AU2006218308B2 - Anti-microbial agent - Google Patents

Abstract

A composition comprising a C8 to C24 polyunsaturated fatty acid such as -linolenic or ester thereof, and terpinene-4-ol or derivative thereof. The composition has activity as a broad-spectrum antibacterial composition.

Description

WO 2006/089348 PCT/AU2006/000068 ANTI-MICROBIAL AGENTS RELATED APPLICATIONS 5 The present application claims priority from Australian Provisional Application No. 2005900247, the entirety of which is incorporated by reference. 10 FIELD The present invention relates to anti-microbial agents. In particular, the present invention relates to broad spectrum anti-microbial agents. 15 BACKGROUND Microorganisms which are resistant to anti-microbial agents are becoming an increasing problem around the 20 world. For example, methicillin-resistant strains of Staphylococcus aureus (MRSA - also referred to as "golden staph") are now multi-resistant to antibiotics and recent isolates show increasing resistance to antibiotics such as Gentamicin, Chloramphenicol, Fusidic Acid, and Rifampicin. 25 Only Vancomycin can currently be relied on for empirical treatment. There is considerable concern that the increasing use of Vancomycin will select Vancomycin resistant strains of MRSA so that in the near future there may no longer be any effective antibiotic therapy against 30 hospital staphylococci. Accordingly, investigations are underway to identify alternative anti-microbial agents, particularly broad spectrum anti-microbial agents. Hospitals are examples of environments where MRSA acquired 35 infections are a significant problem. These infections put the patient at risk, increase the cost of care, and reduce the number of beds available. In other environments such as dental rooms, laboratories, food preparation areas, WO 2006/089348 PCT/AU2006/000068 2 schools and so forth, it is also important to prevent or minimise the growth of such bacteria, together with other microbial agents including fungi and viruses. 5 Hospitals and these other environments requiring high levels of hygiene have a range of strategies to minimise the spread of such microorganisms. Prior to entering into surgery, patients are topically treated with antiseptic solutions such as chlorhexidine or povidone iodine. 10 Carriers of MRSA are treated with such topical antiseptics, or in severe cases, are treated with antibiotics such as Vancomycin. Vancomycin is expensive to administer, and therefore effective, less expensive orally deliverable replacements would be welcomed. 15 The known topical antiseptics cannot be relied upon to provide sufficient protection from such bacteria, especially as more resistant strains develop. They are also not capable of being applied in other presentation 20 forms, such as oral tablets. Such topical antiseptics also may not be effective in preventing or inhibiting the growth of other classes of microorganisms that are present in hospitals and other such environments. Although surfaces in these environments are washed down with 25 sterilising agents and disinfectants including bleaches containing chlorine in the form of hypochlorous acid/hypochlorite ion, these agents are quite corrosive, and other alternatives are desirable. 30 Accordingly, there is a need for new antimicrobial and/or antibacterial agents suitable for use in these and similar applications. All references, including any patents or patent 35 application, cited in this specification are hereby incorporated by reference to enable full understanding of the invention. Nevertheless, such references are not to be read as constituting an admission that any of these -3 documents forms part of the common general knowledge in the art, in Australia or in any other country. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge 5 the accuracy and pertinency of the cited documents. SUMMARY In a first aspect, there is provided a composition comprising a C8 to C24 polyunsaturated fatty acid or ester thereof, and terpinen-4-ol or derivative thereof. 10 Preferably the C8 to C24 polyunsaturated fatty acid or ester thereof is a-linolenic acid, and most preferably the a-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 is form (as defined below). The composition may be in a wide range of presentation forms, including the forms of a body wash, surface spray, a towelette, topical ointment, or nasal ointment, tablet or otherwise. In an embodiment of the first aspect, there is provided a 20 composition comprising: a-linolenic acid or an ester thereof, at a level of between 0.1 and not more than 20% by weight of the composition, wherein the level of C8 to C24 polyunsaturated fatty acids other than a-linolenic acid or 25 an ester thereof is not more than 20% on a weight-to weight basis compared to the a-linolenic acid or ester thereof present in the composition; and a terpinen-4-ol extract of tea-tree oil, at a level of between 0.1 and 5% by weight of the composition, 30 wherein the terpinen-4-ol extract contains no more than 4291329_1 (Gleianss) P54683.AU.1 20% by weight of the non-terpinen-4-ol components of tea tree oil. In a second aspect, there is provided a method of preventing or inhibiting growth of bacteria, the method s comprising the step of contacting a surface with a composition comprising a C8 to C24 polyunsaturated fatty acid or an ester thereof, and terpinen-4-ol or a derivative thereof. In an embodiment of the second aspect, there is provided a 10 method of preventing or inhibiting growth of bacteria, the method comprising the step of contacting an inanimate surface with a composition as defined above in the embodiment of the first aspect of the invention. It is noted that the term "terpinene-4-ol" is also is referred to in the specification as "terpinen-4-ol" and these terms can be used interchangeably. It has been found by the present applicant that a linolenic acid, which is a C8 - C24 polyunsaturated fatty acid, is effective in preventing or inhibiting the growth 20 of bacteria. The activity of this pure a-linolenic acid is similar to polyunsaturated fatty acids closely related to a-linolenic acid, as described in further detail below, a linolenic acid has been found to be effective in preventing or inhibiting the growth of Gram negative and 25 Gram positive bacteria and given this activity against a range of microbes, it is an effective broad-spectrum antimicrobial agent. The present applicants have found that when the fatty acid or ester is used in combination with terpinen-4-ol or a 30 derivative thereof (namely another derivative of 4291328_1 (GHMatters) PS483.AU-1 -5 terpinene), synergistic antimicrobial activity is achieved. Moreover, such a composition has activity against a broader range of microorganisims, including fungi. Indeed, test results show that compositions 5 containing both components have around ten times the activity of compositions containing one component alone. The surface is suitably contacted with a composition comprising terpinen-4-ol or a derivative thereof together with, prior to or following contact with the C8 to C24 10 polyunsaturated fatty acid or ester composition within a timeframe to obtain the synergistic effect. Preferably, the surface is contacted with a composition comprising both the C8 to C24 polyunsaturated fatty acid or ester and terpinen-4-ol or a derivative thereof, such that the time 15 of contact is contemporaneous. Without wishing to be bound by theory, it is believed that the C8 to C24 polyunsaturated fatty acid or ester thereof exerts its anti-microbial effect on the inside of the microorganism by destroying fatty acid metabolism, while 20 visual observation suggests that terpinen-4-ol and derivatives thereof attack the microbe's cell wall. Accordingly, these active agents act via different mechanisms and thus there is a reduced chance of a particular microorganism developing resistance to both of 25 the agents. Moreover, both the C8 to C24 polyunsaturated fatty acid or ester thereof and terpinen-4-ol (or derivative) are volatile agents and therefore are less likely to remain in contact with a microorganism for an extended period of time, thereby further reducing the 30 chance of a microorganism developing resistance to the agents. 