(19) TZZ ¥_Z_T

(11) EP 2 344 170 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 35/64 (2015.01) A61K 31/121 (2006.01) 24.02.2016 Bulletin 2016/08 A61K 31/724 (2006.01) A61P 31/04 (2006.01)

(21) Application number: 09820334.2 (86) International application number: PCT/IB2009/054458 (22) Date of filing: 12.10.2009 (87) International publication number: WO 2010/044042 (22.04.2010 Gazette 2010/16)

(54) ANTIMICROBIAL COMPOSITIONS ANTIMIKROBIELLE ZUSAMMENSETZUNGEN COMPOSITIONS ANTIMICROBIENNES

(84) Designated Contracting States: • LIS BALCHIN M ET AL: "PHARMACOLOGICAL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR AND ANTIMICROBIAL STUDIES ON DIFFERENT HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL TEA-TREE OILS (MELALEUCA ALTERNIFOLIA, PT RO SE SI SK SM TR LEPTOSPERMUM SCOPARIUM OR MANUKA AND KUNZEA ERICOIDES OR KANUKA), (30) Priority: 14.10.2008 JP 2008265342 ORIGINATING IN AUSTRALIA AND NEW ZEALAND",PHYTOTHERAPY RESEARCH, JOHN (43) Date of publication of application: WILEY & SONS LTD. CHICHESTER, GB, vol. 14, 20.07.2011 Bulletin 2011/29 no.8, 1 January 2000 (2000-01-01), pages 623-629, XP009144374, ISSN: 0951-418X (73) Proprietor: Manuka Health New Zealand Limited • PORTER N G ET AL: "Chemical, physical and Te Awamutu (NZ) antimicrobial properties of essential oils of Leptospermum scoparium and Kunzea (72) Inventors: ericoides", PHYTOCHEMISTRY, PERGAMON •KEIJI,Terao PRESS, GB, vol. 50, no. 3, 10 February 1998 Kobe-shi (1998-02-10), pages407-415, XP004290869, ISSN: Hyogo (JP) 0031-9422, DOI: 10.1016/S0031-9422(98)00548-2 • JO, Ayako • FILOCHE S K ET AL: "Antimicrobial effects of Kobe-shi essential oils in combination with chlorhexidine Hyogo (JP) digluconate", ORAL MICROBIOLOGY AND • NAKATA, Daisuke IMMUNOLOGY, MUNKSGAARD, COPENHAGEN, Kobe-shi DK, vol. 20, no. 4, 1 August 2005 (2005-08-01) , Hyogo (JP) pages 221-225, XP009110401, ISSN: 0902-0055 • AL SOMAL N. ET AL: ’Susceptibility of (74) Representative: Casley, Christopher Stuart et al Helicobacter pylori to the antibacterial activity of Mewburn Ellis LLP manuka honey’ JOURNAL OF THE ROYAL City Tower SOCIETY OF MEDICINE vol. 87, no. 1, 1994, pages 40 Basinghall Street 9 - 12, XP009118702 London EC2V 5DE (GB) • MAVRIC E. ET AL: ’Identification and quantification of methylglyoxal as the dominant (56) References cited: antibacterial constituent of Manuka WO-A1-98/50039 WO-A2-2005/039287 (Leptospermum scoparium) honeys from New Zealand’ MOLECULAR NUTRITION AND FOOD RESEARCH vol. 52, no. 4, April 2008, pages 483 - 489, XP002514157

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 344 170 B1

Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 344 170 B1

• VISAVADIA B.G. ET AL: ’Manuka honey dressing: An effective treatment for chronic wound ’ BR J ORAL MAXILLOFAC SURG. vol. 46, no. 1, January 2008, pages 55 - 56, XP022478791

2 1 EP 2 344 170 B1 2

Description (2000-01-01), pages 623-629). Porter N. G., et. al. dis- closed the chemical, physical and antimicrobial proper- FIELD OF INVENTION ties of essential oils of Leptospermum scoparium and Kunzea ericoides (Phytochemistry, vol. 50, no. 3,10 Feb- [0001] This invention relates to antimicrobial composi- 5 ruary 1998 (1998-02-10), pages 407-415). Filoche S K tions. In particular, this invention relates to antimicrobial et al reported the antimicrobial effects of essential oils in compositions containing methylglyoxal and cyclodextrin, combination with chlorhexidine digluconate (Oral Micro- including antimicrobial compositions containing one or biology and Immunology, vol. 20, no. 4, 1 August 2005 more materials with a methylglyoxal presence and one (2005-08-01), pages 221-225). topical application, or for or more materials with a cyclodextrin presence. Particu- 10 thetreatment ofduodenal ulcer, gastriculcer, gastric can- larly contemplated are antimicrobial compositions com- cer and so on caused byHelicobacter pylori, through prising manuka honey as the one or more materials with ingestion. However, for manuka honey to be effective, a methylglyoxal presence, and cyclodextrin. frequent application or ingestion is necessary. For these and other applications it would be desirable to have prod- BACKGROUND OF THE INVENTION 15 ucts and compositions in which the antimicrobial activity is maintained, preferably for long durations. [0002] Manuka honey is the natural honey which is pro- [0007] Accordingly, there is a need for antimicrobial duced by bees which gather nectar from manuka bush compositions, including those suitable for use in the treat- (Leptospermum scoparium) growing throughout New ment of a variety of diseases and disorders in which mi- Zealand. This species is also found in Australia, where 20 crobial activity is associated or implicated, and those suit- the honey is known under different names, such as Jel- able for use in controlling microbial populations, which lybush honey. It contains constituent actives that exhibit are able to support the maintenance of antimicrobial ac- stability in the presence of heat, light, gastric juice, en- tivity or augment antimicrobial activity. zyme, etc. It has been reported that manuka honey in- [0008] It is an object of the present invention to provide hibits the growth of Staphylococcus aureus, Helicobacter 25 antimicrobial compositions, including stable antimicrobi- pylori, and Escherichia coli, etc. J. Roy. Soc. Med. al compositions, or to at least provide the public with a 1994;87:9-12. useful choice. [0003] Identification of the antimicrobial constituent(s) present in manuka honey has been difficult, and it has SUMMARY OF THE INVENTION been referred to as "Unique Manuka Factor" or non-per- 30 oxide activity (NPA). This is the anti-bacterial activity [0009] Accordingly, in a first aspect the invention pro- which exists in manuka honey in addition to the unstable vides an antimicrobial composition comprising methylg- anti-bacterial activity reported in all honeys that is be- lyoxal and cyclodextrin. lieved to be due to hydrogen peroxide. Recently, it has [0010] In one embodiment, the antimicrobial composi- been reported that methylglyoxal is the dominant manu- 35 tion comprises one or more materials comprising meth- ka-specific antibacterial constituent, Mol. Nutr. Food ylglyoxal. Res. 2008 Apr; 52(4) 483-489. [0011] In one embodiment, the material comprising [0004] Methylglyoxal has been suggested as an effec- methylglyoxal is manuka honey. tive antimicrobial ingredient of water-soluble detachment [0012] In one embodiment, the manuka honey has a solution for washing or industrial use disinfectants com- 40 methylglyoxal concentration of greater than about prising food additives or internal agent ingredients, Un- 30mg/kg, than about 38mg/kg, than about 40mg/kg, examined publication JP2008-7408 and Unexamined about 50mg/kg, about 60mg/kg, about 70mg/kg, about publication JPH8-239693. 80mg/kg, about 90mg/kg, about 100mg/kg, about [0005] It has been reported that naturally produced 150mg/kg, about 200mg/kg, about 250mg/kg, about methylglyoxal is contained in dairy products and ferment- 45 300mg/kg, about 350mg/kg, about 400mg/kg, about ed products like beer and wine at the concentration of 3 450mg/kg, about 500mg/kg, about 550mg/kg, about to 11mg/ kg, in roasted coffee at the concentration of 23 600mg/kg, about 650mg/kg, about 700mg/kg, about to 47 mg/kg, and in manuka honey at concentrations of 750mg/kg, about 800mg/kg, about 850mg/kg, about 38 to 761 mg/kg. 900mg/kg, about 950mg/kg, greater than about [0006] Therefore, with this high antimicrobial activity, 50 1000mg/kg, greater than about 1100mg/kg, greater than there has been an expectation that manuka honey can about 1200mg/kg, greater than about 1300mg/kg, great- be used for the treatment of skin and wound infections erthan about1400mg/kg, greater than about1500mg/kg, caused by Staphylococcus aureus by Lis Balchin M., et. greater than about 1600mg/kg, greater than about al. reported the pharmacological and antimicrobial stud- 1700mg/kg, greater than about 1800mg/kg, greater than ies on different tea-tree oils (melaleuca alternifolia, lept- 55 about 1900mg/kg, or about 2000mg/kg. ospermum scoparium or manuka and kunzea ericoides [0013] In one embodiment, the manuka honey has a or kanuka), originating in Australia and new Zealand NPA rating greater than 10, than 15, than 20, than 25, (Phytotherapy Research vol. 14, no. 8, 1 January 2000 26, 27, 28, 29, 30, than 31, 32, 33, 34, or greater than 35.

