HK1117387B - Anti-microbial agent - Google Patents

Description

Antimicrobial agents

RELATED APPLICATIONS

This application claims priority from australian provisional application No. 2005900247, the contents of which are incorporated herein by reference.

FIELD

The present invention relates to antimicrobial agents. In particular, the invention relates to broad spectrum antimicrobial agents.

Background

Microorganisms that are resistant to antimicrobial agents are becoming an increasing problem worldwide. For example, methicillin-resistant strains of Staphylococcus aureus (staphyloccus aureus) (MRSA, also known as "Staphylococcus aureus") are now resistant to a variety of antibiotics, and recent isolates exhibit increasing resistance to antibiotics such as gentamicin, chloramphenicol, fusidic acid, and rifampicin. Only vancomycin currently used for empirical therapy can be relied upon. There is a great concern that the increasing use of vancomycin will select vancomycin-resistant strains of MRSA so that there may no longer be any effective antibiotic treatment against nosocomial staphylococci in the near future. Accordingly, research is ongoing to identify alternative antimicrobial agents, particularly broad spectrum antimicrobial agents.

Hospitals are examples of environments where acquired MRSA infections are a serious problem. These infections put the patient at risk, increase nursing costs, and reduce the number of beds available. In other environments, such as dental studios, laboratories, food preparation areas, schools, etc., it is also important to prevent or minimize the growth of such bacteria, as well as other microorganisms including fungi and viruses.

Hospitals and these other environments that require high levels of hygiene have many strategies to reduce the spread of these microorganisms. Prior to entering the operating room, the patient is treated topically with a disinfecting solution such as chlorhexidine or povidone-iodine. MRSA carriers are treated with these topical antimicrobials or, in severe cases, with antibiotics such as vancomycin. Vancomycin is expensive to administer and, therefore, less expensive and effective alternatives to oral delivery would be welcomed.

Known topical antimicrobial agents cannot be relied upon to provide adequate protection against such bacteria, especially when more resistant strains are present. They cannot be administered in other manifestations such as oral tablets. Such topical antimicrobial agents are also ineffective at preventing or inhibiting the growth of other microbial species present in hospitals and other such environments. Although these environmental surfaces can be washed with disinfectants and disinfectants, including chlorine-containing bleaches in the form of hypochlorous acid/hypochlorite ions, these agents are very corrosive and alternative methods are needed.

Thus, there is a need for new antimicrobial and/or antibacterial agents suitable for these and similar applications.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference to the extent that they are fully understood. However, such references should not be taken as an admission that any of these documents forms part of the common general knowledge in the art, in australia or in any other country. The discussion in the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents.

SUMMARY

In a first aspect, the present invention provides a method of preventing or inhibiting bacterial growth, the method comprising the step of contacting a composition comprising a C8-C24 polyunsaturated fatty acid or ester thereof and terpinen-4-ol or derivative thereof with a surface.

The applicant has found that alpha-linolenic acid, which is a polyunsaturated fatty acid from C8 to C24, is effective in preventing or inhibiting bacterial growth. The activity of this pure alpha-linolenic acid is similar to that of the polyunsaturated fatty acids closely related to alpha-linolenic acid, as further explained below. Alpha-linolenic acid has been found to be effective in preventing or inhibiting the growth of gram-negative and gram-positive bacteria, and is known to have this activity against a wide variety of microorganisms, and is an effective broad-spectrum antimicrobial.

Applicants have found that when a fatty acid or ester is used in combination with terpinen-4-ol or a derivative thereof (i.e., another terpinene derivative), synergistic antimicrobial activity is achieved. In addition, such compositions have activity against a wider range of microorganisms including fungi. In fact, the results of the tests show that the compositions containing both components are approximately 10 times as active as the compositions containing only one of the components.

The surface is suitably contacted with a composition comprising terpinen-4-ol or derivative thereof, either before or after contact with a C8-C24 polyunsaturated fatty acid or ester composition, for a range of times to obtain a synergistic effect. Preferably, the surface is contacted with a composition comprising both a C8-C24 polyunsaturated fatty acid or ester and terpinen-4-ol or derivative thereof such that the contact times are synchronized.

While not wishing to be bound by theory, it is believed that the C8-C24 polyunsaturated fatty acids or esters thereof exert their antimicrobial effects inside microorganisms by disrupting fatty acid metabolism, while visual observation suggests that terpinen-4-ol and its derivatives attack the cell walls of the microorganisms. Thus, these active drugs act through different mechanisms, thus reducing the chance that a particular microorganism will develop resistance to both drugs. Furthermore, both the C8-C24 polyunsaturated fatty acid or ester thereof and the terpinen-4-ol (or derivative) are volatile drugs and, therefore, are less likely to remain in contact with microorganisms for extended periods of time, thereby further reducing the chance of microorganisms developing resistance to such drugs.

