WO2012123924A2 - Antimicrobial composition - Google Patents

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 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 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.

Background to the Invention Throughout history, silver has been regarded by many cultures as a versatile healing tool. Records showed that Hippocrates recognized the role of silver in the prevention of disease and that the Romans stored wine in silver vessels to prevent spoilage.

It is reported that silver exhibits a broad spectrum of antimicrobial activity in vitro. A possible mechanism responsible for such antimicrobial activity involves the binding of silver to microbial DNA thereby preventing bacterial replication, and the binding of silver to the sulfhydryl groups of the metabolic enzymes of the bacterial electron 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 blocking cell transport mechanisms causing inactivation of the microbe.

With the development of mass produced antibiotics, interest in silver as an antimicrobial medicine declined. The widespread use and frequent over-prescribing of antibiotics has led to an increasing incidence of microbes acquiring drug-resistance to current antibiotics. The emergence and spread of antibiotic resistance is an alarming concern in clinical practice.

In addition to silver having broad antimicrobial activities, silver has few, if any, known resistant 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 have antiseptic properties. One of the earliest uses of silver nitrate was in the preparation of infant eye drops in 1881. The use of silver nitrate in this application was highly instrumental for the prophylaxis of gonococcal ophthalmia in newborns. Whilst eye infections and blindness of newborns was reduced by this method, incorrect dosage could cause damage to the eye in extreme cases. Thus, despite the beneficial use of silver nitrate, several notable disadvantages are associated 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 immediate 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 prevention and treatment of infection in burn victims (see U.S. Patent Number 3,761 ,590 to Fox, incorporated herein by reference). Silver sulfadiazine has been associated with necrosis of the skin. In addition hereto, sulfadiazine may accumulate in patients with impaired hepatic and renal function. Moreover, patients allergic to sulfa agents may exhibit cross-hypersensitivity with silver sulfadiazine.

Colloidal silver is widely known and used in the art. A colloidal system consists of two separate phases, namely a dispersed phase and a continuous phase. Colloidal silver comprises a dispersed phase of microscopic silver particles in a continuous phase of water to form a stable suspension of microscopic silver particles which are dispersed evenly throughout the solution.

Said colloidal silver particles carry the same electrical charge (either positive or negative), known as a zeta potential. In terms of the zeta potential, the adjacent, similarly charged particles present in the colloid repel one another and in this way, the colloidal silver particles resist aggregation.

Aggregation of the particles would lead to flocculation and precipitation such that the resultant material would not be regarded as a colloid.

The commonly recommended daily dosage of colloidal silver is about one teaspoon, typically containing 5 to 30 parts per million of silver. Whilst it is claimed 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 have 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 in 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 have 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 present in commercial silver-based products including ionic silver, colloidal silver and protein- based silver colloids.

Table A: Literature data indicating the concentration of silver present in commercial silver- based products including ionic silver, colloidal silver and protein-based silver colloids

While there are compelling arguments both for and against the use of colloidal silver products as an antibiotic, the effectiveness and use of silver colloidal products in the human body to fight severe infections has not been authoritatively accepted. Medical authorities and publications advise against the ingestion of colloidal silver preparations, 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 modern health care, infection remains a serious complication with associated medical procedures. Although many substances have been suggested to guard against prosthesis-related infection, only a few have been demonstrated to be clinically protective. Medical prostheses have been fabricated from pure silver and its alloys, and silver molecules have been incorporated into the surfaces of a large variety of medical devices, including vascular, urinary and peritoneal catheters, 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 resides in their inherent localized and minimum surface area to volume characteristics. In the light of the foregoing, it is clear that the silver-based products known and used in the art have 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 prevention 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 have previously been described.

For purposes of the present specification, the word "aggregation" denotes a plurality of silver particles (atoms, molecules, or macromolecules) loosely bound together via weak intermolecular van der Waals forces. The word "aggregation" is also understood to denote a plurality of silver particles (atoms, molecules, or macromolecules) which are loosely bound together to form one or more weak, friable, crystalline silver structures.

The word "antimicrobial" as referred to herein is understood to encompass antibacterial, antifungal, antiprotozoal and antiviral properties.

In the context of the present 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 present invention provides an antimicrobial composition including at least one or more friable aggregation(s) of silver particles in a liquid medium, wherein the concentration of the composition is 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, have a particle size of 1 x 10^*9 m (1 nm) to 100 x 10^*9 m in diameter, both values inclusive. It will be appreciated that the particle size of each silver particle included in the aggregation(s) does not have 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^~9 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 10^"9 m in diameter, both values inclusive.

In 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^"9 m to 100 x 10^*9 m in diameter (both values inclusive) and/or to form a plurality of silver particles which are 10 x10^"9 m to 100 x 10^"9 m in diameter (again, both values being inclusive).