4291328_1 (GHMANUM) P54630AU.1 - 5a In a third aspect, there is provided a method of preventing or inhibiting growth of both Gram positive and Gram negative bacteria on a surface, the method comprising the step of contacting a surface with a composition 5 comprising a C8 to C24 polyunsaturated fatty acid or ester thereof, and terpinen-4-ol or a derivative thereof. In a fourth aspect, there is provided a use of a CS to C24 polyunsaturated fatty acid or ester thereof and terpinen 4-ol or a derivative thereof in the manufacture of a 10 composition for preventing or inhibiting growth of bacteria. Preferably the composition is a composition for preventing or inhibiting growth of Gram positive and Gram negative bacteria. In an embodiment of the fourth aspect, there is provided is use of a-linolenic acid or an ester thereof and a terpinen-4-ol extract of tea-tree oil in the manufacture of a composition as defined above in the embodiment of the first aspect, for preventing or treating a disease or condition caused by bacteria. 20 In a fifth aspect, there is provided use of composition as defined above in the embodiment of the first aspect, for preventing or treating a disease or condition caused by bacteria. In a sixth aspect, there is provided a method of 25 preventing or treating a disease or condition caused by bacteria, the method comprising the step of administering to a subject in need thereof a composition comprising a C8 to C24 polyunsaturated fatty acid or ester thereof, terpinen-4-ol or a derivative thereof, and a carrier. The 30 mode of administration is preferably topical. 429132_1 (GHMAele) P54683AU.1 -5b In an embodiment of the sixth aspect, there is provided a method of preventing or treating a disease or condition caused by bacteria, the method comprising the step of administering to a subject in need thereof a composition 5 as defined above in the embodiment of the first aspect, and a carrier. The composition may further comprise one or more other active agents. However, according to some embodiments, the only active agents present are the C8 to C24 10 polyunsaturated fatty acid or ester thereof, and terpinen 4-ol or derivative thereof. BRIEF DESCRIPTION Figure 1 is a graph of the log reduction in organism count for a number of microorganisms contacted with a-linolenic 15 acid after 30 minutes. 4291328_1 (GH*aters) PS4683AU.1 WO 2006/089348 PCT/AU2006/000068 -6 Figure 2 is a graph showing the %- reduction of organism count for a number of microorganisms contacted with a composition of one embodiment of the invention. 5 Figure 3 is a graph showing the minimum concentration of a-linolenic acid required to inhibit growth of a range of microorganisms. DETAILED DESCRIPTION 10 In the specification, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, 15 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" 20 include the corresponding plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a virus" includes a plurality of viruses. Similarly, a reference to "a terpinene-4-ol or a derivative thereof" includes a mixture of more than one 25 compound within this class. Compositions comprising a C8 to C24 polyunsaturated fatty acid or ester thereof and terpinene-4-ol or a derivative thereof have been found by the inventors to have broad 30 anti-microbial activity, including activities against Gram-positive and Gram-negative bacteria, including aerobic, anaerobic, and facultative aerobic bacteria. This broad activity provides wide application for the composition. It has also been found that the composition 35 can be used in different forms, which further enhances the applications of the composition. It has been surprisingly found that a synergistic activity is provided when the C8 WO 2006/089348 PCT/AU2006/000068 -7 to C24 polyunsaturated fatty acid or ester thereof is administered with terpinene-4-ol or a derivative thereof. In addition, the combination composition has activity against a broader range of microorganisms including the 5 Gram-positive and Gram-negative bacteria described above, and fungi. C8 to C24 polyunsaturated fatty acids It is explained above that the preferred form of C8 to C24 10 polyunsaturated fatty acid or ester thereof is a-linolenic acid. a-linolenic acid is present in the form of the corresponding glyceride in linseed and other oils, and is usually present in such oils as one of several C8 to C24 polyunsaturated fatty acid components. a-linolenic acid 15 is a C18 fatty acid containing three double bonds (C18:3) at the 9-, 12- and 15- positions. Although the composition in its broadest aspect may comprise a mixture of C8 to C24 polyunsaturated fatty 20 acids or esters thereof, it preferably is 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, preferably the composition used according to the present application comprises a single C8 25 - C24 polyunsaturated fatty acid at a level that is substantially free of other C8 - C24 polyunsaturated fatty acids. Most preferably, the composition comprises a linolenic acid, substantially free of any other C8 to C24 saturated, mono or polyunsaturated fatty acids or esters 30 thereof. By substantially free, it is meant that such components (that the composition is substantially free of) are not present at a level of more than 20% (on a weight to weight basis) of the single fatty acid present, such as a-linolenic acid. 35 C8 to C24 polyunsaturated fatty acid refers to straight chain or branched alkenyl carboxylic acids containing at WO 2006/089348 PCT/AU2006/000068 -8 least 2 degrees of unsaturation, and a hydrocarbon chain length of between 8 and 24 carbon atoms. Preferably the fatty acid is a C18 fatty acid, and more preferably it contains three degrees of unsaturation. Preferably the 5 fatty acid is a cis-fatty acid. The esters of such fatty acids encompass the class of compounds that can be produced by the reaction of such fatty acids with any aliphatic or aromatic organic 10 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 for instance be a polyol such as a diol or triol, and may contain any other functional groups. The alkyl esters 15 are most typical. Terpinene-4-ol and derivatives thereof The C8 to C24 polyunsaturated fatty acid is used in combination with terpinene-4-ol or a derivative thereof. 20 Terpinene-4-ol is a component of tea tree oil. One commercially available terpinene-4-ol mixture derived from tea tree oil is known by the trade mark Melaleucol. The 25 structure of one principal isomer of terpinene-4-ol is as follows: 30 OH 35 The term terpinene-4-ol encompasses single isomers such as that illustrated above or any other individual isomers and mixtures of isomers including the racemic mixture.