According to a second aspect, the present invention also provides a composition comprising a C8-C24 polyunsaturated fatty acid or ester thereof and terpinen-4-ol or derivative thereof. Preferably, the C8-C24 polyunsaturated fatty acid or ester thereof is alpha-linolenic acid, most preferably alpha-linolenic acid is in substantially pure form (as defined below). Preferably the composition comprises terpinen-4-ol. Preferably, the terpinen-4-ol is in substantially pure form (as defined below). The composition may take a wide variety of presentation forms including a body wash, a surface spray, a paper towel, a topical or nasal ointment, a tablet or other form.

In a third aspect, the present invention provides a method of preventing or inhibiting the growth of both gram-positive and gram-negative bacteria on a surface, the method comprising the step of contacting the surface with a composition comprising a C8-C24 polyunsaturated fatty acid or ester thereof and terpinen-4-ol or derivative thereof.

In a fourth aspect, the present invention provides the use of a C8-C24 polyunsaturated fatty acid or ester thereof and terpinen-4-ol or derivative thereof in the manufacture of a composition for use in preventing or inhibiting the growth of bacteria. Preferably the composition is a composition for preventing or inhibiting the growth of gram positive and gram negative bacteria.

In a fifth aspect, the present invention provides a method of preventing or treating a disease or condition caused by bacteria, the method comprising administering to a subject in need of treatment a composition comprising a C8-C24 polyunsaturated fatty acid or ester thereof, terpinen-4-ol or derivative thereof, and a carrier. The mode of administration is preferably topical.

The compositions of the present invention may further comprise one or more other active agents. However, in certain embodiments, the only active drugs present are the C8-C24 polyunsaturated fatty acids or their esters and terpinen-4-ol or its derivatives.

Brief description of the drawings

Figure 1 is a graph of the log reduction in organism count of microbial numbers after 30 minutes of contact with alpha-linolenic acid.

Figure 2 is a graph illustrating the% reduction in organism count of the number of microorganisms contacted with a composition according to one embodiment of the invention.

FIG. 3 is a graph showing the minimum concentration of alpha-linolenic acid required to inhibit the growth of various microorganisms.

Detailed description of the invention

In this specification, except where the context requires otherwise due to express language or necessary implication, "comprise" or "comprises" is used in an inclusive (inclusive) sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a virus" includes a plurality of viruses. Likewise, reference to "terpinen-4-ol (a perpinene-4-ol) or a derivative thereof" includes mixtures of more than one compound within the category.

The present inventors have found that compositions comprising a C8-C24 polyunsaturated fatty acid or ester thereof and terpinen-4-ol or derivative thereof have broad spectrum antimicrobial activity, including activity against gram positive and gram negative bacteria, including aerobic, anaerobic and facultative aerobic bacteria. This broad spectrum of activity provides a broad range of applications for the composition. It has also been found that the composition can be used in different forms which further enhance the use of the composition. It has been unexpectedly found that C8-C24 polyunsaturated fatty acids or esters thereof provide synergistic activity when administered with terpinen-4-ol or a derivative thereof. In addition, the combination composition has activity against a wider range of microorganisms, including the gram positive and gram negative bacteria and fungi mentioned above.

C8-C24 polyunsaturated fatty acids

The preferred form of the C8-C24 polyunsaturated fatty acid or ester thereof is described above as alpha-linolenic acid. Alpha-linolenic acid is present in the corresponding glyceride form in linseed and other oils, usually as one of several C8-C24 polyunsaturated fatty acid components. Alpha-linolenic acid is a C18 fatty acid containing three double bonds (C18: 3) at the 9, 12, and 15 positions.

Although the composition may in its most major aspect comprise a mixture of C8-C24 polyunsaturated fatty acids or esters thereof, it is preferably not present as a component of linseed oil or hydrolysed linseed oil. Preferably it is used in the composition in purified or extracted form. Thus, the compositions used in the present application preferably comprise mono-C8-C24 polyunsaturated fatty acids at a level substantially free of other C8-C24 polyunsaturated fatty acids. Most preferably, the composition comprises alpha-linolenic acid, which is substantially free of any other C8-C24 monounsaturated or polyunsaturated fatty acids or esters thereof. By substantially free, it is meant that such components (components substantially free of in the composition) are not present at levels greater than 20% (weight-to-weight) of the mono-fatty acids, e.g., alpha-linolenic acid, present.

C8-C24 polyunsaturated fatty acids, meaning straight-chain or branched alkenyl carboxylic acids containing a hydrocarbon chain having an unsaturation of at least 2 and 8-24 carbon atoms. Preferably the fatty acid is a C18 fatty acid, more preferably it has an unsaturation of 3. Preferably the fatty acid is a cis fatty acid.