In terms of the present 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 present composition contemplates concentrations of 40 ppm to 500 000 ppm of silver, both values inclusive. In 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 appreciated by the skilled artisan that the surface area of silver in any given composition is directly proportional to its subsequent activity and efficacy as an antimicrobial agent. Particle surface area per unit volume of liquid medium increases as the concentration of silver particles increases. Furthermore, particle 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.

In terms of one embodiment of the invention, the silver aggregations have a surface area of 0.5 cm²/ml at lOOppm to 540 cm²/ml at 100000 ppm. It will be appreciated that the surface area pertaining to each silver particle included in the aggregation(s) does not have 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 appreciated that high particle surface area per unit volume of liquid medium is achieved by increasing the concentration of the silver particles, which in turn form the aggregations present in the antimicrobial composition, to 40 ppm of silver or higher.

When the friable aggregations of silver particles fracture or dissociate under the influence of weak mechanical forces, more surface area is created and hence the surface area is subsequently increased.

This additional surface area consequently increases the particle surface area per gram of silver in the antimicrobial composition of the present invention. Since the particle surface area is increased, further sites on the surface of the silver particles within the friable aggregation(s) are exposed and subsequently available for antimicrobial action.

In terms of a further embodiment of the present invention and under the influence of weak mechanical 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 cm²/ml. The greater the influence of the weak mechanical 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 present invention has sufficient active sites on the surface of the silver particles in order to retain antimicrobial activity in electrolytic environments having an acidic 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. Alternatively, 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 present 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 whilst avoiding the need to administer large volumetric 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, have reduced activity upon being administered, since these compounds react with bodily fluids and electrolytes such as chloride in the stomach to form less soluble silver compounds, such as silver chloride.

While it will be appreciated that any and all substances in the presence of water will have some ionic character, the present composition contemplates the inclusion/presence of no anionic species other than hydroxyl ions.

In an embodiment of the invention, the antimicrobial composition of the present invention may be in the form of a liquid dispersion.

In an alternative embodiment of the invention, the antimicrobial composition may take the form of a dry material. According to this embodiment, the antimicrobial composition may be dried and used/applied directly or further formulated with suitable carrier agents and mediums.

It will be appreciated that, in terms of the present invention, the silver particles 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 structures. By their very nature, van der Waals forces are relatively weaker than those arising from valence bonds. Thus, the weak van der Waals forces acting between the silver particles in conjunction with the friable nature of the crystalline structures of the aggregations will allow the silver aggregations to separate readily into their component particles by mechanical forces such as peristalsis and react, on a cellular level, once introduced to an infected site or body.

Without wishing to be bound by theory, the Inventor believes that the friable aggregations of silver particles present in the antimicrobial composition of the instant invention, together with the high concentration of silver present therein (40 ppm to 500 000 ppm of silver, both values inclusive) affords the superior antimicrobial properties achieved by the present composition in contrast to the products known and used in the art.

The Inventor has found 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 take place 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^"9 m to 100 x 10^"9 m in diameter, both values inclusive;

(ii) allowing said silver particles to aggregate together to form one or more aggregations which are 100 x 10^"9 m to 10 000 x 10^"9 m in diameter, both values inclusive; and (iii) said silver aggregations being friable and able to fracture or dissociate so as to form smaller silver aggregations in the range 10 x 10~⁹ m to 100 x 10^"9 m in diameter, both values inclusive, and/or small silver particles in the range 10 x 10^"9 m to 100 x 10^"9 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 deposition of silver, chemical deposition of silver, photo deposition of silver, plasma deposition of silver and comminution of silver pieces. 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, deposits 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 adherent or coherent mass or plate on the cathode. As electrolysis continues, further deposits of silver are formed on the surface of the deposits, 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 preparation

According to a third aspect of the invention, there is provided an antimicrobial preparation 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, alleviating and curative interventions.

Furthermore, in the context of the present invention, it is to be understood that the antimicrobial composition and/or antimicrobial preparation aids, supports, augments and accelerates the healing processes for, inter alia, wounds and invasive trauma. The term "effective amount" refers to that amount of antimicrobial composition that is required to provide therapeutic benefit. The present invention is not limited by the nature or scope of the therapeutic benefit provided. The degree of benefit may depend on a number of factors, inter alia, 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, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars, polysaccharides or sugar polymers), mineral oils and the like, which can be present singly or in combination. In a further embodiment of this aspect of the invention, there is provided for the antimicrobial preparation to possess activity towards cancer cells.

According to a fourth aspect of the invention, the present 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 preparation, 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 preparation for the treatment, and/or prevention of infections, diseases or disorders.