3 3 EP 2 344 170 B1 4

[0014] In various embodiments, the cyclodextrin is al- powderising, such as by lyophilisation, spray-drying, de- pha-cyclodextrin,or the cyclodextrin isgamma-cyclodex- hydration, freeze-drying, etc. trin, or the cyclodextrin is present as a combination of [0031] In another embodiment, the method comprises alpha-cyclodextrin and gamma-cyclodextrin. the additional step of drying the admixture, such as by [0015] In one embodiment, the cyclodextrin is chemi- 5 lyophilisation, spray-drying, de-hydration, freeze-drying, cally-modified cyclodextrin. etc. [0016] In one embodiment, the composition comprises [0032] In one embodiment, the method comprises the from about 10.0%wt to about 99.0 %wt manuka honey. further step of powderising the dried admixture. [0017] In various embodiments, the composition com- [0033] In one embodiment, the method comprises the prises from about 0.003%wt to about 0.2 %wt methylg- 10 further step of granulating the dried admixture, or of gran- lyoxal, from about 0.003%wt to about 0.15 %wt methyl- ulating the powderised admixture. glyoxal, from about 0.006%wt to about 0.15 %wt meth- [0034] In one embodiment, the method comprises the ylglyoxal, from about 0.01%wt to about 0.15 %wt, from further step of tableting or encapsulating the dried ad- about 0.02%wt to about 0.15 %wt, from about 0.03%wt mixture, or of tableting or encapsulating the powderised to about 0.15 %wt, from about 0.04%wt to about 0.15 15 admixture, or of tableting or encapsulating the granulated %wt, from about 0.05%wt to about 0.15 %wt, from about admixture. 0.06%wt to about 0.15 %wt, from about 0.07%wt to about [0035] In another aspect, the present disclosure pro- 0.15 %wt, from about 0.08%wt to about 0.15 %wt, or from vides a method of treating or preventing a microbial dis- about 0.09%wt to about 0.15 %wt methylglyoxal. ease or disorder, the method comprising administering [0018] In one embodiment, the methylglyoxal content 20 to a subject in need thereof a composition comprising is within the range of 0.006%wt to 0.079 %wt per antimi- methylglyoxal and cyclodextrin. crobial composition. [0036] In another aspect, this disclosure provides a [0019] In one embodiment, the composition is a con- method of promoting wound healing, the method com- sumer good. prising administering to a subject in need thereof a com- [0020] In one embodiment the composition is a food, 25 position comprising methylglyoxal and cyclodextrin. drink, food additive, drink additive, dietary supplement, [0037] In various embodiments, the composition may nutritional product, medical food, nutraceutical, medica- be directly applied to the wound, for example by topical ment or pharmaceutical. application to the wound or surrounding tissue, or indi- [0021] In various embodiments, the composition may rectly applied to the wound, for example by application be formulated for oral, topical, or parenteral administra- 30 to bandages, dressings, surgical equipment. tion. [0038] In a further aspect the present disclosure pro- [0022] In one embodiment, the composition comprises vides a method for controlling one or more microbes, the one or more additional antimicrobial agents. method comprising contacting the one or more microbes [0023] In one embodiment, the composition is a phar- with a composition comprising methylglyoxal and cyclo- maceutical composition. 35 dextrin. [0024] In one embodiment, the composition is or is [0039] In a further aspect the present disclosure pro- present in a medical device or a medical supply, including vides a method for controlling one or more microbes disinfectants, cleaning agents, and surgical wipes, band- present on or in a substrate, whether animate or inani- ages, dressings, and the like. mate, the method comprising contacting the one or more [0025] In one embodiment, the composition is an in- 40 microbes or the substrate with a composition comprising dustrial product, including industrial solutions such as methylglyoxal and cyclodextrin. cleaning or descaling solutions. [0040] In another aspect, the present invention pro- [0026] In a second aspect the invention provides an vides the use of methylglyoxal and cyclodextrin in the antimicrobial composition comprising manuka honey and preparation of a medicament suitable for use in the treat- cyclodextrin. 45 ment of a microbial disease or disorder. [0027] In another aspect, the present invention pro- [0041] In another aspect, the present invention pro- vides a method of preparing a composition comprising vides the use of methylglyoxal and cyclodextrin in the methylglyoxal and cyclodextrin, the method comprising preparation of a medicament suitable for use in promot- admixing methylglyoxal, or a material comprising meth- ing wound healing in a subject in need thereof. ylglyoxal, or both, with cyclodextrin or a material com- 50 [0042] In one embodiment, the use is of a mixture of prising cyclodextrin, or both. methylglyoxal or a material comprising methylglyoxal, [0028] In one embodiment, the admixing is of a pow- and cyclodextrin. derized material with a methylglyoxal presence with cy- [0043] The present invention further provides methyl- clodextrin or a material comprising cyclodextrin, or both. glyoxal and cyclodextrin for the treatment of a microbial [0029] In one embodiment, the admixing is of powder- 55 disease or disorder. ised manuka honey with cyclodextrin. [0044] The present invention further provides methyl- [0030] In one embodiment, the method comprises the glyoxal and cyclodextrin for promoting wound healing in preliminary step of drying manuka honey prior to or during a subject in need thereof.

4 5 EP 2 344 170 B1 6

[0045] It is intended that reference to a range of num- microbial mixture A (Manuka honey 7.14 w/v%, Al- bers disclosed herein (for example, 1 to 10) also incor- pha CD 8.7 w/v%); * Antimicrobial mixture A (Manu- porates reference to all rational numbers within that ka honey 3.27 w/v%, Alpha-CD4.0w/v%); d Antimi- range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5,7, 8, 9 crobial mixture solution E (Manuka honey 7.14w/v%, and 10) and also any range of rational numbers within 5 Alpha-CD8.7 w/v%); + Antimicrobial mixture solution that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) F (MGO Honey 7.14 w/v%, Alpha-CD8, 7w/v%). and, therefore, all sub-ranges of all ranges expressly dis- Figure 5 Antimicrobial activity against Staphylococ- closed herein are hereby expressly disclosed. These are cus aureus of antimicrobial compositions A and an- only examples of what is specifically intended and all timicrobial composition solutions C & D are shown possible combinations of numerical values between the 10 (Test results). ♦ Control; j Manuka honey (7.14 lowest value and the highest value enumerated are to be w/v%); A Antimicrobial mixture A (Manuka honey considered to be expressly stated in this application in a 7.14 w/v%, Alpha-CD8, 7 w/v%); >< Antimicrobial similar manner. mixture A (Manuka honey 3.27 w/v%, Alpha-CD4.0 [0046] In this specification where reference has been w/v%); * Antimicrobial mixture solution C (Manuka made to patent specifications, other external documents, 15 honey 3.27 w/v%, Alpha-CD4.0 w/v%) +α CD4.0 or other sources of information, this is generally for the w/v%; d Antimicrobial mixture solution D (Manuka purpose of providing a context for discussing the features honey 3.27w/v%, Alpha-CD4.0w/v%) + Manuka of the invention. Unless specifically stated otherwise, ref- honey 3.27 w/v%. erence to such external documents is not to be construed as an admission that such documents, or such sources 20 DETAILED DESCRIPTION of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art. [0048] The present invention is based on the finding that by admixing, for example by mixing and powderizing, BRIEF DESCRIPTION OF THE FIGURES materials comprising methylglyoxal and materials com- 25 prising cyclodextrin, the antimicrobial activity of the orig- [0047] inal materials is at least maintained or preferably is en- hanced. Figure 1 Antimicrobial activity against Staphylococ- [0049] Thus, antimicrobial compositions of this inven- cus aureus of antimicrobial composition A are shown tion maintain or enhance the antimicrobial activity of orig- (Test results). ♦ Control; j Alpha-CD (4.0w/v%); m 30 inal methylglyoxal or materials with methylglyoxal con- Manuka honey (3.27w/v%); >< Antimicrobial mixture tained. A (Manuka honey 0.82 w/v%, Alpha CD 1.0 w/v%); [0050] Accordingly, provided that the antimicrobial * Antimicrobial mixture A (Manuka honey 1.64 w/v%, compositions are formulated so as to be suitable for ad- α CD2.0w/v%); d Antimicrobial mixture A (Manuka ministration to a mammalian subject, for example they honey 3.27w/v%, Alpha-CD4, 0w/v%); + Antimicro- 35 consist of materials that are safe to the human body, they bial mixture A (Manuka honey 7.14 w/v%, Alpha- can be used for manufacturing antimicrobial consumer CD8, 7w/v%). goods, such as beverages, foods, and the like, as well Figure 2 Antimicrobial activity against Staphylococ- as pharmaceutical compositions, drugs, and the like. cus aureus of antimicrobial composition A are shown [0051] Since the antimicrobial activity of methylglyoxal (Test results). ♦ Control; j Alpha-CD (4.0w/v%); m 40 or materials with a methylglyoxal presence is maintained Manuka honey (3.27w/v%); >< Antimicrobial mixture in the compositions of the invention for a sustained pe- A (Manuka honey 3.27 w/v%, Alpha-CD 4.0 w/v%); riod, the dosage or frequency of administration of the * Antimicrobial mixture A (Manuka honey 7.14 w/v%, composition can be reduced, or higher efficacy provided Alpha CD8.7w/v%). or both. Figure 3 Antimicrobial activity against Staphylococ- 45 [0052] And further, other embodiments of the invention cus aureus of antimicrobial composition B are shown provide antimicrobial compositions formulated for indus- (Test results). ♦ Control; j Gamma-CD (4.0w/v%); trial use, for example, where compositions comprise in- m Manuka honey (3.27w/v%); >< Antimicrobial mix- dustrially available or acceptable materials. Such com- ture B (Manuka honey 0.82 w/v%, Gamma-CD1.0 positions can be used for industrial products such as dis- w/v%); * Antimicrobial mixture B (Manuka honey50 infectant for cooling water or washing water in the indus- 1.64 w/v%, Gamma-CD2.0w/v%); d Antimicrobial try, or in various industrial processes known to those mixture B (Manuka honey 3.27w/v%, Gamma- skilled in the art. CD4.0/v%). [0053] The phrases "antimicrobial compositions" or Figure 4 Antimicrobial activity against Staphylococ- "compositions having antimicrobial activity" (used inter- cus aureus of antimicrobial composition A and anti- 55 changeably herein) of this invention contemplate any microbial composition solutions E & F are shown kind of compositions, as long as the antimicrobial com- (Test results). ♦ Control; j Manuka honey (7.14 positions are either antimicrobial compositions contain- w/v%); m MGO Honey power (7.14w/v%); >< Anti- ing methylglyoxal and cyclodextrin or antimicrobial com-