Esters of such fatty acids include the class of compounds that can be produced by reacting such fatty acids with any aliphatic or aromatic organic alcohol. It is noted that such products need not be the direct product of such a reaction, and may be formed or derived from different starting materials. The alcohol may be, for example, a polyol, such as a diol or triol, and may contain any other functional group. Alkyl esters are most typical.

Terpinen-4-ols and derivatives thereof

C8-C24 polyunsaturated fatty acids are used in combination with terpinen-4-ol or a derivative thereof.

Terpinen-4-ol is a component of tea tree oil. A commercially available terpinen-4-ol mixture from tea tree oil is known under the trademark Melaleucol. The structure of one of the major isomers of terpinen-4-ol is as follows:

the term terpinen-4-ol includes a single isomer (e.g., the isomers set forth above) or any other individual isomer and mixture of isomers, including racemic mixtures.

Terpinen-4-ol extracts obtained from tea tree oil are usually in the form of a mixture of (+) and (-) enantiomers in a ratio of 1.8-2.4: 1.

Terpinen-4-ols are terpinene derivatives containing a 3-hydro-4-hydroxy group. Terpinen-4-ol derivatives included in this application are other terpinene derivatives in which one or both double bonds are saturated, the compounds being substituted with one or more substituents selected from the group consisting of: hydrogen, alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, amino, alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino, benzylamino, dibenzylamino, acyl, alkenylacyl, alkynylacyl, aroyl, acylamino, diacylamino, acyloxy, alkylsulfonyloxy, arylsulfonyloxy, heterocyclyl, heterocyclyloxy, heterocyclylamino, haloheterocyclyl, alkylsulfinyl, arylsulfinyl, alkoxycarbonyl, aryloxycarbonyl, mercapto, alkylthio, benzylthio, acylthio, and phosphorus-containing groups. Terpinen-4-ols are derivatives containing hydrogen and hydroxyl substituents from one end of the terpinene saturated C3-4 double bond to the other.

However, it is preferred to use terpinen-4-ol (including isomeric mixtures thereof) in substantially pure form. The expression "substantially pure form" means that terpinen-4-ol or a derivative thereof is used in a form substantially free of the other major components of tea tree oil. By substantially it is meant herein that the terpinen-4-ol component used in the composition contains no more than 20% by weight of tea tree oil of a non-terpinen-4-ol component. Thus, this level includes both pure terpinen-4-ol extracts and extracts that retain a small amount of tea tree oil impurities. Of course, terpinen-4-ol or its derivatives may be synthetically produced, in which case any non-terpinen-4-ol component not present in tea tree oil would not be expected.

Components of the composition

The compositions containing C8-C24 polyunsaturated fatty acids used in the present invention preferably contain 0.1% to 50% by weight of C8-C24 polyunsaturated fatty acids. More preferably the composition comprises or is applied at a concentration of 0.1-20% w/w, depending on the form of the composition and its route of administration. At levels below 0.1%, the efficacy of the composition is not as high as desired for a commercially acceptable product. Skin irritation may occur, or be difficult to handle, at levels above 50% C8-C24 polyunsaturated fatty acids (e.g., alpha-linolenic acid). Typically for forms suitable for direct application to the skin, the level of C8-C24 polyunsaturated fatty acids will be below this level-typically no more than 10%. A good balance of antimicrobial properties, ease of use and cost is achieved for compositions comprising 0.1% to 5% C8-C24 polyunsaturated fatty acids, particularly alpha-linolenic acid.

The composition preferably comprises between 0.1% and 5% terpinen-4-ol or derivative thereof, more preferably between 0.25% and 5% terpinen-4-ol or derivative thereof. Preferably, the terpinen-4-ol is itself present in the composition in substantially pure form at the levels given.

For compositions comprising both a C8-C24 polyunsaturated fatty acid or ester thereof (preferably alpha-linolenic acid in substantially pure form) together with terpinen-4-ol or derivative thereof (preferably terpinen-4-ol in substantially pure form), the ratio of these two components in the composition is advantageously between 1: 5 and 5: 1, more preferably between 1: 2 and 2: 1.

The compositions of the present invention typically further comprise a carrier. The carrier may be present in the form of a pharmaceutically acceptable carrier, or it may be a more conventional carrier.

Pharmaceutical carriers for the preparation of Pharmaceutical compositions are well known in the art, as set forth in textbooks such as Remington's Pharmaceutical Sciences, 20th Edition, Williams & Wilkins, Pennsylvania, USA. The carrier will depend on the route of administration and the skilled person will readily be able to determine the most appropriate formulation for each particular situation.

