, incorporated herein by référencé). Silver sulfadiazine has been assodated with necrosîs of the skin. In addition hereto, sulfadiazine may accumulate in patients with impaired hepatic and rénal function. Moreover, patients allergie to sulfa agents may exhibit cross-hypersensitivity with silver sulfadiazine.
Colloïdal silver is widely known and used in the art. A colloïdal system consists of two separate phases, namely a dispersed phase and a continuous phase. Colloïdal silver comprises a dispersed phase of microscopie silver partides in a continuous phase of water to form a stable suspension of microscopie silver partides which are dispersed evenly throughout the solution. Said colloïdal silver particles carry the same electrical charge (either positive or négative), known as a zêta potentlal. In terms of the zêta potential, the adjacent, similarly charged partides présent in the colloid repel one another and in this way, the colloïdal silver particles resist aggregatîon. Aggregation of the particles would lead to flocculation and précipitation such that the résultant material would not be regarded as a colloid.
The commonly recommended daily dosage of colloïdal silver is about one teaspoon, typically containing 5 to 30 parts per million of silver. Whîlst it is ciaimed that the body is readily able to pass or excrete this small amount of silver such that consumption thereof will not likely lead to excessive levels of silver accumulating in the body, it has been reported that this concentration is insufficient to effectively treat severe infections, diseases and/or disorders.
Attempts hâve been made to increase the concentration of silver that is available in silver colloids by incorporating proteins and lipids into the silver colloid. Reports however suggest that although the silver concentration tn protein- or lipid-based silver colloids may be higher than in traditional silver colloids, the introduction of proteins and/or lipids into the silver colloids may lead to bacterial growth. Furthermore, reports suggest that the proteins and/or lipids hâve been found to coat and/or occlude the active surface of the silver particles thereby adversely affecting the efficacy thereof.
The below Table represents data from reported literature indicating the concentration of silver présent in commercial sîlver-based products including ionic silver, colloïdal silver and protein- based silver colloids.
Table A: Literature data indicating the concentration of silver présent in commercial silverbased products including ionic silver, colloïdal silver and protein-based silver colloids
Concentration PPm of silver in Surface area ln cmz/mL
Identlty of commercial silver-based Minimum concentration Maximum concentration Minimum concentration Maximum concentration
product
Ionic 3 200 0.001 0.66
Protein 10 500 0.59 22.52
Colloid 5 30 0.28 104.7
While there are compelling arguments both for and against the use of colloïdal silver products as an antibiotic, the effectiveness and use of silver colloïdal products in the human body to fight severe infections has not been authoritatively accepted. Medical authorities and publications advise against the ingestion of colloïdal silver préparations, because of their lack of proven effectiveness and because of the risk of adverse side effects, such as argyria.
Whilst medical prostheses constitute an indispensible component of modem health care, infection remains a serious complication with associated medical procedures. Although many substances hâve been suggested to guard against prosthesis-related infection, only a few hâve been demonstrated to be clinically protective. Medical prostheses hâve been fabricated from pure silver and its aïloys, and silver molécules hâve been incorporated into the surfaces of a large variety of medical devices, including vascular, urinary and peritoneal cathéters, vascular grafts, prosthetic heart valve sewing rings, sutures and fracture fixation devices. Despite the plethora of such silver-based medical prostheses, their anti-infection efficacy has not been collectively addressed. In particular, a widespread disadvantage associated with silver-based medical prostheses résides in their inhérent localized and minimum surface area to volume characteristics.
In the light of the foregoing, it is dear that the silver-based products known and used in the art hâve limited applications and can be associated with severe adverse effects.
There is thus a need in the art for a silver composition which is suitable for the effective treatment and prévention of a broad range of infections, diseases and/or disorders and which is devoid of the adverse effects and limitations of the silver-based products that hâve previously been described.
For purposes of the présent spécification, the word ‘aggregation dénotés a plurality of silver particles (atoms, molécules, or macromolecules) loosely bound together via weak intermolecular van der Waals forces. The word ‘aggregation’ is also understood to dénoté a plurality of silver particles (atoms, molécules, or macromolecules) which are loosely bound together to form one or more weak, friable, crystalline silver structures.
The word ‘antimicrobiar as referred to herein ts understood to encompass antibacterial, antifungaf, antiprotozoal and antiviral properties.
In the context of the présent invention, pathogenic microorganisms may be eukaryotic or prokaryotic and may include bacteria, fungi, archaea, protista, protazoa, algae, parasites, yeasts and viruses.
Summary of the Invention
According to a first aspect thereof, the présent invention provides an antimicrobial composition including at least one or more friable aggregation(s) of silver particles în a liquid medium, wherein the concentration of the composition Îs from 40 ppm up to, and including, 500 000 ppm of silver.
The invention provides for the silver particles to be elemental silver (Ag) particles.
In an embodiment of the invention, the silver particles, which in combination form the aggregations, hâve a particle size of 1 x 10'9 m (1 nm) to 100 x 10® m in diameter, both values inclusive. It will be appreciated that the particle size of each silver particle induded in the aggregation(s) does not hâve to be identical, such that the plurality of silver particles may demonstrate a variety of particle sizes ranging from 1 x 10'9 m to 100 x 10 e m in diameter.
The invention provides for the aggregations to be friable and to include a plurality of loosely bound silver particles. As mentioned herein before, the plurality of silver particles are loosely bound together via weak intermolecular van der Waals forces. Furthermore, in terms of the invention, the silver particles are loosely bound together to form one or more weak, friable, crystalline silver structures.
In accordance with the invention, the one or more aggregations of silver particles are 100 x 10’9 m to 10 000 x 10s m in diameter, both values inclusive.
ln an embodiment of the invention, the friable aggregations fracture or dissociate under the influence of weak mechanical forces to form a plurality of smaller silver aggregations which are 10 x10'e m to 100 x 10 e m in diameter (both values inclusive) and/or to form a plurality of silver particles which are 10 xlO® m to 100 x 10 e m in diameter (agaîn, both values being inclusive).
In terms of the présent invention, the friable aggregations fracture or dissociate under the influence of weak mechanical forces which forces include, but are not limited to, rubbing, peristalsis, friction, vibration, mechanical movement, sonication or a combination of one or more thereof.
The présent composition contemplâtes concentrations of 40 ppm to 500 000 ppm of silver, both values inclusive, ln one embodiment of the invention, the preferred concentration of the composition is 1 000 ppm to 10 000 ppm of silver, both values inclusive.
It will be appredated by the skilled artisan that the surface area of silver in any given composition is directly proportional to its subséquent activity and efficacy as an antimicrobial agent. Particle surface area per unit volume of liquid medium increases as the concentration of silver partides increases. Furthermore, partide surface area per gram of silver increases as the particle size decreases. Thus high surface areas will give high antimicrobial activity and low surface areas will give correspondingly lower activity.
ln terms of one embodiment of the invention, the silver aggregations hâve a surface area of 0.5 cm2/ml at 100ppm to 540 cm2/m1 at 100000 ppm. It will be appredated that the surface area pertaining to each silver partide induded in the aggregation(s) does not hâve to be identical, such that the aggregations may demonstrate surface areas ranging from 0.5 cm2/ml to 540 cm2/ml for a range of concentrations from 100 to 100000 ppm.
Thus, it will be appredated that high partide surface area per unit volume of liquid medium is achieved by increasing the concentration of the silver partides, which in tum form the aggregations présent in the antimicrobial composition, to 40 ppm of silver or higher.
When the friable aggregations of silver partides fracture or dissodate under the influence of weak mechanica! forces, more surface area is created and hence the surface area is subsequenüy increased.
This additional surface area consequently increases the partide surface area per gram of silver in the antimicrobial composition of the présent invention. Since the partide surface area is increased, further sites on the surface of the silver partides within the friable aggregation(s) are exposed and subsequently available for antimicrobial action.
In terms of a further embodiment of the présent invention and under the influence of weak mechanîcal forces, the surface area of the aggregations in the antimicrobial composition having a concentration of 10000 ppm of silver increases from 54 cm2/ml to 540 00 cm3/ml. The greater the influence of the weak mechanîcal force, such as for instance peristalsis, the greater the increase in surface area pertaining to the aggregations.
According to a further embodiment, the Inventor has found that the antimicrobial composition of the présent invention has sufficient active sites on the surface of the silver partides in order to retain antimicrobial activity in electrolytic environments having an addic or low pH or where such environments are saline.
In an embodiment of the invention, the liquid medium referred to herein above is water, preferably distilled water. Altematively, the liquid medium may be deionised or demineralised water. In a further embodiment of the invention, the liquid medium may be any suitable organic medium, for instance, a suitable alcohol or oil.
Thus according to the présent invention, the antimicrobial composition as described herein contains a higher concentration of silver in comparison to silver products of the type known and described in the art, thereby providing strong antimicrobial activity whîlst avoiding the need to administer large volumétrie dosages of the composition which large dosages are, as is discussed above, most undesirable owing to the associated adverse side effects of the prior art silver products. Accordingly, the Inventor has found that, at the high concentrations of silver disclosed herein, no adverse toxicity has been observed.