5 7 EP 2 344 170 B1 8 positions containing materials with methylglyoxal con- practice of medicine, including for example, disinfect- tained and cyclodextrin. Compositions which maintain or ants, germicides, lavages, solutions, dressings, bandag- enhance the original antimicrobial activity of methylgly- es, and the like. oxal or materials with methylglyoxal contained are par- [0060] As used herein, "microbial disease or disorder" ticularly contemplated. 5 refers to a disease or disorder caused by or exacerbated [0054] The term "and/or" can mean "and" or "or". by one or more microbes, including those diseases or [0055] The term "comprising" as used in this specifi- disorders of which one or more symptoms are caused or cation means "consisting at least in part of". When inter- exacerbated by one or more microbes. preting statements in this specification that include that [0061] As used herein, "manuka honey" refers to a term, the features, prefaced by that term in each state- 10 honey produced by bees from the nectar of flora of Lept- ment, all need to be present but other features can also ospermum spp., in particular Leptospermum scoparium. be present. Related terms such as "comprise" and "com- Other honeys with a methylglyoxal concentration of prised" are to be interpreted in the same manner. above about 15mg/kg, preferably above about 30mg/kg, [0056] The term "control" or "controlling" as used here- more preferably above about 60mg/kg, are contemplated in generally comprehends preventing, reducing, or erad- 15 as suitable for use in embodiments of the present inven- icating microbial or inhibiting the rate and extent tion. This may be honey made from nectars collected by of such infection, or reducing the microbial population, bees both from Leptospermum species and other spe- such as a microbial population present in or on a body cies, or may be honey from species providing such a or structure, surface, liquid, subject, etc, wherein such methylglyoxal content as desired. Alternatively, honeys prevention or reduction in the infection(s) or population(s) 20 (whether manuka or otherwise) augmented with methyl- is statistically significant with respect to untreated infec- glyoxal are contemplated. tion(s) or population(s). Curative treatment is also con- [0062] When used in respect of an agent having anti- templated. Preferably, such control is achieved by in- microbial activity, such as a composition of the invention creased mortality amongst the microbial population. or a component of a composition of the invention, the [0057] An "effective amount" is the amount required to 25 phrase "retaining antimicrobial activity" and grammatical confer therapeutic effect. The interrelationship of dosag- equivalents and derivatives thereof is intended to mean es for animals and humans (based on milligrams per me- that the agent still has useful antimicrobial activity. Pref- ter squared of body surface) is described by Freireich, erably, the retained activity is at least about 35, 40, 45, et al. (1966). Body surface area can be approximately 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% of the determined from height and weight of the subject. See, 30 original activity, and useful ranges may be selected be- e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, tween any of these values (for example, from about 35 New York, 1970, 537. Effective doses also vary, as rec- to about 100%, from about 50 to about 100%, from about ognized by those skilled in the art, dependent on route 60 to about 100%, from about 70 to about 100%, from of administration, excipient usage, and the like. about 80 to about 100%, and from about 90 to about [0058] A "medical device" as used herein includes, for 35 100%). For example, to be useful in the present invention example but is not limited to, any temporarily or perma- a composition should retain antimicrobial activity, that is, nently implanted device into or on a human or animal retain at least about 35, 40, 45, 50, 55, 60, 65, 70, 75, host, for example stents, balloons, prosthetic heart 80, 85, 90, 95, 99 or 100% of the antimicrobial activity of valves, annuloplasty rings, grafts, shunts, sewing rings the original antimicrobial agent, for example the material having silicone or polyurethane inserts, polyester fabric 40 comprising a methylglyoxal presence. Similarly, pre- encasements, stents, medical leads, orthopedic plates, ferred compositions of the invention are capable of sup- catheters, pacemakers, sutures, and/or any one or more porting the maintenance of useful antimicrobial activity of the foregoing medical devices can include a fabric of the antimicrobial agent (s) they comprise, and can be overlayer of any type, including for example a sheath, an said to retain antimicrobial activity, ideally until applied encasement, a layer, or a coating, such that the fabric 45 using the methods contemplated herein. overlayer is in contact with body tissue or fluids such as [0063] When used in respect of a composition of the blood. Alternatively, instead of the medical device includ- invention or a component of a compositon of the inven- ing a fabric overlayer, the medical device may include a tion, the phrase "enhancing antimicrobial activity" and mesh, coil, wire, inflatable balloon, bead, sheet, or any grammatical equivalents and derivatives thereof is in- other structure which is capable of being positioned or 50 tended to mean that when present in the composition, implanted at a target location, including intravascular tar- an equivalent amount or concentration of the antimicro- get locations, intraluminal target locations, target loca- bial agent has increased antimicrobial activity compared tions within solid tissue, and the like. Implantation at to that of the agent in the absence of the composition wounds, such as surgical wounds, is particularly contem- (such as the isolated agent). Preferably, the enhanced plated. 55 activity is at least about 105, 110, 115, 120, 125, 130, [0059] A "medical supply" and grammatical equiva- 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, lents as used herein includes, for example but is not lim- 190, 195,200%, ormore of the original activity, and useful ited to, any consumable product commonly used in the ranges may be selected between any of these values

6 9 EP 2 344 170 B1 10

(for example, from about 35 to about 100%, from about significant amount. 50 to about 100%, from about 60 to about 100%, from [0069] The term "(s)" following a noun contemplates about 70 to about 100%, from about 80 to about 100%, the singular or plural form, or both. and from about 90 to about 100%). In certain embodi- [0070] The term "subject" is intended to refer to an an- ments, compositions of the invention may exhibit en- 5 imal, preferably a mammal, more preferably a mamma- hanced antimicrobial activity, that is, exhibit at least about lian companion animal or human. Preferred companion 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, animals include cats, dogs and horses. Other mamma- 160, 165, 170, 175, 180, 185, 190, 195, 200%, or more lian subjects include an agricultural animal, including a of the antimicrobial activity of the original antimicrobial horse, a pig, a sheep, a goat, a cow, a deer, or a fowl, or agent, for example the material comprising a methylgly- 10 a laboratory animal, including a monkey, a rat, or a oxal presence. Similarly, preferred compositions of the mouse. invention are capable of supporting the maintenance of [0071] The term "treat" and its derivatives should be enhanced antimicrobial activity of the antimicrobial agent interpreted in their broadest possible context. The term (s) they comprise, and can be said to retain enhanced should not be taken to imply that a subject is treated until antimicrobial activity, ideally until applied using the meth- 15 total recovery. Accordingly, "treat" broadly includes main- ods contemplated herein. The enhanced activity (includ- taining a subject’s disease progression or symptoms at ing enhanced maintenance of activity) may result from a substantially static level, increasing a subject’s rate of synergy amongst the various components of the compo- recovery, amelioration and/or prevention of the onset of sitions of the invention. the symptoms or severity of a particular condition, or ex- [0064] As used herein, the term "stable" when used in 20 tending a patient’s quality of life. The term "treat" also relation to a composition of the invention means a com- broadly includes the maintenance of good health for sen- position capable of supporting antimicrobial activity for sitive individuals and building stamina for disease pre- preferably more than two hours, more than three hours, vention. 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 20 hours, [0072] As used herein, "NPA value" means the meas- more than one day, preferably about two, about three, 25 urement of antibacterial activity determined for a whole about four, preferably about five, more preferably about or sieved honey or fraction thereof determined relative six days, preferably a week, two weeks, three weeks, a to phenol equivalents in an agar plate diffusion assay. month, or longer. It will be appreciated that in certain embodiments, stable compositions include those which Exemplary uses of the invention have antimicrobial activity for a period greater than does 30 the antimicrobial agent alone. [0073] The methods and compositions of the disclo- [0065] The term "oral administration" includes oral, sure may be used in the treatment of microbial diseases buccal, enteral and intra-gastric administration. or disorders, to promote wound healing, and to control [0066] The term "parenteral administration" includes microbial infection. but is not limited to topical (including administration to 35 [0074] In one embodiment, the microbial disease or any dermal, epidermal or mucosal surface), subcutane- disorder is a bacterial disease or disorder. In one em- ous, intravenous, intraperitoneal, intramuscular and in- bodiment the microbial infection is a bacterial infection. tratumoural (including any direct administration to a tu- [0075] In one embodiment, the microbial disease or mour) administration. disorder is a fungal disease or disorder. In another em- [0067] The term "pharmaceutically acceptable carrier" 40 bodiment, the microbial disease is a yeast disease or is intended to refer to a carrier including but not limited disorder. In one embodiment the microbial infection is a to an excipient, diluent or auxiliary that can be adminis- fungal infection. In one embodiment the microbial infec- tered to a subject as a component of a composition of tion is a yeast infection. the invention. Preferred carriers do not reduce the activity [0076] In one embodiment, the microbial disease is a of the composition and are not toxic when administered 45 microbial infection of the skin, lung, buccal cavity, gastro- in doses sufficient to deliver an effective amount of meth- intestinal tract, eye, ear, sinuses, kidney, mucosal sur- ylglyoxl, or, when administered, of another antimicrobial faces, or urinary tract. agent. [0077] In one embodiment, the microbial disease or [0068] The term "promote wound healing" and gram- disorder is a skin disease or disorder or a tissue disease matical equivalents thereof when used with reference to 50 or disorder, such as psoriasis, acne, ulceration, wound the methods and compositions of the present invention infection or refractory wound(s), burn(s), dermatitis, ath- contemplates improved wound healing in the presence letes foot, and eczema. For example, the microbial dis- of a composition of the invention than is or was observed ease or disorder is a bacterial infection, such as a bac- in the absence of a composition of the invention. For ex- terial infection of a wound, including an infection of any ample, a refractory wound - that is, a wound resistant to 55 one or more of the following bacteria:Staphylococcus treatment or healing - exhibits improved healing following aureus, including methicillin-resistant Staphylococcus application of a composition of the present invention. aureus (MRSA), E.coli, or Pseudomonas aeruginosa. Preferably, wound healing is improved by a statistically [0078] In one embodiment, the microbial disease or