Examples of different manifestations are lotions, suspensions, solutions, sprays, emulsions, pastes, foams, eye washes, ointments, liquid soaps, creams, solid soaps, mouthwashes, pastilles or lozenges, gels, tissues, hair tonics, shampoos or jellies.

In the case of compositions of the invention in the form of a surface spray, the carrier is suitably water and the composition may further comprise a preservative such as benzyl alcohol (e.g. in an amount of 0.1 to 5 wt%), a co-solvent such as propylene glycol (e.g. in an amount of 1 to 20 wt%) and a surfactant such as Cremophor Rh40 (e.g. in an amount of 1 to 20 wt%).

Where the composition of the invention is in the form of a lotion or body wash, it may comprise an emulsion of a C8-C24 polyunsaturated fatty acid or ester and optionally terpinen-4-ol in water, in the presence of a suitable emulsifier. Examples of suitable emulsifiers are cetostearyl alcohol, Emulgade, SLEX (Texapon), betaine derivatives such as those listedEmpigen B range of surfactants and Tween (e.g. Tween 80)ICI Chemicals)。Tween 80Is a surfactant comprising polyoxyethylene 20 sorbitan monooleate. The composition may further comprise emollients and fragrances (including oils such as sweet almond oil), preservatives (such as benzyl alcohol at levels of e.g. 0.1-10%) and humectants and/or emollients and/or excipients (such as glycerol at levels of e.g. 0.5-10% and cyclomethicone at levels of 0.1-20%).

When the composition of the present invention is in the form of a wet wipe, it may contain the above-mentioned carriers, preservatives, emulsifiers, softeners and/or fragrances.

When the composition of the present invention is in the form of an ointment, it may contain a conventional ointment base to which the active ingredient is added. The ointment base may be a paraffin, such as soft paraffin, or a combination of soft paraffin and liquid paraffin. Other ointment bases, such as polyalkylene glycol (e.g., polyethylene glycol or polypropylene glycol) bases, may also be used. Other possible components of the ointment composition include emulsifying waxes (e.g., in an amount of 1-40%, preferably 5-40%) and preservatives.

When the composition of the present invention is in the form of a liquid soap, any known liquid soap may be used. Preferred liquid soaps are those based on coconut oil. The pH of the liquid soap base may be adjusted to a pH suitable for topical use.

When the composition of the present invention is in the form of a cream, any suitable cream base may be used. The preferred cream base is a sorbolene cream. Other cream bases may also be used.

When the composition is in the form of a solid soap, it is preferred to use a synthetic soap base. Synthetic soap bases are preferred because of their properties that are more suitable for sustained application to weakened skin and their pH is more easily controlled. Examples of synthetic soap bases suitable for use in the compositions of the present invention include: synbase A and Synbase BW available from Witco (USA), Synbase Commercial Bar from dove (USA), Tensia from Dermactif (Belgium) and Syndet Base pH 6-7 from Zetesap (Germany). As an example of ingredients for such a soap base, Syndbase BW comprises disodium myristamidomea sulfosuccinate (disodiummyristotanido MEA sulfosuccinate), stearic acid, sodium cocoyl isethionate (sodium cocoyl isethionate), paraffin, PEG 75, zinc stearate and water.

When the composition is in the form of a lozenge, it may comprise a flavouring base, for example sucrose and acacia or tragacanth.

When the composition is in the form of an eye wash, it may comprise a suitable sterile aqueous or non-aqueous vehicle. Additives such as buffers, preservatives (including bactericides and fungicides, such as phenylmercuric acetate or nitrate, benzalkonium chloride or chlorhexidine) and thickeners (such as hypromellose) may also be included.

The composition may further comprise other agents in addition to any of the ingredients listed above. For example, agents such as binders, sweeteners, thickeners, flavoring agents, disintegrants, coatings, preservatives, lubricants and/or retarding agents. Such suitable examples are well known in the art.

C18-C24 polyunsaturated fatty acids are known to be readily oxidized by air. The present inventors have empirically found that the compositions of the present invention have a useful life of at least 2 years. Which can be extended by adding an antioxidant to the composition.

The compositions of the present invention may include or be used in conjunction with agents such as therapeutic, antibacterial and/or antibacterial agents. A "therapeutic agent" is an agent used to treat a disease or disorder. Suitable therapeutic agents are antibiotics such as vancomycin, penicillin and related antibiotics, methicillin, gentamicin, chloramphenicol, fusidic acid, rifampin, and the like. Other therapeutic agents include surgical implants, prostheses, and catheters.