WO 2006/089348 PCT/AU2006/000068 -9 Generally extracts of terpinene-4-ol from tea tree oil are in the form of an isomeric mixture of the (+) and (-) enantiomers in a ratio of between 1.8-2.4:1. 5 Terpinene-4-ol is a 3-hydrogeno 4-hydroxy containing derivative of terpinene. The derivatives of terpinene-4 ol encompassed by the present application are the other derivatives of terpinene in which one or both double bonds are saturated, and the compound is substituted with one or 10 more substituents selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, 15 nitroheterocyclyl, amino, alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino, benzylamino, dibenzylamino, acyl, alkenylacyl, alkynylacyl, arylacyl, acylamino, diacylamino, acyloxy, alkylsulphonyloxy, arylsulphenyloxy, heterocyclyl, 20 heterocycloxy, heterocyclamino, haloheterocyclyl, alkylsulphenyl, arylsulphenyl, carboalkoxy, carboaryloxy, mercapto, alkylthio, benzylthio, acylthio, and phosphorus containing groups. Terpinene-4-ol is the derivative containing hydrogen and hydroxy substitution across the 25 saturated C3-4 double bond of terpinene. Preferably, however, terpinene-4-ol (including isomeric mixtures thereof) is used in a substantially pure form. The expression "substantially pure form" means that the 30 terpinene-4-ol, or derivative thereof, is used in a form that is substantially free of the other principal components of tea tree oil. In this context, substantial means that the terpinene-4-ol used in the composition contains no more than 20% (weight to weight) of the non 35 terpinene-4-ol components of tea-tree oil. Thus, this level covers pure terpinene-4-ol extracts, and extracts retaining small levels of impurities from tea-tree oil.

WO 2006/089348 PCT/AU2006/000068 - 10 Of course, the terpinene-4-ol or derivative thereof may be produced synthetically, in which case it would not be expected that any of the non-terpinene-4-ol components of tea-tree oil would be present. 5 Components of compositions The C8 - C24 polyunsaturated fatty acid-containing compositions used in accordance with the present invention preferably comprise between 0.1% to 50% by weight C8 - C24 10 polyunsaturated fatty acid. More preferably, the composition comprises or is applied at a concentration of between 0.1 and 20% w/w, depending upon the form of the composition and its route of administration. At levels below 0.1%, the effectiveness of the composition is not as 15 high as would be desired for a commercially acceptable product. At levels above 50% the C8 - C24 polyunsaturated fatty acid such as a-linolenic acid is likely to cause skin irritation or be difficult to handle. Generally for presentations suitable for direct skin applications the 20 level of C8 - C24 polyunsaturated fatty acid will be lower than this - generally at a level of not more than 10%. A good balance between antimicrobial properties, ease of use and cost is obtained for compositions comprising between 0.1% and 5% C8 - C24 polyunsaturated fatty acid, and 25 particularly a-linolenic acid. The composition preferably comprises between 0.1% and 5% terpinene-4-ol or a derivative thereof, more preferably between 0.25% and 5% terpinene-4-ol or a derivative 30 thereof. Preferably, the terpinene-4-ol itself in a substantially pure form is present in the composition at the given levels. For compositions comprising both C8 - C24 polyunsaturated 35 fatty acid or ester thereof (preferably a-linolenic acid in substantially pure form), together with terpinene-4-ol or a derivative thereof (preferably terpinene-4-ol in WO 2006/089348 PCT/AU2006/000068 - 11 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. 5 The compositions of the present invention will generally further comprise a carrier. Depending on the presentation form, the carrier may be a pharmaceutical carrier, or it may be a more general carrier. 10 Pharmaceutical carriers for preparation of pharmaceutical compositions are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 20th Edition, Williams & Wilkins, Pennsylvania, USA. The carrier will depend on the route of administration, and 15 again the person skilled in the art will readily be able to determine the most suitable formulation for each particular case. Examples of different presentation forms are lotion, 20 suspension, solution, spray, emulsion, paste, foam, eyewash, ointment, liquid soap, cream, solid soap, mouthwash, pastille or lozenge, gel, a towelette, hair tonic, shampoo or jelly. 25 In the case where the composition is in the form of a surface spray, the carrier is suitably water, and the composition may further comprise preservative such as benzyl alcohol (for example, in an amount of from 0.1 - 5 wt%), cosolvents such as propylene glycol (for example, in 30 an amount of from 1 - 20wt%), and surfactants such as Cremophor Rh40 (for example, in an amount of from 1 20wt%). In the case where the composition is in the form of a 35 lotion or body wash, it may comprise an emulsion of C8 C24 polyunsaturated fatty acid or ester, and optionally terpinene-4-ol, in water in the presence of a suitable WO 2006/089348 PCT/AU2006/000068 - 12 emulsifying agent. Examples of suitable emulsifying agents are cetosteryl alcohol, Emulgade, SLEX (Texapon), Betaine derivatives such as the Empigen B range of surfactants, and Tween (eg Tween 80@ ICI Chemicals). 5 Tween 80@ is a surfactant which includes polyoxyethylene 20 sorbitan mono-oleate. The composition may further comprise emollients and fragrances (including oils such as sweet almond oil), preservatives (such as benzyl alcohol at levels of, for instance, 0.1 - 10%-) and humectant 10 and/or emollient and/or excipients (such as glycerol, for instance at levels of 0.5 - 10%- and cyclomethicone at levels of between 0.1 - 20%). When the composition is in the form of a moistured 15 towelette it may come comprise carriers, preservatives, emulsifiers, emollients and/or fragrances as described above. When the composition is in the form of an ointment, it may 20 comprise 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 and liquid paraffin. Other ointment bases may also be used such as polyalkylene glycol (such as polyethylene or polypropylene 25 glycol) base. Other possible components of an ointment composition include emulsifying wax (for example, in an amount of between 1 - 40%, preferably 5 - 40%), and preservative. 30 When the composition is in the form of a liquid soap, any known liquid soap may be used. A preferred liquid soap is a coconut oil-based liquid soap. The pH of the liquid soap base may be adjusted to that which is suitable for topical use. 35 When the composition is a cream, any suitable cream base may be used. A preferred cream base is sorbolene cream.