It is known that ionic silver compounds, particularly with reference to silver antibiotic products of the type known in the art, hâve reduced activity upon being administered, since these compounds react with bodify fluids and electrolytes such as chloride in the stomach to form less soluble silver compounds, such as silver chloride.
Whiie it will be appredated that any and ail substances in the presence of water will hâve some ionic character, the présent composition contemplâtes the inclusion/presence of no anionic spedes other than hydroxyl ions.
In an embodiment of the invention, the antimicrobîal composition of the présent invention may be in the form of a liquid dispersion.
In an alternative embodiment of the invention, the antimicrobîal composition may take the form of a dry material. According to this embodiment, the antimicrobîal composition may be dried and used/applied directly or further formulated with suitable carrier agents and médiums.
It will be appredated that, in terms of the présent invention, the silver partides are held together to form the aggregation(s) by means of van der Waals forces and/or are loosely bound together to form one or more weak, friable, crystalline strudures. By their very nature, van der Waals forces are rolatively weaker than those arising from valence bonds. Thus, the weak van der Waals forces acting between the silver partides in conjunction with the friable nature of the crystalline structures of the aggregations will allow the silver aggregations to separate readily into their component partides by mechanical forces such as peristalsis and react, on a cellular Ievel, once introduced to an infeded site or body.
Without wishing to be bound by theory, the Inventor believes that the friable aggregations of silver partides présent in the antimicrobîal composition of the instant invention, together with the high concentration of silver présent therein (40 ppm to 500 000 ppm of silver, both values inclusive) affords the superior antimicrobial properties achieved by the présent composition în contrast to the products known and used in the art.
The Inventer believes that the bond strength between the silver particles, which silver particles in combination form the aggregations, is such that the fracturing of the aggregations, under the action of weak mechanical forces, to generate new sites on the surface of the silver particles is not instantaneous and can form over a period of time.
According to a second aspect thereof, the invention provides a process for preparing the antimicrobial composition, substantially as herein described, said process including the steps of:
(i) providing silver particles having a particle size of 1 x 10 ® m to 100 x W® m in diameter, both values inclusive;
(iî) allowing said silver particles to aggregate together to form one or more aggregations which are 100 x 10 ® m to 10 000 x 10-® m in diameter, both values inclusive; and (iïi) said silver aggregations being friable and able to fracture or dissociate so as to form smaller silver aggregations in the range 10 x 10 ® m to 100 x W® m in diameter, both values inclusive, and/or small silver particles in the range 10 x 10'9 m to 100 x 10‘8 m in diameter; and (iv) providing a sufficient quantity of a liquid medium so as to achieve a concentration of 40 ppm up to, and including, 500 000 ppm of silver.
In an embodiment of the invention, the silver aggregations may be prepared by any suitable method of the type known and described in the art including, but not limited to, electrolytic déposition of silver, chemical déposition of silver, photo déposition of silver, plasma déposition of silver and comminution of silver pièces.
In one embodiment of the invention, the silver aggregations may be electrically prepared by employing an electrolytic cell, a silver anode and an inert cathode. In terms of this embodiment, the anode is etched by Direct Current in distilled water. During electrolysis, deposîts of silver are formed on the cathode. These deposits are loosely bound to each other and do not chemically cohere to the cathode. In this way, said silver deposits do not form an adhèrent or cohérent mass or plate on the cathode. As electrolysis continues, further deposits of silver are formed on the surface of the depos'rts, already formed. In this way, the silver particles form aggregations which are then removed from the cathode by various methods, including sweeping or brushing techniques of the type known in the art.
Antimicrobial préparation
According to a third aspect of the invention, there is provided an antimicrobial préparation for use in the treatment of infections, diseases and/or disorders, comprising a therapeutically effective amount of the antimicrobial composition, as described and identified herein, in combination with one or more suitable/acceptable excipients, additives or carriers.
The expression “treatment of infection, diseases and/or disorders’ as used herein is intended to be understood as covering prophylactic, alleviatîng and curative interventions.
Furthermore, in the context of the présent invention, it is to be understood that the antimicrobial composition and/or antimicrobial préparation aids, supports, augmente and accelerates the healing processes for, inter alla, wounds and invasive trauma.
The term “effective amount refers to that amount of antimicrobial composition that ts required to provide therapeutic benefit. The présent invention is not limited by the nature or scope of the therapeutic benefit provided. The degree of benefit may dépend on a number of factors, inter alla, the severity of the infection, disease and/or disorder and the immune status of the individual.
In an embodiment of this aspect of the invention, the excipients, additives and carriers may include, but are not limited to including, proteins, peptides, amino acids, lipids, carbohydrates (e.g. sugars, indudîng monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars, polysaccharides or sugar polymers), minerai oils and the like, which can be présent singly or in combination.
In a further embodiment of this aspect of the invention, there is provided for the antimicrobial préparation to possess activity towards cancer cells.
According to a fourth aspect of the invention, the présent invention provides a method of treating a patient suffering from an infection, disease and/or disorder comprising the step of administering to such patient a therapeutically effective amount of the antimicrobial composition, as described and identified herein, or an antimicrobial préparation, as described and identified herein.
According to a fifth aspect of the invention, there is provided the use of the antimicrobial composition, as described and identified herein, in the manufacture of an antimicrobial préparation for the treatment, and/or prévention of infections, diseases or disorders.
The invention further provides for the use of the antimicrobial composition, as described and identified herein, for the treatment, dîagnosis and/or prévention of diseases and/or disorders.
According to the invention, one or more agents may be used in combination with the présent antimicrobial composition.
The above mentioned agent(s) can be any compound, chemical, therapeutic agent, antbiotic agent, drug, biologicai molécule, antibody, protein, lipid, nucleic add, vector, plasmid, steroid, enzyme, co-enzyme, carbohydrate, co-factor, anesthetic agent, or any other agent that has an effect in the body.
In addition to the above, the invention also contemplâtes the use of nutritional agents in combination with the antimicrobial composition of the présent invention. Such nutritional agents indude, but are not limited to, nutrients, nutriceuticals, minerais, vitamins, amino adds and essential fats.
It is envisaged that such combination will serve to increase the spectrum of activity of the antimicrobial composition and croate a synergistic effect.
Non-limiting exemples of the agents contemplated for use with the antimicrobial composition of the présent invention include acné préparations such as isotretinoin, benzoyl peroxide, salicylic acid and tétracycline; anesthetics for topical administration such as dibucaine, lidocaine, benzocaine, tetracadne, deperodon and pramoxine hydrochloride; anti5 infiammatory agents such as betamethasone benzoate, betamethasone valerate, desonide, fluodnolone acetonide, halcinonide, hydrocortisone; antiperspirants; antipruritic and external analgésie agents such as camphor, menthol, salicylic acid, methylsalicylate; deansing agents; pigmenting agents; anabolic steraids for building up tissues under wound healing such as methandienone;
proteolytic agents for the décomposition of fibrin such as trypsin; vasodilating substances for improving the flow of blood during wound healing such as tolazoline; thrombosis-hampering substances such as heparin; certain biologically active substances which affect tissue formation and tissue stabilization such as ascorbic add and EGF (epidenmal growth factor); antibiotics; analgésies; immune agents such as immune regulatory proteins and 15 immunotherapy drugs; and chemotherapeutic agents.
Without wishing to be bound by theory, the Inventor believes that when the aforementioned agents are used in combination with the antimicrobial composition, the silver partides présent in the antimicrobial composition augment the effect of the said agent. In this way, it 20 is believed that the silver partides may extensively amplify the effect of the agent and/or serve to croate a synergistic effect.
In situations where the antimicrobial composition is used to treat a disease which has an abundance of dead tissue (e.g., a fungating tumor or a decubitus ulcer), the silver particles 25 présent in the antimicrobial composition function to prevent secondary infection at the diseased site.
Administration
Administration of the antimicrobial composition and/or antimicrobial préparation of the présent invention can be done in any acceptable manner known in the medical arts, v--—
Spécifie, non-limiting examples of administration methods which may be used in accordance with embodiments of the présent invention include oral administration, injection, topical administration, intravenous administration, rectal administration, transdermal administration, ophthalmic administration, lymphatic administration and nasal administration.
Though not limited to any particular means of application, the antimicrobial composition and/or antimicrobial préparation can be applied using gloved hands or by an applicator. Likewise, the composition and/or antimicrobial préparation can be applied to the surface of a dressing, which can then be applied topically.
Topical administration can be carried out using, inter alia, sprays, mists, lotions, creams, ointments, or gels which are formulated to include the antimicrobial composition and/or antimicrobial préparation of the présent invention. Submersion of the diseased or otherwise infected tissue is also an acceptable means of topical administration.
Ophthalmic infections can be treated using standard procedures in the art, such as by pulling down the lower eyelid to form a pocket and applying the antimicrobial composition and/or antimicrobial préparation thereto.