The invention further provides for the use of the antimicrobial composition, as described and identified herein, for the treatment, diagnosis and/or prevention of diseases and/or disorders.

According to the invention, one or more agents may be used in combination with the present antimicrobial composition.

The above mentioned agent(s) can be any compound, chemical, therapeutic agent, antbiotic agent, drug, biological molecule, antibody, protein, lipid, nucleic acid, 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 contemplates the use of nutritional agents in combination with the antimicrobial composition of the present invention. Such nutritional agents include, but are not limited to, nutrients, nutriceuticals, minerals, vitamins, amino acids and essential fats.

It is envisaged that such combination will serve to increase the spectrum of activity of the antimicrobial composition and create a synergistic effect.

Non-limiting examples of the agents contemplated for use with the antimicrobial composition of the present invention include acne preparations such as isotretinoin, benzoyl peroxide, salicylic acid and tetracycline; anesthetics for topical administration such as dibucaine, lidocaine, benzocaine, tetracacine, deperodon and pramoxine hydrochloride; anti-inflammatory agents such as betamethasone benzoate, betamethasone valerate, desonide, fluocinolone acetonide, halcinonide, hydrocortisone; antiperspirants; antipruritic and external analgesic agents such as camphor, menthol, salicylic acid, methylsalicylate; cleansing agents; pigmenting agents; anabolic steroids for building up tissues under wound healing such as methandienone; proteolytic agents for the decomposition 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 acid and EGF (epidermal growth factor); antibiotics; analgesics; immune agents such as immune regulatory proteins and immunotherapy drugs; and chemotherapeutic agents.

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 present in the antimicrobial composition function to prevent secondary infection at the diseased site.

Administration

Administration of the antimicrobial composition and/or antimicrobial preparation of the present invention can be done in any acceptable manner known in the medical arts.

Specific, non-limiting examples of administration methods which may be used in accordance with embodiments of the present 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 preparation can be applied using gloved hands or by an applicator. Likewise, the composition and/or antimicrobial preparation 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 preparation of the present 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 preparation thereto. By way of further illustration, infections of the mouth can be treated by applying the antimicrobial composition and/or antimicrobial preparation with a sponge applicator or a toothbrush.

The mode of administration can be dependent 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 preparation of the present invention can be administered to in vitro and in vivo systems.

The present invention also contemplates cells that have been altered by the antimicrobial composition and/or antimicrobial preparation of the present invention and the administration of such cells to other cells or tissues, in in vitro or in vivo methods.

The antimicrobial composition and/or antimicrobial preparation of the present invention can be administered for clinical use in humans and for veterinary use, such as with domestic animals, in manners known in the art and similar to other therapeutic agents.

It is envisaged that the present antimicrobial composition and/or antimicrobial preparation may be administered for horticulture as well as agriculture use. It is further envisaged that the present antimicrobial composition and/or antimicrobial preparation may be administered for the treatment of raw materials including, but not limited to, food and water.

Formulation

The antimicrobial composition and/or antimicrobial preparation of the present invention can be incorporated with other ingredients to form a variety of products for administration, as detailed herein below.

The antimicrobial composition and/or antimicrobial preparation 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 excipients such as fillers, lubricants, disintegrants, taste masking agents and the like.

The antimicrobial composition and/or antimicrobial preparation of the present invention may be used in cosmetics and personal care products to make said products resistant to antimicrobial contamination. Non-limiting examples thereof include creams, ointments, sunscreens, mouth rinses, toothpastes, dental flosses, gels, moisturizers, foams, powders, liquid and powder makeup foundations, powder and cream blushes, lipsticks and lip-glosses, lip pencils, mascaras, eye liners, eye shadows, perfumes, colognes, deodorants, toners, wipes for skin application, dermal patches, shaving creams, shampoos, conditioners and various hair treatments like mousses and sprays. In another embodiment of the invention, the antimicrobial composition and/or antimicrobial preparation may be incorporated in aerosols and sprays for topical or inhalation application.

In addition hereto, the antimicrobial composition and/or antimicrobial preparation 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 present invention may be incorporated into medical devices including, but not limited to medical implants and wound care devices and can be used to impart antiseptic 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 include, but are not limited to, urinary and intravascular catheters, dialysis shunts, wound drain tubes, endotracheal 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, burn dressings, biological graft materials, tape closures and dressings and surgical drapes. Dosage