WO 2006/089348 PCT/AU2006/000068 - 13 Other cream bases may also be used. When the composition is in the form of a solid soap, it is preferred that a synthetic soap base is used. Synthetic 5 soap bases are preferred because their properties are more suitable for sustained application to debilitated skin and their pH is more readily controlled. Examples of synthetic soap bases suitable for use in a composition of the invention include Synbase A and Synbase BW available 10 from Witco (USA), Synbase Commercial Bar from Dove (USA), Tensia from Dermactif (Belgium) and pH 6-7 Syndet Base from Zetesap (Germany). An as example of the constituents of such soap bases, Syndbase BW comprises disodium myristanido MEA sulphosuccinate, stearic acid, sodium 15 cocyl isethianate, paraffin, PEG 75, zinc stearate, and water. When the composition is in the form of a lozenge, it may comprise a flavoured base, such as sucrose and acacia or 20 tragacenth gum. When the composition is in the form of an eyewash, it may comprise a suitable sterile aqueous or non-aqueous vehicle. Additives such as buffers, preservatives 25 including bactericidal and fungicide agents, such as phenyl mercuric acetate or nitrate, benzalkonium chloride or chlorhexidine and thickening agents such as hypromellose may also be included. 30 In addition.to any of the ingredients listed above, the composition may further comprise other agents. For example, agents such as binders, sweeteners, thickeners, flavouring agents, disintergrating agents, coating agents, preservatives, lubricants, and/or time delay agents. 35 Suitable examples of these are well known in the art. C18 to C24 polyunsaturated fatty acids are known to be WO 2006/089348 PCT/AU2006/000068 - 14 readily oxidised by air. The present inventors have found by experience that the composition of the invention has a shelf-life of at least 2 years. This may be extended by adding an anti-oxidant to the composition. 5 The composition may include or be used with agents such as therapeutic agents, antiseptic agents and/or antibacterial agents. A "therapeutic agent" is an agent used to treat a disease or condition. Suitable therapeutic agents are 10 antibiotics such as vancomycin, penicillin and related antibiotics, methicillin, gentamycin, chloramphenicol, Fusidic acid, rifampicin and so forth. Other therapeutic agents include surgical implants, prosthesis, and catheters. 15 Suitable antispectic or antibacterial agents include Triclosan @, Sapoderm, Chlorhexidine, Povidone-Iodine, Cetrimide, Hexachlorphene, hypochlorite-based antiseptic agents, or mixtures thereof. The other agent(s) may be 20 present in the composition in an amount up to 20% by weight, more preferably up to 15% by weight, of the total composition. The amount of the other agent will depend upon the activity of the other agent; however, a person skilled in the art will be readily able to determine a 25 suitable amount. The composition of the invention preferably has 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. At these pH ranges forms of the 30 composition that can be applied to the skin cause little irritation to the skin or mucous membranes of a subject following application. Methods 35 The present invention provides a method of preventing or inhibiting growth of a microorganism. As used herein "preventing" means to kill the microorganism. In WO 2006/089348 PCT/AU2006/000068 - 15 contrast, "inhibiting" means to decrease, or limit the growth of the microorganism. For example, a bacteriostatic agent inhibits bacterial growth but does not kill bacteria, while a bactericidal agent kills 5 bacteria. The compositions of the invention may be described as antibacterial agents. The compositions may be described as disinfectants when formulated for application to surfaces or for dilution in aqueous solvents. The composition may be described as an 10 antiseptic when formulated for application to a subject, including human or non-human animals. "Growth" means an increase in the size and/or numbers of a microorganism. The growth of a microorganism may be 15 determined by any method known in the art, including both direct and indirect methods. For example, direct methods include measuring the numbers of microorganisms, such as by serial dilutions and plate counts. Indirect methods include estimating the number of microorganisms from the 20 turbidity or metabolic activity of the culture. Alternatively, the dry weight of the culture may be used to determine growth of the microorganism. A "microorganism" or "microbe" is any organism of 25 microscopic size. Microorganisms include bacteria, viruses, fungi such as yeasts and molds, algae, and the like. "Bacteria" are single-celled organisms which are enclosed 30 by a cell wall and whose genetic material is not enclosed in a nuclear membrane. Bacteria may be Gram-positive or Gram-negative, aerobic, anaerobic, or facultative anaerobes, and may have one of several shapes, such as rod-like (bacillus), spherical or ovoid (coccus), 35 corkscrew (spiral), starshaped or square shaped. Individual bacteria may form pairs, chains, clusters or other groupings.