By way of further illustration, infections of the mouth can be treated by applying the antimicrobial composition and/or antimicrobial préparation with a sponge applicator or a toothbrush.
The mode of administration can be dépendent on the disease or infection being treated and the formulated potency of the composition.
In accordance with a further embodiment, the antimicrobial composition and/or antimicrobial préparation of the présent invention can be administered to in vitro and in v/vo Systems.
The présent invention also contemplâtes cells that hâve been altered by the antimicrobial composition and/or antimicrobial préparation of the présent invention and the administration of such cells to other cells or tissues, in in vitro or in vivo methods.
The antimicrobîal composition and/or antimicrobîal préparation of the présent invention can be administered for clînical use in humans and for veterinary use, such as with domestic animais, in manners known in the art and similar to other therapeutic agents.
lt îs envisaged that the présent antimicrobîal composition and/or antimicrobial préparation may be administered for horticulture as well as agriculture use.
lt is further envisaged that the présent antimicrobial composition and/or antimicrobîal préparation may be administered for the treatment of raw materials induding, but not limited to, food and water.
Formulation
The antimicrobial composition and/or antimicrobial préparation of the présent invention can be incorporated with other ingrédients to form a variety of products for administration, as detaîled herein below.
The antimicrobial composition and/or antimicrobial préparation may be made up in any suitable dosage formulation and may be prepared by conventional techniques.
In an embodiment of the invention, the dosage formulation comprises tablets, capsules, caplets, syrups, beverages, powders, granulates, lozenges or the like. In each instance, the formulation may contain any suitable exdpients such as fillers, lubricants, désintégrants, taste masking agents and the like.
The antimicrobial composition and/or antimicrobial préparation of the présent invention may be used in cosmetics and personal care products to make said products résistant to antimicrobial contamination. Non-limiting examples thereof indude creams, ointments, sunscreens, mouth rinses, toothpastes, dental flosses, gels, moisturizers, foams, powders, liquid and powder makeup foundations, powder and cream blushes, lipsticks and lipglosses, lip pendis, mascaras, eye liners, eye shadows, perfumes, colognes, déodorants, toners, wipes for skin application, dermal patches, shaving creams, shampoos, conditioners and various hair treatments like mousses and sprays. *j—·
In another embodiment of the invention, the antimicrobial composition and/or antimicrobial préparation may be incorporated in aérosols and sprays for topîcal or inhalation application.
In addition hereto, the antimicrobial composition and/or antimicrobial préparation may be used in or applied to, inter alia, bandage dressings, sponges, surgical and examination gloves, combs, brushes, cotton swabs, razors and toothbrushes.
In one embodiment of the invention, the antimicrobial composition of the présent invention may be incorporated Into medical devices including, but not limited to medical implants and wound care devices and can be used to impart antiseptie and disinfectant properties to medical appliances and utensils. In a further embodiment of the invention, the antimicrobial composition may be used in applications to sterilize surfaces.
Medical implants indude, but are not limited to, urinary and intravascutar cathéters, diaîysis shunts, wound drain tubes, endotrachéal breathing tubes, skin sutures, vascular grafts and implantable meshes, intraocular devices and heart valves. The composition of the invention can also be used in, inter alia, bone prostheses and reconstructive orthopaedic surgery.
Wound care devices include, but are not limited to, general wound dressings, non-adherent dressings, bum dressings, biological graft materials, tape dosures and dressings and surgical drapes.
Posage
The appropriate dosage of the antimicrobial composition, antimicrobial préparation and/or formulation of the présent invention, as indentified herein, will dépend on, inter alia, the type of infection, disease or disorder to be treated, as is defined herein below; the severity and course of the infection; whether the antimicrobial composition, antimicrobial préparation and/or formulation is administered for therapeutic or préventive purposes; previous therapy and the patient’s dinical history and response to the composition.
The antimicrobial composition, antimicrobial préparation and/or formulation of the présent invention is/are suitably administered to a patient at one time or over a sériés of treatments.
In one embodiment, the administration can occur one or more times daily for a period of 1 day to 360 days. In another embodiment, the administration can occur one or more times daily for a period of 1 to 7 days. In another embodiment, the administration can occur one or more times for a period of 4 hours to 24 hours. In a further embodiment of the invention, single administration will suffice.
The disease symptoms and parameters for assessing improvement and the progress of the therapy can be readiiy monitored by conventional methods and assays known to the physician or other persons of skill in the art.
For example, when administered topically using a spray or submersion administration mode for topical local effect, the amount of antimicrobial composition, antimicrobial préparation and/or formulation may not be as important, but rather, the concentration thereof and the frequency of administration may be more significant.
Infections, diseases and/or disorders
The présent invention relates to methods of treating (inciuding prophylactically treating) viral infections, bacterial infections, fungal infections, parasitic infections and/or cancer. The invention is envisaged to hâve a regenerative effect on damaged tissue thereby factlitating and expediting the healing process.
Examples of viral infections which may be treated using the methods and/or composition, préparation and/or formulation of the présent invention include, without limitation, HlV/(Human immunodeficiency Virus) infection, herpes virus infection, vira! dysentery, flu, bronchitis, pneumonia, measles, rubéfia, chickenpox, mumps, polio, rabies, sinusitis, tonsillitis, mononudeosis, ebola, respiratory syncytial virus, croup, SARS, dengue fever, yellow fever, lassa fever, arena virus, bunyavirus, filovirus, flavivirus, hantavirus, rotavirus, viral meningitis, H5N1 virus (bird flu), arbovirus, parainfluenza, smallpox, epstein-barr virus, dengue hémorrhagie fever, cytomégalovirus, infant cytomegalic virus, progressive multifocal leukoencephalopathy, viral gastroenteritis, hepatitis, cold sores, meningitis, encephalitis, shingles, warts, human papaloma virus, viral ear and eye infections.
Examples of bacterial infections which can be treated and prevented using the methods and/or composition, préparation and/or formulation of the présent invention include, without limitation, tuberculosis, choiera, syphilis, bacterial pneumonia, Escherichia coli (e. coli) infections, candida infection, MRSA methïciîlin résistant Staphylococcus aureus (S. aureus) infection - strain ATCC #43300, vancomycin résistant Enterococcus faecalis (E. faecalis) infection - strain #1061, Salmonella enteritidis (S. enteritidis) infection - strain ATCC #13076, clostridium difficile (C. difficile) infection - strain ATCC #9689 and pseudomonas aeruginosa (P. aeruginosa) infection - hospital clinical strain.
HIV is distinguished from AIDS (Acquired Immune Deficiency Syndrome), în that HIV is a virus whilst AIDS dénotés the condition whereby a déficient or compromised immune system results from the damage caused by HIV to the immune system. As an HIV infection progresses, there is ongoing damage to the immune defense cells whereby the body becomes increasingly less able to fïght off infection. Individuals with advanced HIV deveîop the AIDS condition and become progressively and increasingly susceptible to infections. These infections are called opportunistic infections and may be bacterial in nature. Thus, in terms of an embodiment of the invention, the présent invention relates to a method of reducing and/or controlling opportunistic infections which are associated with compromised (impaired) immune Systems. Said compromised (impaired) immune Systems may be indicated by low CD4 cell counts.
Examples of fungal infection which can be treated and prevented using the methods and/or composition, préparation and/or formulation of the présent invention include, without limitation, thrush, candidiasis, cryptococcosis, histoplasmosis, blastomycosis, aspergillosis, coccidioîdomycosis, paracoccidiomycosis, sporotrichosis, zygomycosis, chromoblastomycosis, lobomycosis, mycetoma, onychomycosis, piedra pityriasis versicofor, tinea barbae, tinea capitis, tinea corporis, tinea cruris, tinea favosa, tinea nigra, tinea pedis, otomycosis, phaeohyphomycosis and rhinosporidiosis. Yeast infections can also be treated and prevented.
Examples of parasitic infections which can be treated and prevented using the methods and/or composition, préparation and/or formulation of the présent invention indude, inter alia, malaria (induding congénital and cérébral malaria), ringworm, tapeworm, lice, typhoid fever and typhus.
The présent invention also provides methods for treating canceraus tissue in a subject. The présent invention has been shown to be effective in redudng the size of and even elimtnating cancerous tumors. The types of cancers which can be treated using the methods of the présent invention indude, without limitation, melanomas of the skin, lung and/or bronchus cancers, colon and rectum cancers, urinary bladder cancer, pancreatic cancer, ovarian cancer, thyroid cancer, stomach cancer, brain cancer, cervical cancer, testicular cancer, lymphomas, breast cancer, prostate cancer, cancers of the blood, cancer of the bones and joints, and the Iike. Further, the invention contemplâtes methods for treating cancerous tissue in a subject in conjunction with chemotherapies and radiothereapies.
The présent invention further provides methods for treating respiratory diseases in a subject including, but not limited to, asthma, Tuberculosis (TB) and pulmonary fibrosis.