7 11 EP 2 344 170 B1 12 disorder is a lung disease or disorder, such as chronic Candida spp., including , Candida utilis, obstructive pulmonary disease (COPD, also referred to Chlamydophila spp., including Chlamydophila pneumo- as chronic obstructive respiratory disease (CORD)), tu- niae, Escherichia spp., including Escherichia coli, Hae- berculosis, or emphysema. For example, the microbial mophilus spp., including Haemophilus influenzae, Heli- disease or disorder is a bacterial infection of Mycobac- 5 cobacter spp., including Helicobacter pylori, Klebsiella teria tuberculosis, or Mycobacteria paratuberculosis. spp., including Klebsiella pneumoniae, Listeria spp., in- [0079] In one embodiment, the microbial disease or cluding Listeria monocytogenes, Micrococcus spp., in- disorder is an oral disease or disorder, such as dental cluding Micrococcus fiavus, Moraxella spp., including caries, gingivitis, ulcers. For example, the microbial dis- Moraxella catarrhalis, Mycobacteria spp., including My- ease or disorder is a bacterial infection of any one or10 cobacteria tuberculosis, Mycobacteria paratuberculosis, more of the following bacteria: Streptococcus salivarius, Mycoplasma spp., including Mycoplasma pneumoniae, S. mitis, S. mutans, S. rattus, S. cricetus, S. sobrinus, S. Pasteurella spp., including Pasteurella multocida, Peni- ferus, S. macacae, or S. downei, Luctobacillus spp., in- cillium spp., including Penicillium chrysogenum, Proteus cluding Lactobacillus caseii. spp., including Proteus mirabilis and Proteus vulgaris, [0080] In one embodiment, the microbial disease or 15 Pseudomonas spp., including Pseudomonas aerugino- disorder is a gastro-intestinal disease or disorder, such sa/ pyocyanea, Salmonella spp., including Salmonella as gastro-enteritis, ulcers including peptic ulcers, chronic typhi, Sarcinalutea spp., Serratia spp., including Serratia gastritis, and duodenitis. For example, the microbial dis- marcescens, Shigella spp., including Shigella boydii, ease or disorder is a bacterial infection of any one or Shigella fiexneri, and Shigella sonnei, Staphylococcus more of the following bacteria: Helicobacter spp., includ- 20 spp., including Staphylococcus albus, and Staphylococ- ing H. acinonychis, H. anseris, H. aurati, H. bilis, H. biz- cus aureus including methicillin-resistant Staphylococ- zozeronii, H. brantae, H. canadensis, H. canis, H. chole- cus aureus, Streptococcus spp., including Group B cystus, H. cinaedi, H. cynogastricus, H. felis, H. fennel- Streptococci, Streptococcus faecalis, Streptococcus liae, H. ganmani, H. hepaticus, H. mesocricetorum, H. pneumoniae, and Streptococcus pyogenes, and Vibrio marmotae, H. muridarum, H. mustelae, H. pametensis, 25 spp., including Vibrio cholerae. H. pullorum, H. pylori, H. rappini, H. rodentium, H. salo- [0084] In various embodiments the microbial disease monis, H. trogontum, H. typhlonius, H. winghamensis, or disorder is, or the fungal infection is of any one or more Campylobacter spp., including C. coli, C. concisus, C. of the following fungi: Candida spp., including Candida curvus, C. fetus, C. gracilis, C. helveticus, C. hominis, C. albicans, Candida utilis, Aspergillus spp., Penicilliium hyointestinalis, C. insulaenigrae, C. jejuni, C. lanienae, 30 spp. C. lari, C. mucosalis, C. rectus, C. showae, C. sputorum, [0085] In various embodiments, the microbial disease C. upsaliensis. or disorder is selected from the group comprising bums, [0081] In one embodiment, the microbial disease or venous leg ulcers, leg ulcers of mixed aetiology, diabetic disorder is an eye disease or disorder, such as blephar- foot ulcers, pressure ulcers, unhealed graft donor sites, itis, conjunctivitis, keratitis including fungal keratitis. For 35 abscesses, boils, pilonidal sinuses, infected wounds in- example, the microbial disease or disorder is a microbial cluding those from lower limb surgery, necrotising faciitis, infection of any one or more of the following microbes: neonatal postoperative wound infection, and gangrene Staphylococcus spp., Aspergillus fumigates, Fusarium including Fournier’s gangrene. spp. and Candida spp. [0086] In one embodiment administration of the inven- [0082] In one embodiment, the microbial disease or 40 tive composition reduces microbial infection, such as disorder is an ear or sinus disease or disorder, such as bacterial or fungal infection, by 5, 10, 15, 20, 25, 30, 35, Otitis externa, Otitis media, sinusitis including acute si- 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, nusitis, chronic sinusitis and -refractory chronic 99, or 100%. sinusitis. For example, the microbial disease or disorder [0087] In embodiments relating to promoting wound is a microbial infection of the ear or sinus, including an 45 healing, the wound may be selected from the group com- infection of any one or more of the following microbes: prising burns, venous ulcers including venous leg ulcers, Staphylococcus spp. including Staphylococcus aureus, leg ulcers of mixed aetiology, diabetic ulcers including Pseudomonas aeruginosa, Aspergillus spp., including diabetic foot ulcers, pressure ulcers, unhealed graft do- Aspergillus fumigates, Streptococcus spp. including nor sites, abscesses, boils, pilonidal sinuses, cancer Streptococcus pneumonia, Haemophilus influenza,50 wounds, surgical wounds, infected wounds including Moraxella catarrhalis, Mycobacterium tuberculosis, and those resulting from surgery such as lower limb surgery, Candida spp. including Candida albicans. necrotising faciitis, neonatal postoperative wound infec- [0083] In various embodiments the microbial disease tion, and gangrene including Fournier’s gangrene. or disorder is, or the microbial infection is of any one or [0088] In one embodiment administration of the inven- more of the following microbes: Aspergillus spp., includ- 55 tivecomposition promoteswound healing(with reference ing Aspergillus flavus, Aspergillus fumigatus, Aspergillus to untreated wound) by 5, 10, 15, 20, 25, 30, 35, 40, 45, niger, Bacillus spp., including Bacillus subtilis, Bacillus 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or cereus, Boretella spp., including Boretella pertussis, 100%.