WO 2006/089348 PCT/AU2006/000068 - 16 Examples of bacteria whose growth may be prevented or inhibited according to the invention include Escherichia coli, Klebsiella pneumoniae, Listeria monocytogenes, 5 methicillin-resistant, Neisseria flavescens, Pseudomonas aeruginosa, Salmonella salford, Salmonella cholerae, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermis, Streptococcus faecalis, Streptococcus faecium and Moraxella catarrhalis. Some characteristics of these 10 bacteria are provided in Table 1. Table 1. Bacteria Gram stain Oxygen requirement E. coli -ve Aerobe K. pneumoniae -ve Aerobe L. monocytogenes +ve Aerobe MRSA +ve Facultative anaerobe N. flavescens -ve Aerobe P. aeruginosa -ve Anaerobe S. Salford -ve Aerobe S. cholerae -ve Aerobe S. marcescens -ve Aerobe S. aureus +ve Facultative anaerobe S. epidermis +ve Aerobe S. faecalis +ve Facultative anaerobe S. faecium +ve Facultative anaerobe "Viruses" are very small, requiring an electron microscope 15 to view, and they are not cellular. A virus particle contains a core made of only one type of nucleic acid, either DNA or RNA, which is surrounded by a protein coat. The coat may be surrounded by a lipid envelope. Viruses WO 2006/089348 PCT/AU2006/000068 - 17 can only reproduce inside the cells of other organisms. "Fungi" are microorganisms whose cells have a distinct nucleus that contains the cell's genetic material and are 5 surrounded by a nuclear membrane. Fungi may be unicellular or multicellular. The unicellular form of fungus is yeast. Where fungi are referred to, yeast is the preferred form of fungi. Yeasts are oval-shaped microorganisms which are larger than bacteria. The most 10 typical fungi are molds. Molds form mycelia, which are long, branched filaments that intertwine. Examples of fungi suitable for use with the invention include A. niger and C. albicans. 15 Growth of the microorganism is prevented or inhibited by contacting the microorganism, or a surface on which the microorganism may come in contact, with a composition comprising a C8 to C24 polyunsaturated fatty acid or ester thereof in combination with terpinene-4-ol or a derivative 20 thereof, or by administering the composition to a subject. The contacting may be by any suitable means, and may have a direct or indirect affect on the microorganism. For example, the composition per se may affect the growth of the microorganism. Alternatively, the composition may 25 decrease the adhesion of the microorganism to a surface or influence another component of the microorganism culture, thereby affecting the growth of the microorganism. The surface may be any suitable surface, such as a human 30 or animal body, or an inanimate object. Suitably the composition is contacted with the microorganism by administering the composition topically and/or orally to a subject in need thereof. 35 Alternatively, the composition may be applied to an inanimate surface comprising the microorganism or suspected or at risk of comprising the microorganism. The WO 2006/089348 PCT/AU2006/000068 - 18 term "surface" is used in its broadest sense, and should not be read as implying any specific physical dimensions. The composition may be administered by any suitable route, 5 and the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated. Dosage will be at the discretion of the attendant physician or veterinarian, and will depend on the nature and state of the condition to be 10 treated, the age and general state of health of the subject to be treated, the route of administration, and any previous treatment which may have been administered. The composition may be administered to a subject in need 15 thereof periodically or repeatedly, and may be administered to the site of actual or possible infection. For example, if infection of a surgical wound has occurred or is desired to be prevented, the composition may be administered on or to the wound and around the wound. If 20 infection of the nasal passages may occur or has occurred or is desired to be prevented, administration of the composition to the nasal passages may be appropriate, and the composition may be in the form of a nasal ointment or nasal spray. If infection of the throat has occurred or 25 is desired to be prevented, the composition may be in the form of a throat gargle, lozenge, or spray. A suitable prophylactic treatment regime includes washing the patient with the composition, optionally following a 30 separate antiseptic treatment (depending on the components in the composition). This may be conducted on a periodic or repeating basis. The composition may be in the form of a body wash, or otherwise a lotion may be applied followed by gauze. The composition is allowed to dry. The procedure 35 may be repeated a number of times a day, with three times a day being suitable. The nasal ointment or spray may also be applied.

WO 2006/089348 PCT/AU2006/000068 - 19 "Disease" is a general term used to refer to any departure from health in which a subject suffers. A "condition" refers to an abnormal functioning of a function or part of 5 a body. Examples of diseases or conditions which may be prevented or treated by the composition comprising a C8 to C24 polyunsaturated fatty acid or ester thereof include cellulitis, necrotizing subcutaneous infections such as necrotizing fasciitis, abscesses, bacterial infections, 10 pruritus, dermatitis, eczema, psoriasis, acne, boils, bed sores, heat rash, skin blemishes, ichthyosis, tinea, and ulcers, fungal diseases, herpes i.e. cold sores, chicken pox, and shingles, and parasitic infections. The method may also be used to improve the appearance of dry, flaky, 15 wrinkled, aged, or photodamaged skin. It may also be used to soothe or alleviate the associated effects of the disease or condition such as pain, itching, swelling, etc. The "subject" may be a mammal. The mammal may be a human, 20 or may be a domestic or companion animal. While it is particularly contemplated that the compounds of the invention are suitable for use in humans, they are also applicable to veterinary use, including treatment of companion animals such as dogs and cats, and domestic 25 animals such as horses, cattle and sheep, or zoo animals such as non-human primates, felids, canids, bovids, and ungulates. Unless otherwise indicated, the present invention employs 30 conventional chemistry and microbiology techniques within the capacity of those skilled in the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. 35 It is to be clearly 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 WO 2006/089348 PCT/AU2006/000068 - 20 that the terminology used herein is for the purpose of describing particular embodiments only, and it is not intended to limit the scope of the present invention, which will be limited only by the appended claims. 5 Where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all values in between these limits. 10 Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described in detail by way of reference only to 15 the following non-limiting examples and drawings. Example 1. Minimum inhibitory concentration test for a-linolenic acid 20 The following tests were conducted to determine the minimum concentration of a-linolenic acid required to inhibit growth of three strains of bacteria which are representative of microorganisms desired to be inhibited 25 by antimicrobial compositions. The test results show that low levels corresponding to compositions having as little as 0.1%. A sample of 90% a-linolenic acid was diluted in accordance 30 with the following protocol to test the minimum effective concentration against the bacteria. The a-linolenic acid was diluted to the dilutions shown in Tables 2A to 2F using Tryptone Soya broth Sigma Aldrich. Each diluted sample was inoculated with approximately 106 test 35 microorganisms of the class and incubated for 48 hours at 37 0 C. The test microorgansisms were S. aureus, S. cholerae, MRSA, E. coli, S. pyogenes and M. catarrhalis.