The présent invention yet further provides methods for treating skin conditions, infections and diseases in a subject induding, inter alia, eczema, acné, vitiligo, warts, bum wounds, blisters and scars.
Thus the présent invention contemplâtes treating and preventing chronic and acute infections, diseases and/or disorders as well as supplementing and supporting the immune system.
These and other objects, features and advantages of the invention will become apparent to those skilled in the art following the detailed description of the invention.
Brief Description of the Figures
Figure 1A is a graph depicting particle size distribution of the silver aggregations of the présent invention measured at 15 minute intervals in accordance with the analysis conducted in Example (ii);
Figure 1B is a pre-sonication photograph showing the particle size range and geometries of the friable silver aggregations under an optical magnification of x400 in accordance with the analysis conducted in Exampie (ii);
Figure 1C shows photographe depicting a comparative analysis to demonstrate the efficacy of the friable silver aggregations as antimicrobial agents when compared to a commercial silver colloid product;
Figure 2 is a graph depicting the results of a dise diffusion test for the inhibition of the growth of E.coli by the silver aggregations prepared in accordance with Example (i)(a) ofthe présent invention;
Figure 3 is a graph depicting the results of a dise diffusion test for the inhibition of the growth of P. aeruginosa by the silver aggregations prepared in accordance with Example (i)(a) of the présent invention;
Figure 4 is a graph depicting the results of a dise diffusion test for the inhibition of the growth of MRSA by the silver aggregations prepared in accordance with Example (i)(a) ofthe présent invention;
Figure 5 îs a photograph of the silver aggregations (20.0% w/v) applied to a preseeded lawn of MRSA demonstrating bacterial growth inhibition (after ovemight incubation);
Figure 6A: is a photograph depicting the results of a disk diffusion test using E. faecalis with the silver aggregations at 1.0% w/v (in tri plicate) with zones of inhibition;
Figure 6B: is a photograph depicting the results of a disk diffusion test using E. faecalis with PBS/Tween in triplicate with two other controls (marked with arrows);
Figure 7: is a photograph depicting the results of a disk diffusion test to compare the silver aggregations employed in Example (iii) and the silver aggregation employed in Example (iv) in their activity against S. enteritidis wherein PBS/Tween was the négative control;
Figure 8: is a photograph depicting the results of a disk diffusion test using S. enteritidis grown on Mueller-Hinton agar wherein the silver aggregations were used at 1.0 and 5.0 % w/v;
Figure 9: is a graph depicting a biodde challenge assay against S. enteritidis’,
Figure 10: is a photograph depiding the results of a biodde challenge assay showing the effects on S. enteritidis after 20 minutes of contact with the silver aggregations. The plates show the effect of the silver aggregations at 1.0% w/v (far left) and 0.1 w/v (middle). The control plate on the far right illustrâtes the growth of S. enteritidis without the addition of the silver aggregations of the présent invention;
Figure 11: is a photograph depicting an agar plate showing the effect of a silver colloid préparation on baderial growth inhibition (filter paper 1) when compared to varying concentrations of the antimicrobîal composition of the présent invention, when prepared in accordance with Formulation A, (filter paper 2 to filter paper 4). Filter paper 5 represents the control;
Figure 12: is a graph depicting the results of a test patient’s CD4 cell count whilst receiving treatment comprising 3 ml of Formulation A, three times per day; M
Figure 13: is a graph depicting the results of a test patient’s white blood cell (WBC) count whilst receiving treatment comprising 3 ml of Formulation A, three times per day;
Figure 14: is a graph depicting the results of a test patient’s viral load whilst receiving treatment comprising 3 ml of Formulation A, three times per day;
Figure 15:
Figure 16:
is a graph depicting the results of a test patient’s érythrocyte sédimentation rate (ESR) whilst receiving treatment comprising 3 ml of Formulation A, three times per day;
is a graph depicting the dose-response plot for Sample 1 used in the in vitro anti-malaria! essayas screened on 07/06/2011;
Figure 17: is a graph depicting the dose-response plot for Sample 2 used in the in vitro anti-malarial assay as screened on 07/06/2011;
Figure 18: is a graph depicting the dose-response plot for Sample 1 used in the in vitro anti-malarial assay as screened on 15/06/2011 ;
Figure 19: is a graph depicting the dose-response plot for Sample 2 used in the in vitro anti-malarial assay as screened on 15/06/2011;
Figure 20: is a graph depicting the dose-response plot for the chloroquine sample used in the in vitro anti-malarial assay as screened on 07/06/2011 ; and
Figure 21: are photographs taken during the anti-cancer assay showing malignant skin melanoma cancer before and after treatment with the antimicrobial composition of the présent invention.
The presently disclosed subject matter, including the préparation of the silver aggregations, Formulation A and Samples 1 and 2, will now be described more fully hereînafter with reference to the accompanying Examples, in which représentative embodiments are shown.
The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complété, and will fully convey the scope ofthe embodiments to those skilled in the art.
Examples
The Examples of the invention are divided into the following parts: (I) Process for preparing the antîmicrobial composition of the présent invention; (ii) Formulations including the antimicrobial composition thus prepared and analyses of the properties of the antimicrobial composition; (iii) ln vitro analysis to demonstrate bacteriostatic properties of the silver aggregation, as prepared in accordance with Example (i)(a); (îv) Further in vitro analysis to demonstrate bacteriostatic properties of the silver aggregation; (v) Comparative analysis; and (vi) Therapeutic use.
Each of these parts will be discussed in tum.
Example (i): Process for preparing the antimicrobial composition ofthe présent invention (a) Préparation of silver aggregations
Eiectroiysis was conducted employing an anode and an inert cathode. The electrolytic cell was filled with distilled water. However, it will be appreciated that deionised or demineralised water can also be used.
Direct Current was supplied to the électrodes. It will be appreciated that regulated or pulsed DC are also suitabie. The voltage was set in the range of 30 to 60 volts.
It will be appreciated that the current to the electrolytic cell can be adjusted by varying the voltage, by moving the anode and cathode doser or further apart or by increasing the number of plates. For purposes of this Example, the current density was set in the range of 0.0008 to 0.002 amps/sq cm. In the case of a preferred array of CACACACAC (where C=cathode and A = anode), a desired current of 4 to 10 amps is empioyed. v
The anode employed comprises fine silver sheets having a purity of 99.9% or higher. Silver sheets were sized according to a 250 mm x 250 mm square dimension. Depending on the nature (for instance, the geometry) of the silver employed, the size thereof will be manipulated to complément the apparatus employed. The silver sheets were suspended from current-carrying rods or clips and were arranged in parallel connection to the voltage source. Both sides of the silver sheet can be active during electrolysis. In certain arrangements, multiple anodes may be arranged within the electrolytic cell.
The cathode employed may comprise an inert conducting material. For purposes of this Example, the cathode employed is in the form of stainless steel. However, it will be appreciated that any other suitable inert material can be employed, for instance, carbon. Furthermore, in certain arrangements, the cathode may comprise silver. The dimensions of the cathode were similar to those provided for the dimensions of the anode. The one or more cathodes were suspended within the electrolytic cell so as to altemate with the anodes. The cathodes were suspended from current-carrying rods or clips and were arranged in parallel connection to the voltage source. In certain arrangements, multiple cathodes may be arranged within the electrolytic cell.
During electrolysis, deposits of silver formed on the cathode. It was observed that the deposit did not adhéré to the cathode and the deposit appeared as a loosely bound, amorphous parti cul a te deposit
The loose particulate deposit was removed from the cathode, either by way of being swept, brushed or otherwise removed there from and collected.
The collected product comprised aggregations of silver particles, said aggregations being 100 x 10'® m to 10000 x10’9m in size as measured by Dynamic Light Scattering analysis (DLS).
The resulting aggregations were friable to the touch and when applied wet as a drop to an absorbent surface showed a cortex or corona displaced from the centre, indicating the ease with which the aggregations fracture to smaller entities. —17317
The aggregations were preferably kept wet aithough they may be dried for alternative use such as blends with petroleum products, e.g. Vaseline, to produce creams and ointments.
(b) Préparation ofthe antimicrobial composition
Using the silver aggregations prepared in accordance with Example (i)(a) above, a so-called standard batch’ of the antimicrobial composition was prepared such that the concentration of silver in distilled water was 100 g of silver in 500 ml of distilled water.
This standard batch could then be used and further diluted with distilled water to produce some ofthe formulations described herein below.
Exampie (11): Formulations including the antimicrobial composition ofthe présent invention It will be appreciated by the skilled artisan that there are numerous formulations that can be made from the antimicrobial composition prepared according to the steps above. The below mentioned formulations are merely illustrative of some of the types of formulations that can be so prepared and should not be construed as limiting the scope of the présent application in any way.
1. A range of concentrations of the antimicrobial composition can be prepared by dilution of the standard batch that may be taken orally, topically etc. For example, 5 ml of the standard batch at 100:500 silver to distilled water is diluted with 95 ml of distilled water to achieve a concentration of 1 g of silver in 100 ml of distilled water. This formulation is herein referred to as Formulation A. This formulation can then be used orally in doses of 1 ml in a small glass of water (50 ml) three times a day to treat infection.