Suitable antibacterial or antianaphylactic agentsThe bacterial agent comprises TriclosanSapoderm, chlorhexidine, povidone iodine, cetyltrimethylammonium bromide, hexachlorophene, hypochlorite-based antibacterial agents, or mixtures thereof. Other agents may be present in the composition in an amount up to 20% by weight of the total composition, more preferably up to 15% by weight. The amount of the other agent will depend on the activity of the agent; however, suitable amounts will be readily determined by those skilled in the art.

The compositions of the present invention preferably have a pH in the range of 4.8 to 7.5. More preferably, the pH is in the range of 5.8 to 7.0. Within these pH ranges, the form of the composition that can be applied to the skin produces little irritation to the skin or mucosa of the subject after application.

Method of producing a composite material

The present invention provides methods of preventing or inhibiting the growth of microorganisms. As used herein, "preventing" means killing the microorganism. By contrast, "inhibit" means reduce or limit microbial growth. For example, bacteriostatic agents inhibit the growth of bacteria but do not kill bacteria, while bacteriocidal agents kill bacteria. The compositions of the present invention may be described as antibacterial agents. When formulated for application to a surface or for dilution in an aqueous solvent, the composition may be described as a disinfectant. When formulated for administration to a subject, including a human or non-human animal, the composition may be described as an antibacterial agent.

By "growth" is meant an increase in the size and/or number of microorganisms. Microbial growth can be measured by any method known in the art, including both direct and indirect methods. For example, direct methods include measuring microbial numbers, such as by serial dilution and plate counting. Indirect methods include estimating the number of microorganisms from the turbidity or metabolic activity of the culture. Alternatively, the dry weight of the culture can be used to determine microbial growth.

A "microorganism" (microbe or microbe) is any organism that is visible in size under a microscope. Microorganisms include bacteria, viruses, fungi (e.g., yeast and mold), algae, and the like.

"bacteria" are unicellular organisms that are surrounded by a cell wall, the genetic material of which is not surrounded by a nuclear membrane. The bacteria may be gram positive or gram negative, aerobic, anaerobic or facultative anaerobic and may be in one of several shapes, such as rods (bacilli), spheres or ellipsoids (cocci), helices (spirochetes), stars or squares. The various bacteria may form pairs, chains, clusters, or other groups.

Examples of bacteria whose growth can be prevented or inhibited according to the present invention include: escherichia coli (Escherichia coli), Klebsiella pneumoniae (Klebsiella pneumoniae), Listeria monocytogenes (Listeria monocytogenes), methicillin-resistant bacteria, Neisseria shalensis (Neisseria flavescens), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Salmonella salfordii (Salmonella salford), Salmonella cholerae (Salmonella cholerae), Serratia marcescens (Serratia marcocens), Staphylococcus aureus, Staphylococcus epidermidis (Staphylococcus epidermidis), Streptococcus faecalis (Streptococcus faecalis), Streptococcus faecium (Streptococcus faecalis), and Moraxella catarrhalis (Moraxella catarrhalis). Table 1 provides some of the characteristics of these bacteria.

Table 1.

The "virus" is very small and requires an electron microscope for observation, and is acellular. The virion contains a core consisting of only one type of nucleic acid (DNA or RNA), surrounded by a protein capsid. The capsid may be surrounded by a lipid envelope. Viruses can only multiply within cells of other organisms.

A "fungus" is a microorganism whose cells have an apparent nucleus containing the cytogenetic material and surrounded by a nuclear membrane. The fungus may be unicellular or multicellular. The fungus in the form of a single cell is a yeast. When referring to fungi, yeast is the preferred form of the fungus. Yeast is an oval-shaped microorganism larger than bacteria. The most typical fungus is a mold. The mold forms a mycelium, which is a long, branched filament that is intertwined. Examples of fungi suitable for use in the present invention include aspergillus niger (a. niger) and candida albicans (c. albicans).

Microbial growth can be prevented or inhibited by contacting a composition comprising a C8-C24 polyunsaturated fatty acid, or ester thereof, in association with terpinen-4-ol or a derivative thereof, with a microorganism or a surface with which a microorganism can come into contact, or by administering the composition to a subject. The contacting can be in any suitable manner, and can have a direct or indirect effect on the microorganism. For example, the composition itself may affect microbial growth. Alternatively, the composition may reduce the attachment of the microorganism to a surface, or affect another component of the microorganism culture, thereby affecting microorganism growth.

The surface may be any suitable surface, such as a human or animal body, or an inanimate object.

The microorganism may suitably be contacted with the composition by topical and/or oral administration of the composition to a subject in need of treatment. Alternatively, the composition may be applied to an inanimate surface that contains a microorganism or is suspected of or at risk of containing the microorganism. The term "surface" is used in its broadest sense and should not be construed to imply any particular physical dimension.