The appropriate dosage of the antimicrobial composition, antimicrobial preparation and/or formulation of the present invention, as indentified herein, will depend 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 preparation and/or formulation is administered for therapeutic or preventive purposes; previous therapy and the patient's clinical history and response to the composition. The antimicrobial composition, antimicrobial preparation and/or formulation of the present invention is/are suitably administered to a patient at one time or over a series 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 readily 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 preparation 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 present invention relates to methods of treating (including prophylactically treating) viral infections, bacterial infections, fungal infections, parasitic infections and/or cancer. The invention is envisaged to have a regenerative effect on damaged tissue thereby facilitating and expediting the healing process. Examples of viral infections which may be treated using the methods and/or composition, preparation and/or formulation of the present invention include, without limitation, HIV/AIDS infection, herpes virus infection, viral dysentery, flu, bronchitis, pneumonia, measles, rubella, chickenpox, mumps, polio, rabies, sinusitis, tonsillitis, mononucleosis, 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 hemorrhagic fever, cytomegalovirus, 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, preparation and/or formulation of the present invention include, without limitation, tuberculosis, cholera, syphilis, bacterial pneumonia, Escherichia coli (e. coli) infections, Candida infection, RSA methicillin resistant Staphylococcus aureus (S. aureus) infection - strain ATCC #43300, vancomycin resistant 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. Examples of fungal infection which can be treated and prevented using the methods and/or composition, preparation and/or formulation of the present invention include, without limitation, thrush, candidiasis, cryptococcosis, histoplasmosis, blastomycosis, aspergillosis, coccidioidomycosis, paracoccidiomycosis, sporotrichosis, zygomycosis, chromoblastomycosis, lobomycosis, mycetoma, onychomycosis, piedra pityriasis versicolor, 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, preparation and/or formulation of the present invention include, inter alia, malaria (including congenital and cerebral malaria), ringworm, tapeworm, lice, typhoid fever and typhus.

The present invention also provides methods for treating cancerous tissue in a subject. The present invention has been shown to be effective in reducing the size of and even eliminating cancerous tumors. The types of cancers which can be treated using the methods of the present invention include, 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 like. Further, the invention contemplates methods for treating cancerous tissue in a subject in conjunction with chemotherapies and radiothereapies.

The present invention further provides methods for treating respiratory diseases in a subject including, but not limited to, asthma, Tuberculosis (TB) and pulmonary fibrosis.

The present invention yet further provides methods for treating skin conditions, infections and diseases in a subject including, inter alia, eczema, acne, vitiligo, warts, burn wounds, blisters and scars.

Thus the present invention contemplates 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 present invention measured at 15 minute intervals in accordance with the analysis conducted in Example (ii);

Figure 1 B 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 Example (ii);

Figure 1C shows photographs 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 disc diffusion test for the inhibition of the growth of E.coli by the silver aggregations prepared in accordance with Example (i)(a) of the present invention; Figure 3 is a graph depicting the results of a disc diffusion test for the inhibition of the growth of P. aeruginosa by the silver aggregations prepared in accordance with Example (i)(a) of the present invention;

Figure 4 is a graph depicting the results of a disc diffusion test for the inhibition of the growth of MRSA by the silver aggregations prepared in accordance with Example

(i)(a) of the present invention;

Figure 5 is a photograph of the silver aggregations (20.0% w/v) applied to a pre-seeded lawn of MRSA demonstrating bacterial growth inhibition (after overnight 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 triplicate) 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 negative 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 biocide challenge assay against S. enteritidis;

Figure 10: is a photograph depicting the results of a biocide 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 illustrates the growth of S. enteritidis without the addition of the silver aggregations of the present invention;

Figure 11 : is a photograph depicting an agar plate showing the effect of a silver colloid preparation on microbial growth inhibition (filter paper 1 ) when compared to varying concentrations of the antimicrobial composition of the present 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;

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: is a graph depicting the results of a test patient's erythrocyte sedimentation 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 antimalarial assay as screened on 07/06/2011 ; is a graph depicting the dose-response plot for Sample 2 used in the in vitro antimalarial assay as screened on 07/06/2011 ; is a graph depicting the dose-response plot for Sample 1 used in the in vitro antimalarial assay as screened on 15/06/2011 ; is a graph depicting the dose-response plot for Sample 2 used in the in vitro antimalarial assay as screened on 15/06/2011 ; 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 are photographs taken during the anti-cancer assay showing malignant skin melanoma cancer before and after treatment with the antimicrobial composition of the present invention.

The presently disclosed subject matter, including the preparation of the silver aggregations, Formulation A and Samples 1 and 2, will now be described more fully hereinafter with reference to the accompanying Examples, in which representative 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 complete, and will fully convey the scope of the embodiments to those skilled in the art.

The Examples of the invention are divided into the following parts: (i) Process for preparing the antimicrobial composition of the present invention; (ii) Formulations including the antimicrobial composition thus prepared and analyses of the properties of the antimicrobial composition; (iii) In vitro analysis to demonstrate antimicrobial properties of the silver aggregation, as prepared in accordance with Example (i)(a); (iv) 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 turn.