2. For topical infection, Formulation A can be applied directly or with a swab to the site to be treated or lésion;
3. A cream can be prepared by blending 1 g of the silver aggregation, in dry form, (as mentioned in Example (i)(a) herein above) with 10 g of petroleum jelly to produce an ointment or cream. This may then be applied topically or applied to a bandage, plaster, etc.;
4. Said silver aggregation, in dry form, can be blended with a setting resin plastic gel and applied to a prosthesis or device as a thin film to become intégral with the device. For example, 1 g of the antimicrobial composition, in dry form, is blended with 10 g of silicone paste. The resulting formulation is then applied to a plastic medical tube and is allowed to set to form a thin coating;
5. The antimicrobial composition, presented as a dry material in dry form, can be tabletted at 0.01 g per tablet in an inert carrier of the type known in the art; and
6. The antimicrobial composition, either presented as a wet or dry material where appropriate, can be blended to form creams, émulsions, etc.
Analyses ofthe properties ofthe antimicrobial composition
1) Surface area
The surface area of three samples of the silver aggregation, in dry form (as mentioned in Example (i)(a) herein above) was measured by BET and the following values were obtained: sample (1) 0.513 m2/g; sample (2) 0.522 m2/g; and sample (3) 0.588 m2/g. An average BET surface area value for the silver aggregations, in dry form, prepared in accordance with Example (i)(a) of 0.54 m2/g was therefore obtained, and used to calculate surface areas of varying concentrations of Formulation A as is indicated in Table B.
Table B:
Surface area values obtained for the silver aggregation, in dry form, and calculated for varying silver concentrations of Formulation A
Silver Aggregation In Dry Form Varvina Silver Concentrations of Formulation A
Surface Area 100 ppm 1000 ppm 10000 ppm 100000 ppm
Measured m2/g 0.54
cm2/g 5400
Calculated g/ml 0.0001 0.001 0.01 0.1
Calculated cm2/ml 0.5 5.4 54 540
The silver aggregations présent in a diluted sample of Formulation A (10:1 water to Formulation A), was subjected to mechanicai agitation by sonication in order to demonstrate the aggregations' ability to dissociate/fracture into smaller silver particulates.
The results indicated that under mechanicai agitation, the silver aggregations fractured/dissodated into silver particulates such that a partide diameter (d nm) (dix 10 e m) change from 400 to 40 d nm was observed using Dynamic Light Scattering analysis (DLS). Only partide diameter sizes below 1000 d nm were considered.
In terms of this Example, it will be appredated that the qualitative relationship between surface area and partide diameter is cubîc. The increase by such mechanicai agitation can thus be qualitatively considered to be a fador of (400:40)3 i.e. 1000:1.
It is appredated that the silver particulates do not possess idéal geometries. A factor of 10% (100:1) ofthe above value is considered conservative, espedally when it is appredated that the initial silver aggregations of Formulation A prior to agitation are shown by DLS to hâve diameters averaging 4000 d nm and above, which would further augment the potential increase in surface area on fracturing.
Fracturing/dissodation of the silver aggregations of a 10:1 dilution of Formulation A following agitation thereof gives the following calculated surface areas as indicated in Table C:
Table C: Surface area values at varying silver concentrations of Formulation A following the influence of mechanical agitation
Varying Silver Concentrations of Formulation A
100 ppm 1000 ppm 10000 ppm 100000
ppm
Calculated Surface Area (cm’/ml) 0.5 5.4 54 540
100% Fractured Surface Area Following Agitation (cm2/ml) 500 5400 54000 540000
10% Fractured Surface Area Following Agitation (cm2/ml) 50 540 5400 54000
Form the above Table, it can cleariy be seen that following mechanical agitation, by for example sonication or peristalsis, the silver aggregations fracture/dissociate into smaller silver particulates whereby the surface area of the resulting particulates is substantially increased.
By way of example, Formulation A has a surface area of 5400 cm2/ml following mechanical agitation when compared to the highest value of 104.7 cmz/ml for a commercial silver colloid, as is indicated in Table A presented herein before. This demonstrates a greater than 50 times increase in surface area. Further potential fracturing up to 100% gives an increase of 500 times in surface area.
2) Dissodation/fracturing of aggregations into smaller particulates
A 1000 ppm stock solution of the antimicrobial composition was prepared by 10:1 dilution of Formulation A.
An aliquot ofthe 1000 ppm stock solution was evaluated by DLS. The results reveal that the silver aggregations hâve an average size diameter of 5500 nm.
A second aliquot of the 1000 ppm stock solution was analyzed using sonication. The ultra sonie was initiated and five consecutive measurements of the aliquot were taken at 15 minute intervals.
Partide size was measured at 15 minute intervals using DLS in conduction with sonication as is represented as Records 6 to 9 in Figure 1A. In terms of this Figure, sizes above 1000 nm are not displayed.
Record 6 in Figure 1A indicates that the silver aggregations below 1000 nm in the aliquot hâve an initial size of 400 nm. After sonication for 15 minutes (Record 7), the silver aggregations hâve started to fracture/dissociate with diminution of the 400 nm peak and the formation of smaller silver particulates around 80 nm. After further sonication (Records 8 and 9), a further size réduction at 400 nm and further fracturing from 80 nm down to 40 - 50 nm is observed. This colfectively demonstrates the fracturing of the silver aggregations at 400 nm into silver particulates at 40 to 50 nm.
Figure 1B is a pre-sonication photograph showing the typical range of aggregation sizes and geometries of the friable silver aggregations under an optical magnification of x400.
These results thus dearly reveal that the silver aggregations of the microbial composition are friable and dissodate/fracture into smaller silver particulates by the action of mechanîcal forces. It is contemplated that other forms of mechanîcal adion, such as peristalsis, friction, abrasion, vibration etc, will effect similar results. —
3) Plate analysis of the antimicrobial composition
A plate analysis was conducted in order to détermine the efficacy of the antimicrobial composition when in solution.
Selected concentrations of Formulation A were tested against a microbial culture of Botrytis cinerea in solution. The analysis was repeated using adjusted pH and salinity levels. It is to be noted that adjusted pH and salinity levels were chosen in order to represent the potential electroiyte levels found in ambient bodily fluids, this being an environment where the antimicrobial composition of the instant invention is required to be effective. Furthemore, a comparative evalution was made by employing a commercial silver colloid product. The plate analysis is depicted in the photographs represented in Figure 1C.
For purposes of this analysis, 100 ppm, 1000 ppm and 10000 ppm solutions were prepared from appropriate dilutions of Formulation A. Said solutions were thereafter shaken and left to stand for 5 minutes.
A 10 ppm commercial silver colloid product was employed for purposes of the comparative evalution. A laboratory produced stock solution of the aforesaid microbial culture was used to dose the aliquots, as indicated in Table D below.
pH and salinity were adjusted for test samples 7 to 12 to correspond qualitatively to conditions that can exist in ambient bodily fluids such as the stomach (typically a pH of 2 to
4) .
Individual 0.1 ml samples were drawn from the aforesaid prepared solutions and striped down pétri dishes, pre-prepared with nutrient agar.
The plates were left to mature for 2 days and were thereafter photographed in order to observe the growth of the microbial culture (Figure 1C).
Table D: Samples used for purposes of the plate analysis of the antimicrobial
composition
Test DIstilled Microbial Silver 100 1000 10000 PH NaCl Comment
Sample Water Culture Colloid ppm ppm ppm 1N
Number ml Solution ml ml ml ml ml
ml
1 1 1 Control
6
2 1 1 6
3 0.1 1 6
4 1 1 6
5 1 1 6
6 1 1 6
7 1 1 Control
2 .2
8 1 1 2 .2
9 0.1 1 2 .2
10 1 1 2 .2
11 1 1 2 .2
12 1 1 2 .2
Observation of Figure 1C dearly reveals that for test samples 1 to 6, only high silver concentrations of 1000 and 10000 ppm, as prepared from Formulation A, control the growth of the microbe. This is indicated dearly in test 5 and 6.
There is a very slight observable diminution in microbial growth for the commerdal silver colloid product when the microbial culture solution is diluted by 10:1 in test 3 as compared with the 1:1 mix of test 2.
Furthemore, the presence of salinity and/or acidic pH inhibits the antimicrobial activity as evidenced by the minimal microbiall growth in test 5 compared with test 11 where extensive microbial growth is observed. However, it is observed that in the presence of salinity and/or acidic pH, the 10000 ppm concentration still effectively inhibits microbial growth which is in direct contrast to test 9, being the commerda! silver colloid product.
Example (iii): In vitro analysis to demonstrate bacteriostatic properties ofthe silver aogregations, as prepared in accordance with Example fi)fa) above (a) Methods and Materials
0.5 g of the silver aggregations, as prepared in Example (i)(a) above, was provided in dry form.