8 13 EP 2 344 170 B1 14

Methylglyoxal and materials comprising methylglyoxal livery properties of the composition. [0095] In particularly contemplated embodiments, the [0089] Methylglyoxal (CAS number 78-98-8), also manuka honey, or manuka honey with additional com- called pyruvaldehyde or 2-oxo-propanal, is the aldehyde pounds, can be further processed to optimise the drug form of pyruvic acid. Methylglyoxal is reportedly formed 5 delivery properties of the resulting composition. For ex- in organisms from several sources: from 3-amino ace- ample, the manuka honey powder can be cut to obtain tone, through lipid peroxidation, and from non-enzymatic a particle size distribution that enables ready admixture dephosphorylation from glyceraldehyde phosphate and with the other components of the composition, or ease dihydroxyacetone phosphate during glycolysis. of administration to a subject, etc. [0090] Methylglyoxal and materials with methylglyoxal 10 [0096] In one embodiment the additional compounds contained in the antimicrobial compositions of this inven- are added prior to powdering, so as to improve the pow- tion are commercially available, eg, Methyglyoxal (Aque- dering process. ous solution) of Nacalai Tesque Co., Ltd as methylgly- [0097] An exemplary process for powdering manuka oxal, in fermented products like dairy products, beer, honey is performed by first heating the honey to kill bac- wine, roasted coffee and manuka honey as exemplary 15 teria, protozoa, yeast, fungi and other organisms that are materials with methylglyoxal contained. Alternatively, naturally present in the manuka honey. The honey is then methylgloxal or a material with methylglyoxal contained powdered by methods well known in the art. Once pow- may be prepared independently. dered, the resulting manuka powder can be stored for [0091] Preferred sources of methylgloxal for use in the admixture, or admixed directly. invention include methylglyoxal 550 (Manuka Health20 [0098] It will be appreciated that in certain embodi- New Zealand Ltd), manuka honey containing 600 mg/g to ments of the invention, the material comprising methyl- 800mg/g of methylglyoxal and methylglyoxal honey pow- glyoxal, such as manuka honey, is admixed with one or der mixed with dextrin 50%wt. Those skilled in the art will more cyclodextrins prior to drying or powderising. Exam- recognise that other sources may be appropriate, having ples of such admixing methods and of the resulting ad- regard to the teaching herein. 25 mixtures are presented herein. [0092] Manuka honey is available from a wide number of sources in New Zealand and elsewhere, commonly as Cyclodextrins and materials comprising cyclodex- a liquid or creamed form. Preferred sources are those trin obtained from bee-hives with the resulting honey held in storage, for example to assess the methylglyoxal con- 30 [0099] Cyclodextrins are cyclic molecules composed tent. Those skilled in the art will recognise that for use in of glucopyranose ring units which form toroidal struc- the present invention, manuka honey may be processed tures. The interior of the cyclodextrin molecule is hydro- to a form suitable for admixture, for example with cyclo- phobic and the exterior is hydrophilic, making the cyclo- dextrin, while maintaining the bioactive ingredients. Typ- dextrin molecule water soluble. The degree of solubility ically the manuka honey, or an extract thereof, is proc- 35 can be altered through substitution of the hydroxyl groups essed to a fine particulate form. Various methods of pre- on the exterior of the cyclodextrin. Similarly, the hydro- paring active manuka honey, or an extract thereof, to a phobicity of the interior can be altered through substitu- particulate form are known. For example, PCT interna- tion, though generally the hydrophobic nature of the in- tional publication WO 05/120250 discloses a freeze dry- terior allows accommodation of relatively hydrophobic ing method. Fractions can be prepared by using chroma- 40 guestswithin thecavity. Accommodation of onemolecule tography (such as HPLC) using, for example, a size ex- within another is known as complexation and the result- clusion matrix or a reverse phase matrix. A typical solvent ing product is referred to as an inclusion complex. Cy- for use in such processes is water. clodextrins are typically identified with reference to the [0093] In one embodiment the manuka honey is pow- number of monomeric units that comprise the molecule, dered without the addition of any additional compounds. 45 wherein alpha-cyclodextrin (α -cyclodextrin) comprises [0094] In an alternate embodiment powdered manuka six monomeric units, beta-cyclodextrin (β -cyclodextrin) honey is combined with other compounds that enhance comprises seven monomeric units, and gamma-cyclo- the properties of manuka honey, for example a com- dextrin γ ( -cyclodextrin) comprises eight monomeric pound that enhances the ease of formulation or admin- units. Larger cyclodextrin molecules have been de- istration, or that enhances antimicrobial activity, or that 50 scribed, including a well-characterised cyclodextrin con- enhances the stability of one or more antimicrobial ac- taining 32 1,4-anhydroglucopyranoside units tivities present in manuka honey. An example of addi- [0100] Cyclodextrin molecules may conveniently be tional compounds are those that improve the therapeutic derivatised, by for example chemical modification, for ex- benefits of the manuka honey. For example, mannitol ample to alter one or more of the physicochemical prop- could be added to enhance the diuretic properties of the 55 erties thereof. Examples of cyclodextrin derivatives in- resulting composition. Alternatively or additionally other clude methylated cyclodextrins, sulfobutylcyclodextrin, compounds such as excipients, and/or propellants could maltosylcyclodextrin, hydroxypropylcyclodextrin, and be added to improve the dosing, manufacturability or de- salts thereof. Those skilled in the art will recognise that

9 15 EP 2 344 170 B1 16 various derivates of cyclodextrin may be suitable for par- methylglyoxal contained and cyclodextrin are independ- ticular purposes, for example, certain derivatives of cy- ently dissolved in water, medium, etc. and then admixed, clodextrin may not be acceptable for administration to for example kneaded, and further those in which powder human subjects, but are suitable for use in compositions prepared as described herein is admixed with cyclodex- of the present invention targeted to industrial uses and 5 trin and/or methylglyoxal, then added to water, medium, applications. In certain embodiments, chemically-modi- etc and further mixed, for example kneaded. In certain fied cyclodextrins are particularly contemplated for such embodiments, antimicrobial compositions prepared as industrial uses and applications. powders as described above may be preferred, for ex- [0101] Cyclodextrins contained in the antimicrobial ample because they may maintain stronger antimicrobial compositions of the present invention may be commer- 10 activity or may maintain antimicrobial activity for a longer cially available, or may be prepared independently by periodthan that of solutions of antimicrobial compositions methods well known to those skilled in the art. It will be prepared as described above. apparent to those skilled in the art that cyclodextrins used [0107] The content of methylglyoxal or materials with in the antimicrobial compositions for administration to a methylglyoxal contained and cyclodextrin of the present subject, for example a cyclodextrin for manufacturing a 15 invention can be at any level as long as the expected beverage, food, or pharmaceutical of the invention antimicrobial activity is realized, such as from about should be safe to human body, and preferably is a phar- 0.003 %wt to about 0.015%wt of methylglyoxal, or from maceutically acceptable cyclodextrin. about 0.006 %wt to 0.079 %wt of methylglyoxal, 10.0 to [0102] Preferably alpha- or gamma-cyclodextrin or 99.0 %wt of materials with methylglyoxal contained, eg, combinations thereof are used. In embodiments where 20 manuka honey and 1.0 to 90.0%wt of cyclodextrin. The alpha-cyclodextrin is used, antimicrobial activity may be content can be adjusted by dissolution in or dilution with especially enhanced, as presented herein in the exam- water, medium, etc. For example, the antimicrobial com- ples. In embodiments where gamma-cyclodextrin is position A prepared as the example below utilizes manu- used, the enhancement in antimicrobial activity may be ka honey as the material with methylglyoxal contained less pronounced than that by alpha-cyclodextrin. How- 25 and its concentration is adjusted with water in the exam- ever, compositions comprising gamma-cyclodextrin may ple. provide enhanced mouth feel or palatability, for example [0108] Medium prepared so that the concentration of compositions comprising gamma-cyclodextrin and manuka honey can be 7.14% w/v and that of alpha-cy- manuka honey exhibit a stronger tendency to maintain clodextrin can be 8.37%w/v prevented the growth of in- the sweetness of the manuka honey. 30 oculated Staphylococcus aureus more than 96 hours of [0103] This means that gamma-cyclodextrin may be incubation and is found to be very suited concentration preferred for use in compositions, such as beverages for microbial growth prevention. and foods, in which palatability, and particularly sweet [0109] In case of the antimicrobial composition B, taste, is important. manuka honey is used as the material with methylglyoxal [0104] In case of the antimicrobial compositions for in- 35 contained , which is dissolved in water to the intended dustrial products like as disinfectants for cooling water concentration. Medium prepared so that the concentra- or washing water in industry, chemically modified cyclo- tion of manuka honey can be 3.27% w/v and that of gam- dextrins can be used. ma-cyclodextrin can be 4.0%w/v prevented the growth [0105] Cyclodextrins suitable for use in the present in- of inoculated Staphylococcus aureus more strongly than vention can be obtained from commercial sources, or 40 the concentration of 3.27%w/v of only manuka honey. can prepared independently by methods well known in This means that the concentration was adjusted to the the art, such as from starch by enzymatic conversion. favorable level. Preferably CAVAMAX W6 Food as alpha-cyclodextrin [0110] Other antimicrobial substances generally (CycloChem Co.,Ltd.) and CAVAMAX W8 FOOD (Cy- known can be combined with the antimicrobial composi- cloChem Co., Ltd.) as gamma-cyclodextrin are used. 45 tions of this invention, depending upon the application to which the composition is to be put. Compositions of the invention [0111] Without wishing to be bound by any theory, the applicants believe that the enhanced antimicrobial activ- [0106] Exemplary antimicrobial compositions of the ity observed in exemplary compositions of the present present invention include a powder that was spray-dried 50 invention maybe due at leastin part to a synergybetween after mixing methylglyoxal or materials with methylgly- methylglyoxal, particularly when present as manuka hon- oxal contained with cyclodextrin, then adding water and ey, and cyclodextrin, particularly alpha-cyclodextrin and homogenizing the composition. Other exemplary antimi- to a lesser extent gamma-cyclodextrin. Again, without crobial compositions of the present invention include so- wishing to be bound by any theory, the applicants ac- lutions, including for example, those in which methylgly- 55 knowledge that there may be a role of other components oxal or materials with methylglyoxal contained and cy- of the exemplary compositions, such as polyphenols clodextrin are mixed and then dissolved in water, medi- present in manuka honey, in achieving the observed en- um, etc., those in which methylglyoxal or materials with hanced antimicrobial activities.