Example (i): Process for preparing the antimicrobial composition of the present invention

(a) Preparation of silver aggregations

Electrolysis 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 electrodes. It will be appreciated that regulated or pulsed DC are also suitable. 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 closer 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 employed.

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 complement 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 alternate 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 adhere to the cathode and the deposit appeared as a loosely bound, amorphous particulate 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^"9 m to 10000 x 10^"9 m 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.

The aggregations were preferably kept wet although they may be dried for alternative use such as blends with petroleum products, e.g. Vaseline, to produce creams and ointments.

(b) Preparation of the 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 of the formulations described herein below. Example (it): Formulations including the antimicrobial composition of the present 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 present 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 lesion;

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 integral 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, emulsions, etc. Analyses of the properties of the 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

The silver aggregations present in a diluted sample of Formulation A (10:1 water to Formulation A), was subjected to mechanical agitation by sonication in order to demonstrate the aggregations' ability to dissociate/fracture into smaller silver particulates.

The results indicated that under mechanical agitation, the silver aggregations fractured/dissociated into silver particulates such that a particle diameter (d nm) (d 1 x 10^"9 m) change from 400 to 40 d nm was observed using Dynamic Light Scattering analysis (DLS). Only particle diameter sizes below 1000 d nm were considered. In terms of this Example, it will be appreciated that the qualitative relationship between surface area and particle diameter is cubic. The increase by such mechanical agitation can thus be qualitatively considered to be a factor of (400:40)³ i.e. 1000:1. It is appreciated that the silver particulates do not possess ideal geometries. A factor of 10% (100:1 ) of the above value is considered conservative, especially when it is appreciated that the initial silver aggregations of Formulation A prior to agitation are shown by DLS to have diameters averaging 4000 d nm and above, which would further augment the potential increase in surface area on fracturing.

Fracturing/dissociation 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

Form the above Table, it can clearly 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 cm²/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) Dissociation/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 of the 1000 ppm stock solution was evaluated by DLS. The results reveal that the silver aggregations have an average size diameter of 5500 nm.

A second aliquot of the 1000 ppm stock solution was analyzed using sonication. The ultra sonic was initiated and five consecutive measurements of the aliquot were taken at 15 minute intervals. Particle size was measured at 15 minute intervals using DLS in conjunction with sonication, using Malvern Instruments Zetasizer, 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 1 A indicates that the silver aggregations below 1000 nm in the aliquot have an initial size of 400 nm. After sonication for 15 minutes (Record 7), the silver aggregations have 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 reduction at 400 nm and further fracturing from 80 nm down to 40 - 50 nm is observed. This collectively demonstrates the fracturing of the silver aggregations at 400 nm into silver particulates at 40 to 50 nm.

Figure 1 B 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 clearly reveal that the silver aggregations of the microbial composition are friable and dissociate/fracture into smaller silver particulates by the action of mechanical forces. It is contemplated that other forms of mechanical action, 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 determine 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 electrolyte levels found in ambient bodily fluids, this being an environment where the antimicrobial composition of the instant invention is required to be effective. Furthermore, a comparative evalution was made by employing a commercial silver colloid product. The plate analysis is depicted in the photographs represented in Figure 1 C.

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 petri 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 NaCI 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 1 C clearly 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 clearly in test 5 and 6. There is a very slight observable diminution in microbial growth for the commercial 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 commercial silver colloid product.

Example (Hi): In vitro analysis to demonstrate bacteriostatic properties of the silver

aggregations, as prepared in accordance with Example (0(a) 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 bacterial strains used in the study were:

1 ) Escherichia coli, a clinical strain^*;

2) Pseudomonas aeruginosa, a clinical strain^*; and

3) Methicillin-resistant Staphylococcus aureus (MRSA), strain ATCC 43300.

* Obtained from the Respiratory Research Laboratory, Department of Medicine, University of Birmingham. Bacterial suspensions in sterile PBS were prepared equivalent to a density of 0.5 on the McFarland scale (approximately 1.5 x 10⁸ colony-forming units per ml) from overnight incubations of bacteria streaked for single colonies. A sterile cotton swab was used to pre-seed Mueller- Hinton agar plates (Oxoid Ltd, UK) with lawns of each of the bacterial strains. (b) Bacteriostasis assay

A stock suspension of the silver aggregations was prepared (20% w/v in sterile phosphate- buffered saline with 0.1% v/v Tween; Sigma-Aldrich Ltd, UK).

Dilutions were made to give 10.0, 2.5, 1.0 and 0.1 % w/v. Sterile filter paper discs 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. Discs were used immediately ('wet') or air-dried for 2 hours prior to use ('dry'). The discs were then placed onto freshly pre-seeded Mueller-Hinton agar plates prepared as described above under "Methods and Materials". The plates were incubated overnight at 37°C and zones of inhibition around the discs 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 photographs.