The bacteriai strains used in the study were:
) Escherichla coli, a dinical strain*;
2) Pseudomonas aeruginosa, a clinical strain*; and
3) Methidllin-resistant Staphylococcus aureus (MRSA), strain ATCC 43300.
* Obtained from the Respiratory Research Laboratory, Department of Medidne, University of Birmingham.
Bacteriai suspensions in stérile PBS were prepared équivalent to a density of 0.5 on the McFariand scale (approximately 1.5 x 10e colony-forming units per ml) from ovemight incubations of bacteria streaked for single colonies. A stérile cotton swab was used to preseed Mueller-Hinton agar plates (Oxoid Ltd, UK) with lawns of each of the bacteriai strains.^ (b) Bacteriostasis assay
A stock suspension of the silver aggregations was prepared (20% w/v in stérile phosphatebuffered saline with 0.1% v/vTween; Sigma-Aldrich Ltd, UK).
Dilutions were made to give 10.0, 2.5,1.0 and 0.1% w/v. Stérile fitter paper dises of 5 mm diameter (filter cards 190005, Shandon Inc., Pittsburgh, USA) were saturated with the suspensions, vortex mixed, and the excess suspension was removed by aspiration. Dises were used immediately fwet’) or air-dried for 2 hours prior to use ('dry'). The dises were then placed onto freshly pre-seeded Mueller-Hinton agar plates prepared as described above under “Methods and Materials. The plates were incubated ovemight at 37°C and zones of inhibition around the dises were measured. The means (and standard errors) of triplicates were calculated.
A 20.0% w/v suspension of the silver aggregations was also applied as a streak across Mueller-Hinton agar plates freshly pre-seeded with the bacteria named under ‘Methods and Materials. The results were recorded as digital photographe.
(c) Qualitv Control and Qualitv Assurance
Filter paper dises treated with PBS only were used as négative contrais. Assays were performed in triplicate.
(d) Results
The silver aggregations were effective in inhibiting the growth of ail the bacteria tested at ait the dilution ranges tested. There were no différences in the effectiveness of dilutions of 1.0% w/v and above. The silver aggregations were less effective at the lowest dilution of 0.1 % w/v for ail bacteria tested (see Table 1 below and Figures 2,3 and 4).
The effect of the growth inhibitor on E. edi was tested with *wet’ and 'dry1 dises. Similar zones of inhibition were observed between the two treatments taking into considération that the experiments were performed on different days (see Table 1, Figure 2). \A---17317
Figure 5 demonstrates the effects of a streak of the silver aggregations (20.0% w/v) on the growth of MRSA. Similar results were observed with E. coli and P. aeruginosa (photographe not shown).
Table 1: Mean dîameters (with standard errors) of the zones of inhibition for each of the bacterial species and dilutions of the silver aggregations tested
Bacteria Dilution of aggregations 70902 (% w/v) Mean diameter of zone of Inhibition (mm (SE))
E.coli fwet’ dises) 0 (PBS control) 0.0
0.1 14.3 (0.17)
1.0 17.5 (0.57)
2.5 16.8 (0.61)
10 17.5 (0.29)
E.coli (‘dry* dises) 0 (PBS control) 0.0
0.1 12.8(1.13)
1.0 18.3 (0.19)
2.5 18.0 (0.37)
10 18.7 (0.19)
P. aeruginosa (*wet’ dises) 0 (PBS control) 0.0
0.1 17.5 (0.76)
1.0 20.0 (0.73)
2.5 19.3(0.60)
10 20.2 (0.67)
MRSA (‘dry1 dises) 0 (PBS control) 0.0
0.1 11.9(0.08)
1.0 16.0(0.00)
2.5 16.0(0.00)
10 16.8 (0.17)
Example (iv): Further in vitro analysis to demonstrate bacteriostatic properties of the silver agareqation, prepared in accordance with Example (Malabove (a) Methods and Materials
0.5 g of the silver aggregations, as prepared in Example (i)(a) above, was provided in dry form.
The bacterial species used in the study were:
1) Clostridium difficile, an anaérobie bacteria, American Type Cell Collection (ATCC) #9689;
2) Vancomycin-resistant Enterococcus faecalis (#1061), an aerobe, obtained from the strain collection of Professor C. Dowson, Dept of Biology, University of Warwick; and
3) Salmonella enteritidis, an aerobe, ATCC #13076.
Disk diffusion bacteriostasis assay
A stock suspension of the silver aggregations was prepared (10% w/v in stérile phosphatebuffered saline with 0.1% v/vTween; Sigma-Aldrich Ltd, UK).
Dilutions were made to give 5,0, 1.0 and 0.1% w/v. Stérile filter paper dises of 5 mm diameter (filter cards 190005, Shandon Inc., Pittsburgh, USA) were saturated with the suspensions, vortex mixed, and the excess suspension was removed by aspiration. Dises were air-dried for at least 2 hours prior to use (‘dry1). The dises were then placed onto freshly pre-seeded agar plates prepared as follows. Bacterial suspensions in stérile PBS were prepared équivalent to a density of 0.5 on the McFarland scale (approximately 1.5 x 10e colony-forming units per ml) from agar plates of bacteria streaked for single colonies. A stérile cotton swab was used to pre-seed agar plates with lawns of each of the bacterial species (anaérobie blood for C. difficile, blood iso-sensitest for E. faecalis, or iso-sensitest for S. enteritidisr, Oxoid Ltd, UK), u—
Anaérobie conditions for incubation were achieved using the AnaeroGen Compact system from Oxoid. The system consists of a plastic pouch and a paper gas-generating sachet. The paper sachet contains ascorbic acid and activated carbon which react on contact with air. Oxygen is rapidly absorbed and carbon dioxide is produced. When the paper sachet is 5 placed in a sealed plastic pouch, the AnaeroGen Compact sachet reduces the oxygen content in the pouch to below 1.0% within 30 minutes. The resulting carbon dioxide content is between 8.0% and 14.0% depending on how many plates are placed in the pouch.
The plates were incubated ovemight at 37°C aerobically or anaerobîcally, as appropriate, 10 and any zones of inhibition around the dises were measured. The means (and standard errors) of triplicates were calculated.
(b) Biocide challenge assay
An ovemight culture of S. enteritidis was grown in brain-heart infusion (BHI) broth and 15 diluted 1/1000 in BHI. The silver aggregations were added to the diluted bacterial suspension to provide a final concentration of 1.0% or 0.1% w/v. The suspensions were incubated at 37°C with shaking. After 20 minutes, the suspensions were centrifuged at low speed to pellet the silver aggregations and 10 pL of the supematant was plated out onto isosensitest agar plates. The plates were incubated ovemight at 37°C and the bacterial 20 colonies were counted. The means (and standard errors) of triplicates were calculated.
(c) Quality Control and Quality Assurance
In the bacteriostasis assay, filter paper dises treated with PBS only were used as négative controls. Assays were performed in triplicate. In the biocide challenge assay, no inoculum 25 (silver aggregations only) and no silver aggregations (inoculum only) controls were included.
(d) Results
The silver aggregations as prepared in accordance with Example (i)(a) above were tested at 1.0 and 0.1% w/v using a disk diffusion method under aérobic conditions (Table 2). The 30 aggregations inhibited the growth of S. enteritidis at 1.0% w/v but not at 0.1% w/v. E faecalis was inhibited by the aggregations at both concentrations (Figures 6A and 6B). —
Bacteriostasis assay
The silver aggregations as prepared in accordance with Example (i)(a) above were tested at 5.0 and 1.0% w/v using the disk diffusion method under anaérobie conditions using a blood anaerobe agar (Table 2). C. difficile was inhibited by the aggregations at both dilutions tested. The zones of inhibition were accompanied with zones of haemolysis (clearing of the blood agar) of identical size.
Table 2: Mean diameters (with standard errors) of the zones of inhibition for each of the bacterial spedes and dilutions of the silver aggregations tested
Bacteria Dilution of compound VS190805 (%w/v) Mean diameter of zone of Inhibition (mm (SE))
S. enteritidis 0 (PBS only control) 0(0)
0.1 0(0)
1.0 7.92 (0.08)
E. faecalis 0 (PBS only control) 0(0)
0.1 7(0)
1.0 8(0)
C. difficile 0 (PBS only control) 0(0)
1.0 10(0)
5.0 10(0)
Tuming to Figures 6A and 6B, zones of inhibition were not as large as those observed in previous experiments (see results obtained from Example (iii)). The batch of silver aggregations used in this Example and the batches of the silver aggregations used in Example (iii) at 1.0% w/v were therefore compared in their activity towards S. enteritidis in a disk diffusion test. The same (small) zones of inhibition were observed with both batches of the silver aggregations (Figure 7).
In order to test whether the growth medium was affecting the size of the inhibition zones, a Mueller-Hinton agar plate was prepared and disks impregnated with PBS/Tween, 1.0 or 5.0% w/v silver aggregations were tested against S. enteritidis (Figure 8). Zones of inhibition were 8 mm for each concentration of silver which is similar to that observed with iso-sensitest agar (Table 2). There was no inhibition zone for PBS/Tween.