The compositions may be administered by any suitable route, and the skilled person will readily be able to determine the most appropriate route and dosage for the condition to be treated. The dosage will be at the discretion of the attendant physician or veterinarian, depending on the nature and state of the condition to be treated, the age and general health of the subject to be treated, the route of administration and the previous treatment which may have been given.

The composition may be administered periodically or repeatedly to a subject in need thereof, and may be administered to the site of actual or possible infection. For example, if an infection has occurred in a surgical wound or it is desired to prevent infection, the composition may be administered over the wound or to the wound and surrounding. If infection of the nasal passage is likely or has occurred, or it is desired to prevent it, it may be appropriate to administer to the nasal passage a composition which may be in the form of a nasal ointment or nasal spray. If an infection of the throat has occurred, or it is desired to prevent it, the composition may be in the form of a throat rinse, lozenge or spray.

Suitable prophylactic treatment regimens include washing the patient with the composition, optionally after separate antibacterial treatment (depending on the components of the composition). This may be done on a periodic or repeated basis. The composition of the invention may be in the form of a body wash, or may be applied to a gauze after application of the lotion. The composition is allowed to dry. This procedure can be repeated several times a day, preferably 3 times a day. Nasal ointments or sprays may also be used.

"disease" is a general term used to refer to any deviation from health that a subject suffers from. "disorder" refers to an abnormality in the function or function of a part of the body. Examples of diseases or conditions that can be prevented or treated by a composition comprising a C8-C24 polyunsaturated fatty acid or ester thereof include: cellulitis, necrotizing subcutaneous infections (e.g., necrotizing fasciitis), abscesses, bacterial infections, pruritus, dermatitis, eczema, psoriasis, acne, boils, bedsores, miliaria, skin blemishes, ichthyosis, tinea and ulcers, mycoses, herpes (i.e., cold sores, chicken pox, and shingles), and parasitic infections. The method of the present invention may also be used to improve the appearance of dry, sloughed, wrinkled, aged or photodamaged skin. It may also be used to alleviate or reduce the associated effects of a disease or condition, such as pain, itching, swelling, and the like.

The "subject" may be a mammal. The mammal may be a human, or may be a domestic or companion animal. Although it is particularly contemplated that the compounds of the invention are suitable for use in humans, they may also find application in veterinary applications, including the treatment of companion animals (e.g., dogs and cats) and domestic animals (e.g., horses, cattle and sheep) or zoo animals (e.g., non-human primates, felines, canines, bovines, and ungulates).

The present invention employs, unless otherwise indicated, conventional chemical and microbiological techniques within the capabilities of those skilled in the art. Such techniques are well known to the skilled person and are explained fully in the literature.

It is to be expressly understood that this invention is not limited to the particular materials and methods described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

When a range of values is recited, it is expressly understood that the range includes the upper and lower limits of the range and all values between these upper and lower limits.

Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described in detail by reference only to the following non-limiting examples and the accompanying drawings.

Example 1.Determination of the minimum inhibitory concentration of alpha-linolenic acid

The following test was conducted to determine the minimum alpha-linolenic acid concentration required to inhibit the growth of three bacterial strains, which are representative microorganisms that are desired to be inhibited with the antimicrobial composition. The test results show that the corresponding compositions have low levels as low as 0.1%.

A 90% sample of alpha-linolenic acid was diluted to test for minimum effective concentration against bacteria as per the following protocol. Alpha-linolenic acid was diluted with soybean tryptone broth from Sigma Aldrich to the dilutions shown in tables 2A-2F. By about 10⁶Each of these test microorganisms was inoculated into each diluted sample and incubated at 37 ℃ for 48 hours. The test microorganisms were staphylococcus aureus, salmonella choleraesuis, MRSA, escherichia coli, streptococcus pyogenes (s.pyogenes), and moraxella catarrhalis. An uninoculated broth was included as a control.

After the incubation period, the respective broths were examined for bacterial growth, expressed by the turbidity of the broth. Each broth sample was subcultured to confirm the presence of the test microorganism. The highest dilution series that achieved inhibition of the test microorganism was considered the Minimum Inhibitory Concentration (MIC).

The turbidity of each dilution series for each test microorganism is shown in tables 2A, 2B, 2C, 2D, 2E and 2F.

Table 2A test microorganisms: staphylococcus aureus ATCC 6538

Table 2B test microorganisms: salmonella choleraesuis ATCC 10708

Table 2C test microorganisms: methicillin-resistant staphylococcus wild strain

Table 2D test microorganisms: escherichia coli ATCC 8739

Table 2E test microorganisms: streptococcus pyogenes (University of Melbourne MDU)

Table 2F test microorganisms: moraxella catarrhalis

Note that: "-" indicates no growth and "+" indicates growth.