(c) Quality Control and Quality Assurance Filter paper discs treated with PBS only were used as negative controls. Assays were performed in triplicate.

(d) Results The silver aggregations were effective in inhibiting the growth of all the bacteria tested at all the dilution ranges tested. There were no differences 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 all bacteria tested (see Table 1 below and Figures 2, 3 and 4). The effect of the growth inhibitor on E. coli was tested with 'wet' and 'dry' discs. Similar zones of inhibition were observed between the two treatments taking into consideration that the experiments were performed on different days (see Table 1 , Figure 2).

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 £ coli and P. aeruginosa (photographs not shown). Table 1 : Mean diameters (with standard errors) of the zones of inhibition for each of the bacterial species and dilutions of the silver aggregations tested

Example (iv): Further in vitro analysis to demonstrate bacteriostatic properties of the silver aggregation, prepared in accordance with Example (iMa)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 bacterial species used in the study were:

1) Clostridium difficile, an anaerobic 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. (b) Disk diffusion bacteriostasis assay

A stock suspension of the silver aggregations was prepared (10% w/v in sterile phosphate- buffered saline with 0.1% v/v Tween; Sigma-Aldrich Ltd, UK).

Dilutions were made to give 5,0, 1.0 and 0.1 % w/v. Sterile filter paper discs 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. Discs were air-dried for at least 2 hours prior to use ('dry'). The discs were then placed onto freshly pre-seeded agar plates prepared as follows. Bacterial suspensions in sterile PBS were prepared equivalent to a density of 0.5 on the McFarland scale (approximately 1.5 x 10⁸ colony-forming units per ml) from agar plates of bacteria streaked for single colonies. A sterile cotton swab was used to pre-seed agar plates with lawns of each of the bacterial species (anaerobic blood for C. difficile, blood iso- sensitest for E. faecalis, or iso-sensitest for S. enteritidis; Oxoid Ltd, UK).

Anaerobic 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 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 overnight at 37°C aerobically or anaerobically, as appropriate, and any zones of inhibition around the discs were measured. The means (and standard errors) of triplicates were calculated.

(c) Biocide challenge assay An overnight culture of S. enteritidis was grown in brain-heart infusion (BHI) broth and 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 μί of the supernatant was plated out onto iso-sensitest agar plates. The plates were incubated overnight at 37°C and the bacterial colonies were counted. The means (and standard errors) of triplicates were calculated. (d) Quality Control and Quality Assurance

In the bacteriostasis assay, filter paper discs treated with PBS only were used as negative controls. Assays were performed in triplicate. In the biocide challenge assay, no inoculum (silver aggregations only) and no silver aggregations (inoculum only) controls were included.

(e) 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 aerobic conditions (Table 2). The 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 anaerobic 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 species and dilutions of the silver aggregations tested

Turning 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 reduction 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

Mean cfu (SE)

VS190805 (% w/v)

S. enteritidis 0 31 ,333 (5512)

0.1 13,233 (1794.75)

1.0 67 (33.33)

No bacteria 0.1 0 (no replicates)

( ) 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 ^' Iso-Sensitest Agar' was developed specifically for antimicrobial susceptibility tests. Its formulation was carefully constructed to give a reproducible, semi-defined medium in which the undefined components were kept to a minimal level. However, it allows the growth of the majority of microorganisms without further supplementation.

Formula gm/litre

Hydrolysed 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

Magnesium glycerophosphate 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

Thiamine 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 internationally recognised standard procedures having a pH of 7.3 ± 0.1.

The major use of Mueller-Hinton Agar is for Antimicrobial Susceptibility Testing (AST). It has become the standard medium for the Bauer-Kirby method and its performance is specified by the NCCLS. Oxoid Mueller-Hinton Agar meets the requirements of WHO. Example (v): Comparative Analysis Comparative Analysis: Colloidal 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 colloidal silver 10 ppm solution applied to test filter paper. Filter paper 2 represents the antimicrobial composition of the present 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 present invention, as prepared in accordance with Formulation A, at 10 times dilution. Filter paper 4 represents the antimicrobial composition of the present 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 colloidal silver preparation (filter paper 1 ). As the concentration of the present 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 present.

The present Example thus indicates the efficacy of the present antimicrobial composition compared with colloidal silver, which shows no visible effect. The Inventor has thus found that the composition of the present invention affords superior results when compared to the use of prior art products, including colloidal silver.

Example (vi): Therapeutic use (a) Oral administration For use during signs of infection, 1 ml of the antimicrobial composition of the present 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 sufficient. The solution may then be swirled in a user's 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. Alternatively 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 dying 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 medication in the form of either antiretroviral or treatment for the thrush and TB.