Biocide challenge assay
S. enteritidis in BHI was exposed to the silver aggregations at 1.0 or 0.1% w/v for 20 minutes (Table 3, Figures 9 and 10). Exposure to the silver aggregations for 20 min at 0.1 or 1.0% w/v resulted in a réduction of bacterial numbers by 57.8% and 99.8% respectively, compared to bacteria not challenged.
Table 3: Mean colony-forming units per mL (cfu/mL), with standard errors, for each of the dilutions of the silver aggregations tested
Dilution of aggregations VS190805 (%w/v) Mean cfu (SE)
S. enteritidis 0 31,333(5512)
0.1 13,233(1794.75)
1.0 67 (33.33)
No bacteria 0.1 0 (no replicates)
(e) Media Formulations
Iso-sensitest agar is a semi-defined medium designed for antimicrobial susceptibility testing. Said medium has a pH of 7.4 ± 0.2.
Oxoid ’lso-Sensitest Agar1 was developed specifically for antimicrobial susceptibility tests. Its formulation was carefully constructed to give a reprodudble, semi-defined medium in which the undefined components were kept to a minimal Ievel. However, it allows the growth of the majority of microorganisms without further supplémentation. —
Formula gm/litre
Hydroiysed casein 11.0
Peptones 3.0
Glucose 2.0
Sodium chloride 3.0
Soluble starch 1.0
Disodium hydrogen phosphate 2.0
Sodium acetate 1.0
Magnésium glycérophosphate 0.2
Calcium gluconate 0.1
Cobaltous sulphate 0.001
Cupric sulphate 0.001
Zinc sulphate 0.001
Ferrous sulphate 0.001
Manganous chloride 0.002
Menadione 0.001
Cyanocobalamin 0.001
L-Cysteine hydrochloride 0.02
L-Tryptophan 0.02
Pyridoxine 0.003
Pantothenate 0.003
Nicotinamide 0.003
Biotin 0.0003
Thîamine 0.00004
Adenine 0.01
Guanine 0.01
Xanthine 0.01
Uracil 0.01
Agar 8.0
Mueller-hinton agar is an antimicrobial susceptibility-testing medium that may be used in intemationally recognised standard procedures having a pH of 7.3 ± 0.1.
Formula gm/litre
Beef, dehydrated infusion from 300.0
Casein hydrolysate 17.5
Starch 1.5
The major use of Mueller-Hinton Agar is for Antimicrobial Susœptibility Testing (AST). It has become the standard medium for the Bauer-Kirby method and its performance is spedfied by the NCCLS. Oxoid Mueller-Hinton Agar meets the requirements of WHO.
Example (v): Comparative Analysis
Comparative Analysis: Colloïdal Silver
A standard agar plate with bacterial lawn was provided, as shown in Figure 11. Various filter papers, numbered 1 to 5 were also provided. Filter paper 1 represents a colloïdal silver 10 ppm solution applied to test filter paper. Filter paper 2 represents the antimicrobial composition of the présent invention, as prepared in accordance with Formulation A, at 100 times dilution applied to test filter paper. Filter paper 3 represents the antimicrobial composition of the présent invention, as prepared in accordance with Formulation A, at 10 times dilution. Filter paper 4 represents the antimicrobial composition of the présent invention, as prepared in accordance with Formulation A, at no dilution and filter paper 5 represents a blank control.
In terms of this Example, the zone of bacterial inhibition, discussed below, is an indicator of bacterial growth inhibition efficacy.
From Figure 11, it can be observed that there is no noticeable zone of bacterial inhibition around the colloïdal silver préparation (filter paper 1). As the concentration of the présent antimicrobial composition (Formulation A) is increased from 100 times dilution to no dilution (filter paper 2 to filter paper 4), the visible inhibition zone becomes more apparent and the size of this inhibition cortex or corona of inhibition increases. Even at 100 times dilution (filter paper 2), there is a small visible zone of inhibition présent
The présent Example thus indieates the effïcacy of the présent antimicrobial composition compared with colloïdal silver, which shows no visible effect.
The Inventor has thus found that the composition of the présent invention affords superior results when compared to the use of prior art products, including colloïdal silver.
Example (vi): Therapeutic use (a) Oral administration
For use during signs of infection, 1 ml of the antimicrobial composition of the présent invention, as prepared in accordance with Formulation A, is introduced to a small glass of water (25 ml) of water. Ordinary tap water or bottled water is suffirent. The solution may then be swiried in a useris mouth for a few seconds before swallowed.
For severe infection, an amount of 3 ml of the antimicrobial composition, as prepared in accordance with Formulation A, can be introduced to said quantity of water.
For optimum results, this solution is to be taken three to four times a day, approximately 5 minutes before meals.
Once the symptoms of the infection are no longer apparent, the dosage may be reduced to 1 ml per day. This may be continued for 10 days thereafter and may be continued for a longer period should the user feel at risk or exposed to further infection.
(b) Skin and Topical Infection
As mentioned herein before, the antimicrobial composition may be applied to plasters and bandages, and then applied to the infected area.
Altematively the antimicrobial composition, as prepared in accordance with Formulation A, can be applied directly to the site of infection.
(c) Test Patient
In October 2008, a test patient, Aaron X, was dytng of HIV/AIDS complicated by TB and candida (thrush) which had infected his mouth, throat and stomach making it almost impossible for him to eat or drink.
After blood work was taken, the hospital advised Aaron X that he had but days to live. Apart from a rehydration drip, Aaron X received no médication in the form of either antirétroviral or treatment for the thrush and TB.
ml of Formulation A was administered to Aaron X, three times per day. ln order to monitor improvements, regular blood tests were conducted and analyzed by Lancet Laboratories, South Africa.
The results of the blood tests are indicated herein below in Table 3 and graphs thereof are depicted in Figures 12 to 15.
Within days, Aaron X*s thrush had cleared completely and he was able to take fluids and solids unassisted. Within a couple of weeks he was able to get out of bed and take light walks. Within a year, his condition steadily improved and today, he is an active citizen enjoying a full and productive life.
Table 4:
Results of blood tests indicating the test patient’s CD4 cell count, white blood cell (WBC) count, viral load and érythrocyte sédimentation rate (ESR) __
Aaron X CD4 Count White Blood Cell Viral Load ESR
2008/10/24 56 422258
2008/12/18 76 4.89 303265 100
2009/01/27 118 5.56 109320
2009/02/02 139 5.81 120954
2009/02/09 166 7.6 101179
2009/02/13 225 8.36 76989
2009/02/20 245 8.15 64021 63
2009/02/27 175 5.93 200307 60
2009/03/06 183 6.28 86231 62
2009/03/13 182 5.99 163563 50
2009/03/20 206 8.16 104126 62
2009/03/27 168 5.78 120688 57
2009/04/03 202 6.68 196141 62
2009/04/09 202 7.3 87376 70
2009/04/17 236 6.27 119982 63
2009/04/24 121 5.65 269666 34
Test Patient Aaron X CD4 Count White Blood Cell Viral Load ESR
2009/04/30 196 6.64 291823 49
2009/05/08 180 7.83 159622 60
2009/05/15 137 7.84 124880 69
2009/05/22 154 6.93 100004 62
2009/05/29 168 7.89 53628 30
2009/06/05 109 7.34 84342 63
2009/06/12 8.36 48
2009/06/19 138 5.74 107901 61
2009/06/26 141 4.42 68376 57
2009/07/03 154 5.63 183480 64
2009/07/10 102 4.09 169857 65
2009/07/17 155 6.15 118914 59
2009/07/24 135 5.62 308225 29
2009/07/31 119 4.63 301794 73
2009/08/07 133 6.16 56808 48
2009/08/14 153 5.79 61584 49
2009/08/21 135 7.59 41460 35
2009/08/28 95 6.48 58279 41
2009/09/04 131 8.18 64333 25
2009/09/11 153 5.31 45846 48
2009/09/18 102 5.54 34343 51
2009/09/25 175 5.68 56696 59
2009/10/02 114 5.45 39606 71
2009/10/09 131 5.07 41239 43
2009/10/16 111 4.57 51442 56
2009/10/26 136 5.58 44801 50
2009/10/30 162 7.13 73847 43
2009/11/13 172 4.82 52140 54
2009/11/20 146 5.15 54868 44
2009/11/27 146 4.65 89326 50
2009/12/04 167 5.34 107455 41
2010/01/15 171 5.67 71289 57
(d) In vitro Anti-malarial Assay
Assay Background
The in vitro anti-malarial activity of test samples against the 3D7 (chloroquine-sensitive) strain of the malaria parasite, Plasmodium falciparum, is measured by assessing parasite survival after drug exposure using a parasite lactate dehydrogenase (pLDH) colorimétrie enzyme assay. pLDH activity is used as a surrogate for parasite levels in the cultures. To distinguish between pLDH and human LDH contaîned in the host red blood cells, APAD (3acetyîpyridine adenine nucléotide) is used as cofactor for the conversion of lactate to pyruvate instead of NAD. The human enzyme is incapable of using APAD. APADH formed in the reaction reduces nitro blue tétrazolium to a purple formazan product which absorbs at 620 nm.