WO 2006/089348 PCT/AU2006/000068 - 21 Uninoculated broth was included as a control. Following the incubation period each broth was examined for bacterial growth, as indicated by turbidity of the 5 broth. Samples from each broth were subcultured to confirm the presence of the test organism. The highest serial dilution which achieved inhibition of the test organism was deemed to be the minimum inhibitory concentration (MIC). 10 The turbidity of each serial dilution for each test organism is shown in Tables 2A, 2B, 2C, 2D, 2E and 2F. Table 2A Test Organism: Staphylococcus aureus ATCC 6538 Yest Dilotion 1:00 :;00 1:400 I1:00 | 01600 I1:3200 I 6400 0:1200 1:25600 I 1:51200 l :102400 I

-

+ |- Not Tested 2 - - - - - - - - - - 4 Table 2B Test Organism: Salmonella cholerae-suis ATCC 10708 Test |"Dilution 1-00 1:200 400 " '100 ii600 ~ 302600 1;6400 i1:10800 1:25600 1:51200 1 1:102400 2 - + + + + + + 4 Table 2C Test Organism: Methicillin Resistant Staphylococcus Wild Strain ITest Dilutiou :100 |1:260 1:400 1:800 1:1600 | :3200 1:6400 |.12800 1:25600 1:51200 1:102400 I - - - - - . - | - | - - + 2 - - . + Table 2D Test Organism: Escherichia coli ATCC 8739 fest Dilution 1:100 1.200 1:400 1:800 1400 1:3200 21:6400 12800 I 1:25600 1:51200 1:102400 -- + 4 + + ++- 4 4 4 2 + + + + + + + + + Table 2E Test Organism: Streptococcus pyogenes University of Melbourne MDU Test Dilution :100 1:200 |1:400 1:L0 1:1600 :3200 4:6400 1:12800 1:25600 1:58200 1:102400 - - . - - - - - 4- + + 2 - " - . .- - | - + Ros -' indicates Slo growtli. '+' indicates growth.

WO 2006/089348 PCT/AU2006/000068 - 22 Table 2F Test Organism: Morazella Catarrhalis stD t :1 1:200 1:4 1 :1600 Q164 1:12890 I U5 1:102400 Mous: '' indoateos no growth, '-Indicates growtL Example 2. Compositions comprising a-linolenic acid 15 Compositions comprising 1% and 2% a-linolenic acid were prepared with the following components: 1% Solution: 20 % -Material 1 a-linolenic acid, 90% purity, from _ -Optigen (South Australia) 3 Cetostearyl Alcohol 5 Sweet Almond Oil 10 Emulgade N1 1000 2 Glycerol 1 Benzyl Alcohol 1 Cyclomethicone (DC 345) 10 SLES (TEXAPON N25) 10 Betaine (Empigen BS AU) 1 Linolenic Acid 56 Purified Water 2% Solution: 91 Material 2 a-linolenic acid, 90% purity, from Optigen (South Australia) 3 Cetostearyl Alcohol 5 Sweet Almond Oil 10 Emulgade N1 1000 WO 2006/089348 PCT/AU2006/000068 - 23 2 Glycerol 1 Benzyl Alcohol 1 Cyclomethicone (DC 345) 10 SLES (TEXAPON N25) 10 Betaine (Empigen BS AU) 1 Linolenic Acid 55 Purified Water Example 3. Anti-microbial activity of compositions comprising a-linolenic acid 5 The 1% solution of Example 2 was subjected to testing to show that such solutions are effective in preventing growth of both Gram positive and Gram negative bacteria. 10 The biocidal effect of the 1% a-linolenic acid solution was tested against MRSA (Gram positive, facultative anaerobe), Salmonella cholerae (Gram negative, aerobe), and Pseudomonas aeruginosa (Gram negative, anaerobe) using a suspension test. The suspension test was performed 15 according to the following procedure: 1. The starting microorganism concentration was determined by standard plate count technique for each of a test microorganism culture comprising 20 1% ca-linolenic acid and a control microorganism culture. The microorganism concentration of each culture was recorded as cfu/mL. 2. At desired time points, a sample of each culture was removed and the microorganism concentration 25 of each culture was determined by the standard plate count technique. The concentration was recorded as cfu/mL.

WO 2006/089348 PCT/AU2006/000068 - 24 3. The % kill caused by the a-linolenic acid was calculated by comparing the difference in the cfu/mL of the test microorganism over time with the difference in the cfu/mL of the control 5 microorganism culture over the same time. The results are presented as geometric means of duplicate tests using fresh bacterial cultures and solutions for each test, as shown in Table 3. 10 Table 3 Biocidal effect of a-linoleuic acid Orgaanism Initia Count Fial CoIt % Kill per ML. pr mL 5 minm 30 ins SlapIPooccus 1L5 x Li ,5 x o10 1.7 KsI0* 89,7 Salmonella chaerae-su 3,3 x 10' 1.8 x 1o 1.8 x Io 45,5 PseudoMonas aerugnosa 71 x 10? 3,2 X 107 3,2x I0 7 54,9 &M:t Resuhs are Geometric means of duplioat tests. The tests were also performed on Streptococcus pyogenes. The results on this microorganism showed a %kill of 87.7% at 30 minutes. Further tests were performed on 15 Escherichia coli (G-ve) and Moraxella catarrhalis (G-ve). These results are presented in Figures 1 and 2 (in two scales - log reduction and % reduction in organism count) with the results of all microorganisms tested in this Example. 20 Example 4. Compositions comprising a-linolenic acid and terpinene-4-ol 25 Compositions containing 1% a-linolenic acid and 1% terpinene-4-ol were prepared in the form of a body wash, WO 2006/089348 PCT/AU2006/000068 - 25 surface spray, topical ointment, and nasal ointment. The components of the compositions are outlined in Tables 4A, 4B, 4C, and 4D. Melaleucol is a commercially available terpinene-4-ol of a minimum 99% purity, and having a 5 chiral ratio of +/- enantiomers of 1.8:1 - 2.4:1.