3 ml of Formulation A was administered to Aaron X, three times per day. In 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 erythrocyte sedimentation rate (ESR)

Aaron X CD4 Count White Blood Viral Load ESR

Cell

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 CD4 Count White Blood Viral Load ESR

Aaron X Cell

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) colorimetric enzyme assay. pLDH activity is used as a surrogate for parasite levels in the cultures. To distinguish between pLDH and human LDH contained in the host red blood cells, APAD (3-acetylpyridine adenine nucleotide) 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 tetrazolium 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 erythrocytes are used as background controls and their mean Abs₆2o value subtracted from those of the 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. log[compound] by non-linear regression using GraphPad Prism software, to derive the 50% inhibitory concentration (IC₅₀). Chloroquine is used as an antimalarial reference standard (IC₅₀ = 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 4°C until use. Eleven 3-fold serial dilutions of Sample 1 and Sample 2 were prepared (50 - 8.47 x 10-4 g/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 07/06/201 1 and 15/06/201 1 ). The IC50 values obtained are summarized in Table 5 below. The IC50 values are expressed as concentration (μς/ητιΙ) and percentage. Chloroquine was only included in the first screen.

Table 4: IC50 values obtained during the in vitro anti-malarial assay

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 present invention are used to define 100% parasite viability). SD represents the standard deviation.

Conclusion

In the present assay, ICso values of 1.0 - 10.0 pg/ml may be regarded as moderate anti-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 depends on several additional factors. For instance, if the sample displays no toxic or other effects on human cells below 100 ig/ \, the therapeutic window may be large enough to warrant further investigation. Moreover, if concentrations of the sample >1 pg/rnl can be maintained (and tolerated) for several hours in the blood of human patients after oral administration, anti-malarial ICso values of 0.1 - 1 pg/ml may be sufficient to result in cure. The latter depends on additional factors, e.g. the stage of the malaria parasite life-cycle that is vulnerable to the sample and how rapidly the sample kills parasites (both may be assessed in in vitro experiments).

(e) Anti-Cancer Assay - malignant melanoma cancer In January 2011 , treatment was administered to a patient for extensive and recurring 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 recurrence. 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 present International application, recurred.

Conclusion

In conclusion, and as is discussed herein before, the administration of silver salts, such as silver nitrates, present severe drawbacks, primarily attributed to the toxicity associated therewith as widely reported in the literature. In order to circumvent the concerns 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 insufficient to effectively treat and prevent severe infections, as is demonstrated in Example (v). The present data provided herein thus demonstrates the limitations and disadvantages associated with silver colloids and reveal the superior advantages afforded by the present invention. Having described the invention in detail and by reference to the aspects and embodiments thereof, the scope of the present invention is not limited only to those described characteristics, aspects or embodiments. As will be apparent to persons skilled in the art, modifications, analogies, variations, derivatives and adaptations to the above-described invention can be made on the base of art-known knowledge and/or on the base of the disclosure (e.g. the explicit, implicit or inherent disclosure) of the present invention without departing from the spirit and scope of this invention.

Claims

Claims

1. An antimicrobial composition including at least one or more friable aggregation(s) of silver particles in a liquid medium, wherein the concentration of the composition is from 40 ppm up to, and including, 500 000 ppm of silver.

2. The antimicrobial composition according to claim 1 , wherein the silver particles are elemental silver (Ag) particles.

3. The antimicrobial composition according to claim 1 or claim 2, wherein the silver particles have a particle size of 1 x 10^"9 m to 100 x 10^*9 m in diameter, both values inclusive.

4. The antimicrobial composition according to claim 1 , wherein the aggregations are friable and include a plurality of loosely bound silver particles.

5. The antimicrobial composition according to claim 4, wherein the plurality of silver particles are loosely bound together via weak intermolecular van der Waals forces.

6. The antimicrobial composition according to claim 4, wherein the silver particles are loosely bound together to form one or more weak, friable, crystalline silver structures.

7. The antimicrobial composition according to any one of claims 4 to 6, wherein the one or more aggregations of silver particles are 100 x 10^"9 m to 10 000 x 10^"9 m in diameter, both values inclusive.

8. The antimicrobial composition according to claim 4, wherein 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^*9 m to 100 x 10^~9 m in diameter (both values inclusive) and/or to form a plurality of silver particles which are 10 x10^"9 m to 100 x 10^"9 m in diameter (both values being inclusive).

9. The antimicrobial composition according to claim 1 , wherein the concentration of the composition is from 1 000 ppm to 10 000 ppm of silver, both values inclusive.