In the standard dose-response assay, 11 x 3-fold serial dilutions of test samples are added to trophozoite-stage parasite cultures (2.0% parasitaemia, 1.0% haematocrit) in 96-well plates (duplicate wells for each compound concentration). After 48h of incubation, pLDH activity in the wells is determined at 620 nm using a multiwall spectrophotometer. Wells containing uninfected érythrocytes are used as background controls and their mean Absa» value subtracted from those ofthe test wells. Percentage parasite viability in the test wells is calculated by reference to control wells containing parasites incubated with compound-free medium. Dose-response curves are fitted to plots of % parasite viability vs. Iog[compound] by non-linear régression using GraphPad Prism software, to dérivé the 50% inhibitory concentration (ICæ). Chloroquine is used as an anti-malarial reference standard (ΙΟ» = 5 12 ng/ml).
Assay conditions
Two samples prepared from Formulation A, herein designated as Sample 1 and Sample 2, were provided as 10 mg/mL suspensions and stored at 4eC until use.
Eleven 3-fold serial dilutions of Sample 1 and Sample 2 were prepared (50 - 8.47 x 10-4 pg/ml). Care was taken to keep the particulates in the Samples in suspension by vigorous shaking/pipetting. The Samples were added to cultures containing trophozoite-stage parasites (2.0% parasitaemia, 1.0%haematocrit).
As a reference standard drug, chloroquine controls were prepared. These are parasite cultures devoid of any samples (100% viability reference).
Results
Samples were screened on two occasions (namely on 07/06/2011 and 15/06/2011). The IC50 values obtained are summarized in Table 5 below. The IC50 values are expressed as concentration (pg/ml) and percentage. Chloroquine was only included in the first screen.
Table 4: IC50 values obtained during the in vitro anti-malaria! assay
Test sample IC 50 (pg/mL) 07/06 IC50 (pg/mL )15/06 Average (pg/mL) IC50 (%) 07/06 IC50 (%) 15/06 Average (%)
1 Sample 1 2.06 1.34 1.7 0.0206 0.0134 0.0170
2 Sample 2 0.221 0.277 0.249 0.0022 0.0028 0.0025
Re f Chloroquin e 0.0166 0.0166
From the above, it is important to note that: 0.017% = 5,882-fold dilution of the original Sample and 0.0025% - 40,000-fold dilution of the original Sample.
The dose-response plots pertaining to Sample 1, Sample 2 and the chloroquine sample are depicted in Figures 16 to 20.
From these plots, it is important to note that %PV represents percentage parasite viability (i.e. parasites not exposed to the antimicrobial composition of the présent invention are used to define 100% parasite viability). SD represents the standard déviation,
Conclusion
In the présent assay, IC50 values of 1.0 - 10.0 pg/ml may be regarded as moderate anti5 malarial activity, 0.1 -1.0 pg/ml as good activity and <0.1 pg/ml as potent activity.
Observation of the results obtained from this assay and the graphs depicted in Figures 16 to 20 reveal that Sample 1 has moderate activity, while Sample 2 displays good activity.
Moderately active samples are unlikely to be promising therapeutics, without further modification.
The therapeutic potential of samples with good activity dépends on several additional factors. For instance, if the sample displays no toxic or other effects on human cells below 15 100 pg/ml, the therapeutic window may be large enough to warrant further investigation.
Moreover, if concentrations of the sample >1 pg/ml can be maintained (and tolerated) for several hours in the blood of human patients after oral administration, anti-malarial IC50 values of 0.1 - 1 pg/ml may be sufficient to resuit in cure. The latter dépends on additional factors, e.g. the stage of the malaria parasite life-cycle that is vulnérable to the sample and 20 how rapidly the sample kills parasites (both may be assessed in in vitro experiments).
(e) Anti-Cancer Assay - maïïanant melanoma cancer
In January 2011, treatment was administered to a patient for extensive and recurring 25 malignant skin melanoma cancer which had persisted for over 10 years.
Up to this point, the melanomas recurred and increased in number and frequency of récurrence. Consequently, said melanomas had to be surgically removed on a regular basis.
One particular cancerous site, as illustrated photographically in Figure 21, was due for surgical excision. Formulation A was orally and topically administered for two weeks. As can be seen from the Figure, the melanomas reduced in size with little scarring and discoloration remaining. The site was medically diagnosed as in remission and surgical intervention was not required. The melanoma has not, at the date of filing of the présent International application, recurred.
Conclusion
In conclusion, and as is discussed herein before, the administration of silver salts, such as silver nitrates, présent severe drawbacks, primarily attributed to the toxicity associated therewith as widely reported in the literature. In order to drcumvent the concems regarding toxicity, silver colloids were explored.
However, silver colloids are predominantly limited to concentrations of between 10 to 20 ppm of silver. Such low concentrations are insuffident to effectively treat and prevent severe infections, as is demonstrated in Example (v). The présent data provided herein thus demonstrates the limitations and disadvantages associated with silver colloids and reveal the superior advantages afforded by the présent invention.
Having described the invention in detail and by reference to the aspects and embodiments thereof, the scope of the présent invention is not limited only to those described charaderistics, aspects or embodiments. As will be apparent to persons skilled in the art, modifications, analogies, variations, dérivatives and adaptations to the above-described invention can be made on the base of art-known knowledge and/or on the base of the disdosure (e.g. the explidt, implidt or inhérent disdosure) of the présent invention without departing from the spirit and scope of this invention.

Claims (10)

1. An antimicrobial composition induding at least one or more friable aggregation(s) of silver partides in a liquid medium, wherein the concentration of the composition is from 40 ppm up to, and induding, 500 000 ppm of silver and wherein the aggregations hâve a surface area of 0.5 cm2/ml to 540 cm2/ml, both values indusive.
2. The antimicrobial composition according to daim 1, wherein the aggregations are friable and indude a plurality of loosely bound silver partides.
3. The antimicrobial composition according to daim 2, wherein the plurality of silver particles are loosely bound together via weak intermolecular van der Waals forces.
4. The antimicrobial composition according to daim 2, wherein the silver partides are loosely bound together to form one or more weak, friable, crystalline silver structures.
5. The antimicrobial composition according to any one of the proceeding daims, wherein the friable aggregations fradure or dissodate under the influence of weak mechanical forces to form a plurality of smaller silver aggregations, such that when the concentration of the antimicrobial composition is 10 000 ppm of silver and under the influence of weak mechanical forces, the surface area of the aggregations increases from 54 cm2/ml to 54 000 cm2/ml, both values indusive.
6. An antimicrobial préparation for use in the treatment of infections, diseases and/or disorders, comprising a therapeutically effective amount of the antimicrobial composition according to any one of daims 1 to 5 in combination with one or more suitable/acceptable excipients, addîtives or carriers.
7. Use of the antimicrobial composition according to any one of claims 1 to 5, in the manufadure of an antimicrobial préparation for the treatment, and/or prévention of infections, diseases or disorders. . »__
8. The antimicrobial préparation and/or use according to claim 6 or 7, wherein the infection, disease and/or disorder is selected from the group consisting of viral infections, bacterial infections, fungal infections, parasitic infections, protozoa infections, cancer and/or respiratory diseases,
9. The antimicrobial préparation and/or use according to daim 8, wherein the viral infection is selected from the group consisting of HIV infection, herpes virus infection, viral dysentery, flu, bronchitis, pneumonia, measles, rubella, chickenpox, mumps, polio, rabies, sinusitis, tonsillitis, mononudeosis, ebola, respiratory syncytial virus, croup, SARS, dengue fever, yellow fever, lassa fever, arena virus, bunyavirus, filovirus, flavivirus, hantavïrus, rotavirus, viral meningitis, H5N1 virus (bird flu), arbovirus, parainfluenza, smallpox, epstein-barr virus, dengue hémorrhagie fever, cytomégalovirus, infant cytomegalic virus, progressive multifocal leukoencephalopathy, viral gastroenteritis, hepatitis, cold sores, meningitis, encephalitis, shingles, warts, human papaloma virus, viral ear and eye infections.
10. The antimicrobial préparation and/or use according to daim 8, wherein the bacterial infection is selected from the group consisting of opportunistic infedions assodated with AIDS inter alia; tuberculosis, choiera, syphilis, bacterial pneumonia, Escherichia coli (e. coli) infections, candida infection, MRSA methicillin résistant Staphylococcus aureus (S. aureus), vancomycin résistant Enterococcus faecalis (E. faecalis), salmonella enteritidis (S. enteritidis), clostridium difficile (C. difficile) and pseudomonas aeruginosa (P.
aeruginosa) infection.
OA1201300384 2011-03-16 2012-03-16 Antimicrobial composition including at least one or more aggregation(s) silver particles. OA17317A (en)

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