Example 2.Compositions comprising alpha-linolenic acid

A composition comprising 1% and 2% alpha-linolenic acid was prepared with the following components:

1% solution:

2% solution:

example 3.Antimicrobial activity of compositions comprising alpha-linolenic acid

The 1% solution of example 2 was tested to demonstrate that such a solution is effective in preventing the growth of both gram positive and gram negative bacteria.

The 1% alpha-linolenic acid solution was tested for its bactericidal effect against MRSA (gram positive, facultative anaerobes), salmonella choleraesuis (gram negative, aerobic) and pseudomonas aeruginosa (gram negative, anaerobic) using the suspension test. The suspension test was carried out according to the following procedure:

1. the initial microbial concentration of each test microbial culture, comprising 1% alpha-linolenic acid and a control microbial culture, was determined using standard plate counting techniques. The microbial concentration of each culture was recorded as cfu/mL.

2. At the desired time point, samples of each culture were taken and the microbial concentration of each culture was determined using standard plate count techniques. Concentrations are reported as cfu/mL.

3. The% killing by alpha-linolenic acid was calculated by comparing the cfu/mL difference over time for the test microorganisms with the cfu/mL difference over the same time for the control microorganism culture.

Fresh bacterial cultures and solutions were used for each experiment and the results are expressed as geometric means of two replicates as shown in table 3.

TABLE 3 Sterilization Effect of alpha-linolenic acid

Note that: results are geometric means of duplicate experiments.

Streptococcus pyogenes was also tested. The results for this microorganism showed that the% kill was 87.7% in 30 minutes. Coli (gram-negative) and moraxella catarrhalis (gram-negative) were further tested. These results and the results for all microorganisms tested in this example are shown in figures 1 and 2 (using two metrics-log reduction and% reduction in organism count).

Example 4.Compositions comprising alpha-linolenic acid and terpinen-4-ol

Compositions containing 1% alpha-linolenic acid and 1% terpinen-4-ol were prepared in the form of body wash, topical spray, topical ointment and nasal ointment. The components of the compositions are summarized in tables 4A, 4B, 4C and 4D. Melaleucol is a commercially available terpinen-4-ol of minimum 99% purity having a chiral +/-enantiomeric ratio of 1.8: 1 to 2.4: 1.

TABLE 4A bath lotion

TABLE 4B surface sprays

TABLE 4C topical ointment

TABLE 4D nasal ointment

Example 5.Antimicrobial activity of compositions comprising alpha-linolenic acid and terpinen-4-ol

The composition of example 4 was tested for antimicrobial activity. The results shown below indicate that the composition is extremely effective in preventing the growth of gram-positive bacteria, gram-negative bacteria and fungi.

The compositions were tested for antimicrobial activity against the following organisms at room temperature using the suspension test described in example 3:

staphylococcus aureus

Escherichia coli

Pseudomonas aeruginosa

Candida albicans

Aspergillus niger

Neisseria subflaveri

MRSA

The results of the body wash are shown in tables 5A and 5B.

TABLE 5A survival results (cfu/mL) for different contact times

CFU (colony Forming Unit) less than

TABLE 5B Log reduction in differential contact time (Log)₁₀)

Less than greater than

The surface spray results are shown in tables 6A and 6B.

TABLE 6A survival results (cfu/mL) for different contact times

CFU (colony Forming Unit) less than

TABLE 6B Log reduction in different contact times (Log)₁₀)

Less than greater than

The nasal ointment results are shown in tables 7A and 7B.

TABLE 7A survival results (cfu/mL) for different contact times

CFU (colony Forming Unit)

TABLE 7B Log reduction in different contact times (Log)₁₀)

Less than greater than

The results of the topical ointment are shown in tables 8A and 8B.

TABLE 8A survival results (cfu/mL) for different contact times

CFU (colony Forming Unit)

TABLE 8B Log reduction in differential contact time (Log)₁₀)

Less than greater than

Example 6.Synergistic activity of alpha-linolenic acid and terpinen-4-ol

The antimicrobial activity of alpha-linolenic acid and terpinen-4-ol, alone and in combination, was determined using the suspension test outlined in example 3 above.

A 1% solution of alpha-linolenic acid in water was prepared to determine the activity of the drug alone. This solution is denoted solution a. The results of the microbial recovery estimates after a given contact time with solution a are shown in table 9A below.

1% terpinen-4-ol (specifically Melaleucol) in water was prepared to determine the activity of the drug alone. This solution is denoted as solution B. The results of the microbial recovery estimates after a given contact time with solution B are shown in table 9B below.

A solution of a combination of 1% alpha-linolenic acid and 1% terpinen-4-ol (specifically Melaleucol) in water was prepared to determine the activity of the combination. This solution is denoted as solution C. The results of the microbial recovery estimates after a given contact time with solution C are shown in table 9C below (in terms of viable microbial recovery levels) and Log₁₀And (4) reducing.