10 17 EP 2 344 170 B1 18

[0112] Compositions suitable for administration to a ticularly contemplated are compositions additionally subject may be formulated as a food, drink, food additive, comprising milk or one or more milk products or compo- drink additive, dietary supplement, nutritional product, nents of milk, such as milk protein, whey protein, colos- medical food, nutraceutical, medical supply, medical de- trums, milk fat, or any fractions of milk or one or more vice, medicament or pharmaceutical. Appropriate formu- 5 milk products or components of milk, such as a milk fat lations may be prepared by an art skilled worker with fraction, a milk protein fraction, a whey protein fraction, regard to that skill and the teaching of this specification. a colostrums fraction, or the like. [0113] In one embodiment the present invention re- [0119] Compositions useful herein may further include lates to use of methylglyoxl, optionally with at least one other factors such as calcium, zinc, magnesium, seleni- antimicrobial agent, in the manufacture of a food, drink, 10 um, , vitamin D, vitamin E, vitamin K2, complex food additive, drink additive, dietary supplement, nutri- carbohydrates, edible or cooking oils including palm, ol- tional product, medical food, nutraceutical, medical de- ive, soybean, canola, com, sunflower, safflower, peanut, vice, medical supply, medicament or pharmaceutical. In grape seed, sesame, nut, almond, cashew, hazelnut, one embodiment, the composition is formulated for oral macadamia, pecan, pistachio, and walnut, and other ed- administration. In another embodiement, the composi- 15 ibles include acai, amaranth, apricot, argan, artichoke, tion is formulated for parenteral, including topical, admin- avocado, babassu, ben, blackcurrant seed, borage seed, istration. Preferably the composition is for inhibiting mi- borneo tallow nut, bottle gourd, buffalo gourd, carob pod crobial growth, treating or preventing a microbial disease (algaroba), cohune, coriander seed, evening primrose, or disorder, or one or more other uses as described false flax, hemp, kapok seed, lallemantia, meadowfoam above. 20 seed, mustard, okra seed (hibiscus seed), perilla seed, [0114] In one embodiment the composition is in the pequi, pine nut, poppyseed, prune kernel, pumpkin seed, form of a powder, a tablet, a caplet, a pill, a hard or soft quinoa, ramtil, rice bran, tea (camellia), thistle, watermel- capsule or a lozenge. on seed, or wheat germ oil, or a combination thereof. [0115] In one embodiment the composition is in the [0120] The compositions useful herein may be formu- form of a cachet, a dispensable powder, granules, a sus- 25 lated to allow for administration to a subject by any cho- pension, an elixir, a liquid, a drink, or any other form that sen route, including but not limited to oral or parenteral can be added to food or drink, including for example water (including topical, subcutaneous, intramuscular and in- or fruit juice. In one embodiment the composition is an travenous) administration. Those skilled in the art will ap- enteral product, a solid enteral product or a liquid enteral preciate that the route of administration to a subject will product. 30 typically take into account the purpose for which the com- [0116] In one embodiment, the composition is in the position is being administered - for example, where a form of a , ointment, a paste, a drop solution in- pharmaceutical composition of the invention is being ad- cluding eye drops or ear drops, an inhaler or as an inhal- ministered to treat a microbial disease or disorder, the able composition, a dressing, a pad, or a spray. route of administration will typically be chosen taking into [0117] In one embodiment the composition further35 account the nature of the microbial disease or disorder. comprises one or more constituents (such as antioxi- Accordingly, exemplary compositions for the treatment dants) which prevent or reduce degradation of the com- of skin infections caused by or exacerbated by Staphy- position during storage or after administration. lococcus aureus may be formulated for topical adminis- [0118] In one embodiment, compositions useful herein tration. include any edible consumer product which is able to40 [0121] In general, for oral administration a dietary (a carry one or more cyclodextrins. When the composition food, food additive or food supplement for example), nu- comprises as the at least one additional antimicrobial traceutical or pharmaceutical composition useful herein agent a proteinaceous factor, the edible consumer prod- may be formulated by a skilled worker according to known uct is one able to carry protein. Examples of suitable ed- formulation techniques. ible consumer products include baked goods, powders, 45 [0122] Thus, a pharmaceutical composition useful ac- liquids, confectionary products, reconstituted fruit prod- cording to the invention may be formulated with an ap- ucts, snack bars, food bards muesli bars, spreads, sauc- propriate pharmaceutically acceptable carrier (including es, dips, dairy products including ice creams, yoghurts excipients, diluents, auxiliaries, and combinations there- and cheeses, drinks including dairy and non-dairy based of) selected with regard to the intended route of admin- drinks (such as milk drinks including milk shakes, and 50 istration and standard pharmaceutical practice. See for yogurt drinks), milk powders, sports or nutritional supple- example, Remington’s Pharmaceutical Sciences, 16th ments including dairy and non-dairy based sports or nu- edition, Osol, A. Ed., Mack Publishing Co., 1980. tritional supplements, food additives such as protein [0123] While the preferred route of administration is sprinkles and dietary supplement products including dai- oral, it should be understood that any mode of adminis- ly supplement tablets. Within this embodiment, a com- 55 tration may be suitable for any composition of the inven- position useful herein may also be an infant formula, in tion, including administration by multiple routes, including powder or liquid form. Suitable nutraceutical composi- different routes for different agents. Therefore, inhalation tions useful herein may be provided in similar forms. Par- (nasal or buccal inhalation) and vaginal and rectal ad-

11 19 EP 2 344 170 B1 20 ministration of any composition of the invention is also semi-permeable matrices of solid hydrophobic polymers contemplated. Intramedullar, epidural, intra-articular, containing methylglyoxal and cyclodextrin, and when and intra-pleural administration of any composition of the present the at least one additional antimicrobial agent. invention is also contemplated. Administration of a com- The matrices may be in the form of shaped articles, e.g., position of the invention, optionally with at least one ad- 5 films, or microcapsules. Examples of sustained-release ditional antimicrobial factor, by a first administration route matrices include polyesters, hydrogels (for example, po- accompanied by separate, simultaneous or sequential ly(2-hydroxyethyl- methacrylate), or poly(vinylalcohol)), administration of one or more other agents, including one polylactides (see US 3,773,919), copolymers of L- or more other antimicrobial agents, by a second admin- glutamic acid and ethyl-L-glutamate, non-degradable istration route is also contemplated; for example, oral ad- 10 ethylene-vinyl acetate, and degradable -glycol- ministration of a composition of the invention accompa- ic acid copolymers such as the LUPRON DEPOT™ (in- niedby topical administration ofthe atleast one additional jectable microspheres composed of lactic acid-glycolic antimicrobial agent. acid copolymer and leuprolide acetate). [0124] The compositions of the invention may also be [0128] Topical formulations comprising methylglyoxal formulated as a dosage form. A dosage form useful here- 15 and cyclodextrin, and when present the at least one ad- in may be administered orally as a powder, liquid, tablet ditional antimicrobial agent, may be prepared as lotions, or capsule. Suitable dosage forms may contain additional creams,ointments, pastes orsalves using knowncarriers agents as required, including emulsifying, antioxidant, for such applications. Such formulations may be admin- flavouring or colouring agents, or have an enteric coating. istered directly, for example, applied directly on to a Suitable enteric coatings are known. Enteric coatings 20 wound, sprayed onto a surgical site, etc, or may be ap- surrounding the active ingredients and prevent the re- plied indirectly, such as by impregnation into a bandage lease of the active ingredients in the stomach but allow or dressing or sprayed onto surgical equipment, dress- release after the dosage form has left the stomach. Dos- ings and the like. age forms useful herein may be adapted for immediate, [0129] The present invention also relates to a parenter- delayed, modified, sustained, pulsed or controlled re- 25 al unit dosage form comprising methylglyoxal and cyclo- lease of the active components. Suitable formulations dextrin, optionally with at least one additional antimicro- may contain additional agents as required, including bial agent. emulsifying, antioxidant, flavouring or colouring agents. [0130] In various embodiments, the at least one addi- [0125] Capsules can contain any standard pharma- tional antimicrobial agent is an antibiotic, such as an ceutically acceptable materials such as gelatin or cellu- 30 aminoglycoside, such as amikacin, gentamicin, kanamy- lose. Tablets can be formulated in accordance with con- cin, neomycin, netilmicin, streptomycin, tobramicin, or ventional procedures by compressing mixtures of the ac- paromomycin; an ansamycin, such as geldanamycin, or tive ingredients with a solid carrier and a lubricant. Ex- herbimycin; a carbacephem, such as loracarbef; carbap- amples of solid carriers include starch and sugar ben- enems, such as, ertapenem, doripenem, imipenem/cil- tonite. Active ingredients can also be administered in a 35 astatin, ormeropenem; cephalosporins(first generation), form of a hard shell tablet or a capsule containing a bind- such as cefadroxil, cefazolin, cefalotin or cefalothin, or er, e.g., lactose or mannitol, a conventional filler, and a cefalexin; cephalosporins (second generation), such as tabletting agent. Pharmaceutical compositions can also cefaclor, cefamandole, cefoxitin, cefprozil, or cefuroxi- be administered via the parenteral route. Examples of me; cephalosporins (third generation), such as cefixime, parenteral dosage forms include aqueous solutions, iso- 40 cefdinir, cefditoren, cefoperazone, cefotaxime, cefpo- tonic saline or 5% glucose of the active agent, or other doxime, ceftazidime, ceftibuten, ceftizoxime, or ceftriax- well-known pharmaceutically acceptable excipient. Sol- one; cephalosporins (fourth generation), such as ubilising agents well-known to those familiar with the art, cefepime; cephalosporins (fifth generation), such as can be utilized as pharmaceutical excipients for delivery ceftobiprole; glycopeptides, such as teicoplanin, or van- of the antimicrobial agent. 45 comycin; macrolides, such as azithromycin, clarithromy- [0126] Injectable dosage forms may be formulated as cin, dirithromycin, erythromycin, roxithromycin, trolean- liquid solutions or suspensions. Solid forms suitable for domycin, telithromycin, or spectinomycin; mono- solution in, or suspension in, liquid prior to injection may bactams, such as aztreonam; penicillins, such as amox- also be prepared. The dosage form may also be emulsi- icillin, ampicillin, azlocillin, carbenicillin, cloxacillin, di- fied. Methylglyoxal, or a material comprising methylgly- 50 cloxacillin, flucloxacillin, mezlocillin, meticillin, nafcillin, oxal, and cyclodextrin or a material comprising cyclodex- oxacillin, penicillin, piperacillin, or ticarcillin; polypep- trin, and when present the at least one additional antimi- tides, such as bacitracin, colistin, or polymyxin b; qui- crobial factor may be mixed with carriers such as, for nolones, such as ciprofloxacin, enoxacin, gatifloxacin, example, water, saline, dextrose, glycerol, ethanol, or levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, or the like and combinations thereof. 55 ofloxacin; sulfonamides, such as mafenide, sulfonami- [0127] Sustained-release preparations may be pre- dochrysoidine (archaic), sulfacetamide, sulfadiazine, pared incorporating methylglyoxal and cyclodextrin. Suit- sulfamethizole, sulfanilimide (archaic), sulfasalazine, able examples of sustained-release preparations include sulfisoxazole, trimethoprim, or trimethoprim-sulfameth-