10. The antimicrobial composition according to claim 1 , wherein the aggregations have a surface area of 0.5 cm²/ml to 540 cm²/ml, both values inclusive.

11. The antimicrobial composition according to claim 10, wherein additional surface area is created and hence the surface area is subsequently increased when the friable aggregations of silver particles fracture or dissociate under the influence of weak mechanical forces.

12. The antimicrobial composition according to claim 1 , wherein the additional surface area consequently increases the particle surface area per gram of silver in the antimicrobial composition.

13. The antimicrobial composition according to claim 11 , wherein the surface area of the aggregations increases from 54 cm²/ml to 54 000 cm²/ml, both values inclusive, under the influence of weak mechanical forces when the concentration of the antimicrobial composition is 10 000 ppm of silver.

14. The antimicrobial composition according to claim 1 having sufficient active sites on the surface of the silver particles in order to retain antimicrobial activity in electrolytic environments having an acidic or low pH or where such environments are saline.

15. The antimicrobial composition according to claim 1 , wherein the liquid is water.

16. The antimicrobial composition according to claim 1 or 15, wherein the liquid is a suitable organic medium.

17. The antimicrobial composition according to claim 1 , characterized in that said composition is in the form of a dry material.

18. A process for preparing the antimicrobial composition according to any one of claims 1 to 17, said process including the steps of:

(i) providing silver particles having a particle size of 1 x 10^"9 m to 100 x 10^"9 m in diameter, both values inclusive;

(ii) allowing said silver particles to aggregate together to form one or more aggregations which are 100 x 10^~9 m to 10 000 x 10^"9 m in diameter, both values inclusive; and

(iii) said silver aggregations being friable and able to fracture or dissociate so as to form smaller silver aggregations in the range 10 x 10^"9 m to 100 x 10^"9 m in diameter, both values inclusive, and/or small silver particles in the range 10 x 10^"9 m to 100 x 10^"9 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.

19. An antimicrobial preparation 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 claims 1 to 17 in combination with one or more suitable/acceptable excipients, additives or carriers.

20. 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 according to any one of claims 1 to 17, or an antimicrobial preparation according to claim 19.

21. Use of the antimicrobial composition according to any one of claims 1 to 17, in the manufacture of an antimicrobial preparation for the treatment, and/or prevention of infections, diseases or disorders,

22. Use of the antimicrobial composition according to any one of claims 1 to 17 for the treatment, diagnosis and/or prevention of diseases and/or disorders.

23. The antimicrobial preparation, method and/or use according to any one of claims 19 to 22, wherein the infection, disease and/or disorder is selected from the group consisting of viral infections, bacterial infections, fungal infections, parasitic infections, cancer and/or respiratory diseases.

24. The antimicrobial preparation, method and/or use according to claim 23, wherein the viral infection is selected from the group consisting of HIV/AIDS infection, herpes virus infection, viral dysentery, flu, bronchitis, pneumonia, measles, rubella, chickenpox, mumps, polio, rabies, sinusitis, tonsillitis, mononucleosis, 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 hemorrhagic fever, cytomegalovirus, infant cytomegalic virus, progressive multifocal leukoencephalopathy, viral gastroenteritis, hepatitis, cold sores, meningitis, encephalitis, shingles, warts, human papaloma virus, viral ear and eye infections.

25. The antimicrobial preparation, method and/or use according to claim 23, wherein the bacterial infection is selected from the group consisting of tuberculosis, cholera, syphilis, bacterial pneumonia, Escherichia coli (e. coli) infections, Candida infection, RSA methicillin resistant Staphylococcus aureus (S, aureus) infection - strain ATCC #43300, vancomycin resistant 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.

26. The antimicrobial preparation, method and/or use according to claim 23, wherein the fungal infection is selected from the group consisting of thrush, candidiasis, cryptococcosis, histoplasmosis, blastomycosis, aspergillosis, coccidioidomycosis, paracoccidiomycosis, sporotrichosis, zygomycosis, chromoblastomycosis, lobomycosis, mycetoma, onychomycosis, piedra pityriasis versicolor, tinea barbae, tinea capitis, tinea corporis, tinea cruris, tinea favosa, tinea nigra, tinea pedis, otomycosis, phaeohyphomycosis and rhinosporidiosis.

27. The antimicrobial preparation, method and/or use according to claim 23, wherein the parasitic infection is selected from the group consisting of malaria (including congenital and cerebral malaria), ringworm, tapeworm, lice, typhoid fever and typhus.

28. The antimicrobial preparation, method and/or use according to claim 23, wherein the cancer is selected from the group consisting of 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 like.

29. The antimicrobial preparation, method and/or use according to claim 23, wherein the respiratory disease is selected from the group consisting of asthma, Tuberculosis (TB) and pulmonary fibrosis.