WO 2006/089348 PCT/AU2006/000068 - 26 Table 4A Body Wash % m ----- - 3,00 .3010_0 Cetoztearyi Alcohol 5000 0S000weet Almond Oil 5.000 0000 Promu Ie N1 1000 Moo 20,00 GlyIcerol 10200 21.000 Benz I Alcohol Odd ~ ~~ hIcone (DC 345) 1000 10000 Metal e2o ;00 - 00 SLES__(TjEXAONE 5)2 10 000 100,000 Beta nelmpigen BS AU) 1.00 10000Lnoei Acid 1,00 1.000 - ~ -~~~~ 000 0 100 -00 -- o-- Table 4B Surface Spray % EL- -- era 66 ~~~0 50.000Crr~o 14 Oi00 1000(ilfucol 87.800 878.000p 1001000 Toa WO 2006/089348 PCT/AU2006/000068 - 27 Table 4C Topical Ointment % m Materi4l 60,000 600000 PEG 400 24.000 240000 PEG 4000 14,000 T40.000 PropyIone G ycol 1.000 10.000 Mela euco 1,000 10.000 id 100 '11000 'Total Table 4D Nasal Ointment % grn Material 29.400 294.000 Emulsifying Wax 49.000 490,000 White Soft Parraffin 19.600 196.000 Liquid Paraffin Heavy 1.000 10.000 Melaleucol 1,000 10.000 Linolenic Acid 100.00 1000 Total Example 5. Antimicrobial activity of compositions comprising a-linolenic acid and terpinene 5 4-ol The antimicrobial activities of the compositions of WO 2006/089348 PCT/AU2006/000068 - 28 Example 4 were subjected to testing. The results presented below show that the compositions are very effective in preventing the growth of Gram positive bacteria, gram negative bacteria and fungi. 5 The compositions were tested for antimicrobial activity using the suspension test in Example 3 outlined at room temperature against the following organisms: S. aureus 10 E. coli P. aeruginosa C. albicans A. niger N. flavescens 15 MRSA The results for the body wash are shown in Tables 5A and 5B.

WO 2006/089348 PCT/AU2006/000068 - 29 Table 5A Recovery Results at Different Exposure Times (cfu/mL) at4~ mism 0 a1 - o min , Kula 4 ~ ~ ~ ~ : x 1012A1 7% nt < . 10 <10 TbeSaB woa R-ut~ tDffrLEpsr Tie -togo 4 x104 ZSx to, 2.3xf01 2. 0 10 <10 4 100 to0 0. Table 5B Lo Reduction at Different Exposure Times (Log) ~~andypaniss -GB. neu so nif 60 mi 9010 i - c t -t2. The results for the surface spray are shown in Tables 6A and 6B.

WO 2006/089348 PCT/AU2006/000068 - 30 Table 6A Recovery Results at Different Ex~posure TiMes (cf u/ mL) 30 tti, ~Owin' 90 MEU 7-TIO mw Ideati wopmum) X4n~ Z.5 X 10' I6A'DXIC 29710 X to, X 13 x 10, ~10 1 <0 O~Ui~fjfl1t 1, X- Table 6B Lo-g Reduction at Different Exposure Times (Loglo) The results or he nasa ointmen are ~wfials7 aXd 2ABS WO 2006/089348 PCT/AU2006/000068 -31 Table 7A Recovery Results at Different Exposure Times (cfu/mL) 'j*O Or94110 30in 60 XV~ Poulin 5Z0bi 41f s.~~ ~ 10~eU 6.8 X'0 10 1.3 100 'x0 ~ mli P.a~ugr~~a~0~O'1.~E~ 90 60 4,5 10 A~nj~ '0 40 50 0 L 00 ~ ~ ~ ~ 1 1~ 1 1 I .

3

I

0 Table 7B Log Reduction at Different Exposure Times (Logio) * 60 Min 90 Main 120UMIA1 1A SA and 4,8495.

WO 2006/089348 PCT/AU2006/000068 - 32 Table 8A Recovery Results at Different Exposure Times (cfu/mL) Tt Orgatms t min 90 Tu2 2 min 24 hr hitia 'XVg ,5.wiu b1JJ 71 4 1,6.x1W 1.5 1t 4 4~0 3, X IQ, 0 <10 10 <t0 10 L3 x 10 ?,30'N~n 23.101 2A x W 14xc10 4 1l'10 .40.k is0 * *80 60 60 20 410 3.1 x 10' I~~ J, <10 1,0 104< 1D <10 <10 <10 < Table 8B Log Reduction at Different Exposure Times (Log o) go 1n 0 min 24 houn 24 2:5 2,4 2.4 E.c1 d * >6 1 >6.1 - >64 >6.,.~ . 6 -, ,S7 >5.7 >5,7 S >5.7 Qq.'thidwks ~ 2.11 ____ Ani~ 3,63,7 42>4.5 A________ 51>. >5.4>5 >. EXAMPLE 6. SYNERGSTIC ACTIVITY OF a-LINOLENIC ACID AND TERPINENE-4-OL 5 The anti-microbial activity of a-linolenic acid and terpinene-4-ol separately and in combination were tested using the suspension test as outlined in Example 3 above. 10 A 1% a-linolenic acid solution in water was prepared to test the activity of this agent alone. This solution was denoted Solution A. The results of the assessment of the WO 2006/089348 PCT/AU2006/000068 - 33 recovery of microorganisms following exposure to Solution A for the given time of contact are presented in Table 9A below. 5 A 1% terpinene-4-ol (specifically, Melaleucol) solution in water was prepared to test the activity of this agent alone. This solution was denoted solution B. The results of the assessment of the recovery of microorganisms following exposure to Solution B for the given time of 10 contact are presented in Table 9B below. A combination solution was prepared containing 1%co a linolenic acid and 1% terpinene-4-ol (specifically, Melaleucol) in water to test the activity of this 15 combination. This solution was denoted Solution C. The results of the assessment of the recovery of microorganisms following exposure to Solution C for the given time of contact are presented in Table 9C (in terms of surviving organism recovery levels) and Logo 20 reductions.