12 21 EP 2 344 170 B1 22 oxazole (co-trimoxazole) (tmp-smx); tetracyclines, such whom it is administered. Preferred therapeutic agents as demeclocycline, doxycycline, minocycline, oxytetra- include therapeutic food factors, immunogenic or immu- cycline, tetracycline; others such as arsphenamine, chlo- nostimulatory agents, wound healing agents, and the ramphenicol, , lincomycin, ethambutol, fos- like. fomycin, fusidic acid, , isoniazid, linezolid, 5 [0135] It should be understood that the additional an- , mupirocin, nitrofurantoin, platensimycin, timicrobial or therapeutic agents listed above (both food pyrazinamide, quinupristin/dalfopristin, rifampicin (ri- based and pharmaceutical agents) may also be em- fampin in US), thiamphenicol, tinidazole, , ployed in a method according to the invention where they clofazimine; or a cyclic lipopeptides, such as daptomycin, are administered separately, simultaneously or sequen- aglycylcycline, suchas tigecycline, or anoxazolidinones, 10 tially with a composition useful herein. such as linezolid. [0136] As will be appreciated, the dose of the compo- [0131] In other embodiments, the at least one addition- sition administered, the period of administration, and the al antimicrobial agent is an , such as a polyene general administration regime may differ between sub- antifungal, such as , rimocidin, filipin, , jects depending on such variables as the severity of , candicin; , such as micona- 15 symptoms of a subject, the type of disorder to be treated, zole, , , , , the mode of administration chosen, and the age, sex , , , , and/or general health of a subject. However, by way of , , or ; , general example, from about 1 mg to about 5000 mg per such as , , isavuconazole, ravu- kg body weight of a composition useful herein is admin- conazole, , , or ; 20 istered, 1 mg to about 4000 mg per kg body weight of a such as ; , such as terbin- composition useful herein is administered, 1 mg to about afine, , , or ; , 3000 mg per kg body weight of a composition useful here- such as , , or ; oth- in is administered, 1 mg to about 2000 mg per kg body ers such as benzoic acid, , , unde- weight of a composition useful herein is administered, 1 cylenic acid, or 5-fluorocytosine, , 25 mg to about 1000 mg per kg body weight of a composition , and sodium bicarbonate; or alternatives such useful herein is administered, per administration or per as allicin, , citronella oil, iodine, lemon grass, day, preferably about 50 to about 1000 mg per kg, pref- oliveleaf, orange oil,palmarosa oil, patchouli, lemon myr- erably per day. In one embodiment, the administration is tle, neem seed oil, coconut oil, zinc, or selenium of from about 0.05 mg to about 250 mg per kg body weight [0132] Alternatively the agent is selected from any of 30 of a composition useful herein. those described herein. [0137] In various embodiments, sufficient composition [0133] The efficacy of a composition useful according is administered to deliver from about 0.001 mg to about to the invention can be evaluated both in vitro and in vivo. 5 mg of methylglyoxal per kg body weight, from about See, e.g., the examples below. Briefly, in one embodi- 0.001 mg to about 4 mg of methylglyoxal per kg body ment the composition can be tested for its ability, to for 35 weight, from about 0.001 mg to about 3 mg of methylg- example, inhibit microbial growth in vitro. For in vivo stud- lyoxal per kg body weight, from about 0.001 mg to about ies, the composition can be fed to or injected into an 2 mg of methylglyoxal per kg body weight, from about animal (e.g., a mouse) and its effects on microbial colo- 0.001 mg to about 1 mg of methylglyoxal per kg body nization, infection, or one or more symptoms of the mi- weight, from about 0.001 mg to about 0.5 mg of methyl- crobial disease or disorder are then assessed. Likewise, 40 glyoxal per kg body weight, from about 0.001 mg to about the ability of the composition to promote wound healing 0.1 mg of methylglyoxal per kg body weight, from about can be assessed using in vitro models of wound healing, 0.001 mg to about 0.05 mg of methylglyoxal per kg body or in vivo. Based on the results, an appropriate dosage weight, from about 0.001 mg to about 0.01 mg of meth- range, frequency, and administration route can be deter- ylglyoxal per kg body weight, or from about 0.001 mg to mined. 45 about 0.005 mg of methylglyoxal per kg body weight, per [0134] The compositions useful herein may be used administration or per day. alone or in combination with one or more other antimi- [0138] It should be appreciated that administration crobial agents, or one or more additional therapeutic may include a single dose, such as a single daily dose, agents. The antimicrobial agent or additional therapeutic or administration of a number of discrete divided doses agent may be or comprise a food, drink, food additive, 50 as may be appropriate. It should be understood that a drink additive, food component, drink component, dietary personof ordinary skill inthe artwill be able without undue supplement, nutritional product, medical food, nutraceu- experimentation, having regard to that skill and this dis- tical, medical device, medical supply, medicament or closure, to determine an effective dosage regime (includ- pharmaceutical. The antimicrobial agent or additional ing dose and timing of administration) for a given condi- therapeutic agent is preferably effective to attenuate one 55 tion. or more microbial diseases or disorders or one or more [0139] The present invention also relates to a dietary, of the symptoms of one or more microbial diseases or nutraceutical or oral pharmaceutical composition com- disorders, or otherwise confer a benefit on the subject to prising, consisting essentially of or consisting of methyl-