TABLE 9 recovery results of contacting solution A composition comprising 1% alpha-linolenic acid alone for various times

CFU (colony Forming Unit) less than

TABLE 9 recovery results for different times of exposure of solution B to solution B

CFU (colony Forming Unit) less than

TABLE 9 recovery results for various times of contact of solution C with solution C

CFU (colony Forming Unit) less than

Table 9D compares Log reduction (Log) after contact with solution C₁₀)

Less than greater than

It was observed that both alpha-linolenic acid and terpinen-4-ol are effective antimicrobial agents. Furthermore, a synergistic antimicrobial effect between alpha-linolenic acid and terpinen-4-ol was observed, since a 2 × log reduction in viable Pseudomonas aeruginosa was observed after 30 minutes₁₀In contrast, 1 XLog was reduced with alpha-linolenic acid or terpinen-4-ol alone₁₀. This corresponds to a 10-fold increase in antimicrobial effect compared to the individual components used alone, greater than the 2-fold effect resulting from the additive effect of using the two components.

It will be apparent to those skilled in the art that, although the present invention has been described in some detail for purposes of clarity and understanding, various modifications and improvements can be made to the embodiments and methods described herein without departing from the scope of the inventive concepts disclosed in the specification.

Claims (26)

1. A composition for preventing or inhibiting bacterial growth comprising alpha-linolenic acid and 0.1% to 5% terpinen-4-ol.

2. The composition of claim 1, further comprising a C8-C24 polyunsaturated fatty acid, or ester thereof, other than alpha-linolenic acid, present at a level no greater than 20% by weight of the alpha-linolenic acid.

3. The composition of claim 1 or 2, wherein the composition comprises 0.1% to 50% by weight of alpha-linolenic acid.

4. The composition of claim 3, wherein the composition comprises alpha-linolenic acid at a concentration of 0.1-20% w/w.

5. The composition of claim 1 or 2 wherein said composition comprises 0.25% to 5% of said terpinen-4-ol.

6. The composition of claim 1 or 2 wherein the ratio of alpha-linolenic acid to terpinen-4-ol is from 1: 5 to 5: 1.

7. The composition of claim 6, wherein the ratio is 1: 2 to 2: 1.

8. The composition of claim 1 or 2, wherein the composition is in the form of a lotion, suspension, solution, spray, emulsion, paste, foam, ointment, liquid soap, cream, solid soap, pastille or lozenge, gel, tissue, hair tonic, or jelly.

9. The composition of claim 8, wherein the composition is in the form of an eye wash, mouthwash or shampoo.

10. The composition of claim 8, wherein the composition is in the form of a body wash, a surface spray, a paper towel, and a topical ointment.

11. The composition of claim 10, wherein the composition is in the form of a nasal ointment.

12. The composition of claim 10 or 11 wherein the composition comprises 0.1% to 5% alpha-linolenic acid and 0.1% to 5% terpinen-4-ol.

13. The composition of claim 1 or 2, wherein the pH is in the range of 4.8-7.5.

14. The composition of claim 1 or 2, wherein the composition is a disinfectant composition.

15. Use of a composition comprising alpha-linolenic acid and terpinen-4-ol in the manufacture of a medicament for preventing or inhibiting bacterial growth.

16. Use according to claim 15, wherein the composition is as defined in any one of claims 1 to 14.

17. The use of claim 15 or 16, wherein the medicament is for preventing or inhibiting the growth of gram-negative and gram-positive bacteria.

18. The use of claim 15 or 16, wherein the medicament is for preventing or inhibiting the growth of methicillin-resistant staphylococcus aureus.

19. Use of alpha-linolenic acid and terpinen-4-ol in the manufacture of a composition for preventing or inhibiting bacterial growth.

20. The use of claim 19, wherein the composition is a composition for preventing or inhibiting the growth of gram positive and gram negative bacteria.

21. The use of claim 19 or 20, wherein the composition further comprises a C8-C24 polyunsaturated fatty acid, or ester thereof, other than alpha-linolenic acid, not present at a level greater than 20% by weight of the alpha-linolenic acid.

22. The use of claim 19 or 20, wherein the bacterium is methicillin-resistant staphylococcus aureus.

23. Use of a composition comprising alpha-linolenic acid and terpinen-4-ol in the manufacture of a medicament for the prevention or treatment of a disease or condition caused by bacteria.

24. Use according to claim 23, wherein the composition is as described in any one of claims 1 to 14.

25. The use of claim 23 or 24, wherein the composition is administered topically.

26. The use of claim 23 or 24, wherein the medicament is for the prevention or treatment of a disease or condition caused by methicillin-resistant staphylococcus aureus.