WO 2006/089348 PCT/AU2006/000068 -34 Table 9A Recovery Results at Different Exposure Times to Solution A composition comprising 1% a-linolenic acid alone ~~?,9xt0 6 ~~~~ 1: 1 i.~ n;) 06 Ldb 80 O Y. IaIOUS, 3.6 x 1V 1.6x 106 1.3 Xc 10, L&SS tua .3.9X .Nfla Vesdits, 13&iO 3.7j10 1,9 X 04 , 6.10 75 0 Table 9B Recovery Results at Different Exposure Times to Solution S *Suri'vinfg organisms (CfwIVIL.)/ "1,8x 10' IOXG104 3.7 z1 01 9.1 k~0 2 fe B"OX*10 .Y-rdu X0 0 *4.5~ % Vj 2,6 x 10 s,2 x10 P~eino 11 ~. .3x O 3.9 X 10' L.li6( 2,2 X 101 Nqm'~~yi <t <I .' <0 <00 :7,5x~ 10 * . ~ ~ ~2X) (412.0Y om. .

WO 2006/089348 PCT/AU2006/000068 - 35 Table 9C Recovery Results at Different Exposure Times to solution C sur~yivig orgaisim (m / mL)/ Test )rganisms Lov Vine Contact Tim (Min'uten_ 30 60 90 120 n MISA 2.0 x0* 3.2x10 A x 10' 7,4 x10 4 6,9 X 10* (6.3) (.61 (5IS) (4.87) (6,84) Q* L x 0* .6 x104 2.6x 10" Jx1 (6.11) (5.18) (4.82) (4.4:) (Gli) aeuioa 5.3 x W0 5.0 X 10' 6.9 x 14 5. 7 x_ 104 3.5 X 10* (4,72) (4.70) (4.84) (4.76) (O.S4) fravesces <z100 <100 <100 <100 6.x * (<2.0) (<2.0) (<2.0) (<2.0) (62) Table 9D Comparison of Log Reductions (Log1O) Following Exposure to Solution C ~Ws i Orgaisms Contact Time 30miu 60mn 90min 120 mn ___A 0.5 1.3 1.7 2,0 9, aureus 0.5 4 L2. A OerugfhOsw ('CC 15442) 0.6 0.4 0.6 <0.6 Results from 0403747 (AMWC 9027})818 70 N. flamcens >48 >4.8 >4.8 >4 <-Leis t >a ete tban It can be seen that both a-linolenic acid and terpinene-4 ol are effective anti-microbial agents. Moreover, it can be seen that there is a synergistic anti-microbial effect 5 between X-linolenic acid and terpinene-4-ol as a 2 x logo reduction in the survival of P. aeruginosa was observed after 30 mins, compared with a 1 x logio reduction using WO 2006/089348 PCT/AU2006/000068 - 36 either a-linolenic acid or terpinene-4-ol separately. This corresponds to a ten-fold increase in antimicrobial effect compared to each component alone, which is greater than the two-fold effect that would follow from the 5 additive effect of the use of the two components. It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various 10 modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

Claims (21)

1. A composition comprising: a-linolenic acid or an ester thereof, at a level of between 0.1 and not more than 20% by weight of the 5 composition, wherein the level of C8 to C24 polyunsaturated fatty acids other than a-linolenic acid or an ester thereof is not more than 20% on a weight-to weight basis compared to the a-linolenic acid or ester thereof present in the composition; and 10 a terpinen-4-ol extract of tea-tree oil, at a level of between 0.1 and 5% by weight of the composition, wherein the terpinen-4-ol extract contains no more than 20% by weight of the non-terpinen-4-ol components of tea tree oil. 15

2. The composition of claim 1, wherein the composition comprises between 0.25% and 5% of the terpinen-4-ol extract.

3. The composition of claim 1 or 2, wherein the ratio of a-linolenic acid or an ester thereof to terpinen-4-ol 20 extract is between 1:5 and 5:1.

4. The composition of claim 3, wherein the ratio is between 1:2 and 2:1.

5. The composition of any one of claims 1 to 4, wherein the composition is in the form of a lotion, suspension, 25 solution, spray, emulsion, paste, foam, eyewash, ointment, liquid soap, cream, solid soap, mouthwash, pastille or lozenge, gel, a towelette, hair tonic, shampoo or jelly. 42013281 (GHMafles) P54683AU.1 - 38

6. The composition of claim 5, wherein the composition is in the form of a body wash, surface spray, a towelette, topical ointment, or nasal ointment.

7. The composition of any one of the preceding claims, s wherein the composition comprises between 0.1% and 5% of a-linolenic acid or an ester thereof, and between 0.1% and 5% terpinen-4-ol extract.

8. The composition of any one of the preceding claims, wherein the pH is in the range of 4.8 to 7.5. 10

9. The composition of any one of the preceding claims, wherein the composition is a disinfectant composition.

10. A method of preventing or inhibiting growth of bacteria, the method comprising the step of contacting an inanimate surface with a composition according to any one is of claims 1 to 9.

11. The method of claim 10, wherein the method prevents or inhibits the growth of Gram negative and/or Gram positive bacteria.

12. The method of claim 10 or 11, wherein the method 20 prevents or inhibits the growth of methicillin-resistant Staphylococcus aureas.

13. Use of a-linolenic acid or an ester thereof and a terpinen-4-ol extract of tea-tree oil in the manufacture of a composition as defined in any one of claims 1 to 9, 25 for preventing or treating a disease or condition caused by bacteria.

14. Use of a composition as defined in any one of claims 1 to 9, for preventing or treating a disease or condition caused by bacteria. 429132L (GHMate9s) PS4683AU.1 - 39

15. Use of claim 13 or 14, wherein the composition is for preventing or inhibiting growth of Gram positive and/or Gram negative bacteria.

16. Use of any one of claims 13 to 15, wherein the 5 bacteria is methicillin-resistant Staphylococcus aureas.

17. Use of any one of claims 13 to 16, wherein the composition is administered topically.

18. A method of preventing or treating a disease or condition caused by bacteria, the method comprising the 10 step of administering to a subject in need thereof a composition as defined in any one of claims 1 to 9, and a carrier.

19. The method of claim 18, wherein the composition is administered topically. 15

20. The method of claim 18 or claim 19, wherein the method prevents or treats a disease or condition caused by methicillin-resistant Staphylococcus aureas.

21. A composition as defined in claim 1, or uses or methods involving the composition, substantially as herein 20 described with reference to the accompanying Examples and/or Figures. 4291328_1 (GMMatofs) P54M83.AU.1