13 23 EP 2 344 170 B1 24 glyoxal or a material comprising methylglyoxal in combi- ministration of a single dosage form that comprises all nation with cyclodextrin. Preferably the composition con- components or the administration of separate dosage sists essentially of about 0.1 to 99 wt % methylglyoxal or forms at substantially the same time. Sequential admin- a material comprising methylglyoxal and about 0.1 to 99 istration includes administration according to different wt % cyclodextrin. More preferably the composition con- 5 schedules, preferably so that there is an overlap in the sists essentially of about 0.5 to 10 wt % methylglyoxal or periods during which the composition useful herein and a material comprising methylglyoxal and about 10 to 99 other therapeutic agent are provided. wt % cyclodextrin. Most preferably the composition con- [0143] Additionally, it is contemplated that a composi- sists essentially of about 1 wt % methylgloxal and about tion in accordance with the invention may be formulated 20 wt % cyclodextrin. 10 with additional active ingredients which may be of benefit [0140] In one embodiment a composition of the inven- to a subject in particular instances. For example, thera- tion comprises manuka honey or a manuka honey frac- peutic agents that target the same or different facets of tion. In one embodiment the composition comprises at the disease process may be used. least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, [0144] Accordingly, "foods and beverages comprising 60, 65, 70, 75, 80, 85, 90, 95 or 99% by weight manuka 15 antimicrobial compositions" of this invention can be used honey or a manuka honey fraction, and useful ranges for general foods and health food. Since the antimicrobial may be selected from any of these values (for example, compositions of the present invention maintain sweet from about 1 to about 99% by weight, from about 5 to taste of manuka honey, they can be eaten as they are or about 99% by weight, from about 10 to about 99% by in the form of powder like honey or may be used or con- weight, from about 15 to about 99% by weight, from about 20 sumed in the same manner as honey, including being 20 to about 99% by weight, from about 25 to about 99% eaten as a spread, for example by spreading them on by weight, from about 30 to about 99% by weight, from bread or cracker, or mixing with yogurt, or may be used about 35 to about 99% by weight, from about 40 to about as a preservative or marinade, for example, as a pre- 99% by weight, from about 45 to about 99% by weight, servative or marinade for meat. They can be used as an from about 50 to about 99% by weight, from about 55 to 25 ingredient or raw material for cake, biscuit, cookie, choc- about 99% by weight, from about 60 to about 99% by olate, sweets and other confectionary, including drops or weight, from about 65 to about 99% by weight, from about chewing gum. The compositions of the invention may be 70 to about 99% by weight, from about 75 to about 99% added to water as a drink, can be used as sweetener for by weight, from about 80 to about 99% by weight, from beverages such as milk, tea, coffee, hot chocolate, etc., about 85 to about 99% by weight, from about 90 to about 30 and as an ingredient or raw material for fruit juice bever- 99% by weight, or from about 95 to about 99% by weight). ages, sports drink, etc. [0141] In one embodiment a composition of the inven- [0145] "Drugs including antimicrobial compositions" of tion comprises cyclodextrin, preferably alpha-cyclodex- this invention provide the examples of drugs for the treat- trin, gamma-cyclodextrin, or both alpha-cyclodextrin and ment of microbial diseases or conditions, including skin gamma-cyclodextrin. In one embodiment the composi- 35 infection, such as that caused by Staphylococcus aureus tion comprises at least about 1, 5, 10, 15, 20, 25, 30, 35, and ones for the prevention or treatment of other condi- 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% by tions such as duodenal ulcer, gastric ulcer, gastric cancer weight cyclodextrin, and useful ranges may be selected and so caused by Helicobacter pylori. from any of these values (for example, from about 1 to [0146] Exemplary drugs of this invention can be ad- about 99% by weight, from about 5 to about 99% by40 ministered transdermally or orally so long as the antimi- weight, from about 10 to about 99% by weight, from about crobial composition is safe to human body. 15 to about 99% by weight, from about 20 to about 99% [0147] The compositions of the invention may be or by weight, from about 25 to about 99% by weight, from may be incorporated into or onto medical devices and about 30 to about 99% by weight, from about 35 to about medical supplies. The invention particularly contem- 99% by weight, from about 40 to about 99% by weight, 45 plates, but is not limited to, medical devices or supplies from about 45 to about 99% by weight, from about 50 to used in the treatment of external wounds, surgical about 99% by weight, from about 55 to about 99% by wounds, and the like, but those skilled in the art will rec- weight, from about 60 to about 99% by weight, from about ognise numerous applications. Exemplary medical de- 65 to about 99% by weight, from about 70 to about 99% vices that are intended as tissue implants include, for by weight, from about 75 to about 99% by weight, from 50 example but are not limited to, brachytherapy sources, about 80 to about 99% by weight, from about 85 to about embolization materials, tumor-bed implants, intra-joint 99% by weight, from about 90 to about 99% by weight, implants, materials to minimize adhesions, and the like. or from about 95 to about 99% by weight). Exemplary stents include, for example but are not limited [0142] When used in combination with another antimi- to, intravascular stents that include for example both bal- crobial agent or therapeutic agent, the administration of 55 loon-expandable stents and self-expanding stents. Bal- a composition useful herein and the other antimicrobial loon-expandable stents are available from a number of agent or therapeutic agent may be simultaneous or se- commercial suppliers, including Cordis. Self-expanding quential. Simultaneous administration includes the ad- stents are typically composed from a shape memory alloy

14 25 EP 2 344 170 B1 26 and are available from suppliers, such as Instent. In the powder. case of stents, a balloon-expandable stent is typically [0153] 60.0g of manuka honey and 61.3g of alpha-cy- composed of a stainless steel framework or, in the case clodextrin were added to 1L of a beaker, to which 128.7ml of self-expanding stents, from nickel/titanium alloy. Ex- of water was added and the composition was homoge- emplary balloons include, for example but are not limited 5 nized at 6000rpm for 5 minutes. After that, water was to, balloon catheters including inflatable and self inflata- added so that the solid content was 20.0%wt. ble balloon catheters. The inflatable balloon may be a [0154] The resultant suspension was spray-dried (dry- non-dispensable balloon, typically composed of polyeth- ing gas temperature:160°C) to obtain powder of Antimi- yleneterephthalate, or it may be an elastic balloon, typi- crobial composition A. cally being composed of latex or silicone rubber. 10 [0148] "Medical supplies including antimicrobial com- Example 2 - Antimicrobial composition B (gamma- positions" of this invention provide the examples of ger- cyclodextrin containing powder composition) micide and disinfectant used for medical devices or fa- cilities. Exemplary medical supplies include dressings, [0155] Manuka honey was mixed with gamma-cyclo- disinfectants, bandages, lavages, and the like. 15 dextrin at the ratio of 45.0%wt/ 55.0%wt and was made [0149] The medical devices or supplies may be coated to a powder (ie, as Antimicrobial composition B) in the or impregnated with compositions of the invention by well same way as that of Antimicrobial composition A as de- recognized methods. One exemplary method of coating scribed above as Example 1. a surface of a medical device with the composition of the invention comprises contacting the surface with the com- 20 Example 3 - Antimicrobial composition solution C position of the invention so as to cause the surface to be (alpha-cyclodextrin containing solution) coated with the particular composition of the invention. Coating of the artificial surface may be accomplished us- [0156] 14.5454g of Antimicrobial composition A and ing standard methods well known to those of ordinary 8.0g of alpha-cyclodextrin were dissolved in water to skill in the art. For example, coating a surface with com- 25 make 100mL to produce Antimicrobial composition so- position of the invention can be achieved by bathing the lution C. artificial surface, either by itself or within a device, in a [0157] The manuka honey content in Antimicrobial solution containing the composition of the invention. composition solution C (from Antimicrobial composition [0150] Exemplary antimicrobial compositions of the in- A) was 6.55%w/v. vention, and methods for preparing such compositions 30 [0158] Alpha cyclodextrin which came from Antimicro- will now be described with reference to the following ex- bial composition A, was 8.0%w/v and the separately add- amples. ed alpha-cyclodextrin was 8.0%w/v. Thus total alpha cy- clodextrin was 16.0 %w/v. Example 4 - Antimicrobial EXAMPLES composition solution D (alpha cyclodextrin - higher 35 honey content) 1. Material [0159] 14.5454g of Antimicrobial composition A and 6.54g of manuka honey were dissolved in water to make [0151] 100mL to produce Antimicrobial composition solution D. [0160] Alpha cyclodextrin content in Antimicrobial 1) manuka honey:Manuaka honey MGO™55040 composition solution D (from Antimicrobial composition (Manuka Health New Zealand Ltd.) A) was 8.0%w/v. Manuka honey which came from Anti- 2) Methylglyoxal honey powder (Manuka Health microbial composition A was 6.55%w/v and separately New Zealand Ltd.) added manuka honey was 6.55%w/v. The total manuka 3) Alpha-cyclodextrin:CAVAMAX W6 Food (Cy- honey content was 13.10%w/v. cloChem Co., Ltd.) 45 4) Gamma-cyclodextrin: CAVAMAX W8 Food (Cy- Example 5 - Antimicrobial composition solution E cloChem Co., Ltd.) [0161] 14.29g of manuka honey and 17.4g of alpha- 2. Manufacturing method of antimicrobial composi- cyclodextrin were dissolved in water to make 100ml to tions 50 produce Antimicrobial composition solution E, in which the ratio of manuka honey and alpha-cyclodextrin was Antimicrobial compositions containing manuka honey 45% to 55% by weight.

Example 1 - Antimicrobial composition A (alpha-cy- Example 6 - Antimicrobial composition solution F clodextrin containing powder composition) 55 [0162] 14.29g of methylglyoxal honey powder and [0152] Manuka honey was mixed with alpha-cyclodex- 17.4g of alpha-cyclodextrin was dissolved in water to trin at the ratio of 45.0%wt/ 55.0%wt and was made to a make 100mL to produce Antimicrobial composition so-

15 27 EP 2 344 170 B1 28 lution F. [0172] Each filtrate was added to heart infusion broth [0163] Manuka honey content was 14.29%w/v and al- with the ratio of (1:1). pha-cyclodextrin content was 17.4%w/v. 4) Reference samples Example 7 - Antimicrobial Activity of the composi- 5 tions A to F (a.) Manuka honey aqueous solution

[0164] Compositions A to F were prepared as de- [0173] 14.29% w/v of manuka honey aqueous solution scribed in Example 1 to 6 above. was prepared by dissolving manuka honey MGO550™ [0165] Reagents used were as follows: 10 (Cosana Co., Ltd.) with water.

(1) Heart infusion broth (Eiken Chemical Co., Ltd.). (b.) Cyclodextrin aqueous solution Bacto™ Heart Infusion Broth (beef heart infusion (from 500g) - 10.0g, Tryptose - 10.0 g, Sodium Chlo- [0174] 8% w/v of cyclodextrin aqueous solution was ride - 5.0g/per litre) was prepared according to the 15 prepared by dissolving either CAVAMAXC W6 Food or manufacturer’s instructions. CAVAMAX W8 Food with water. (2) Methicillin-sensitive Staphylococcus au- reus.MSSA, Strain IFO12732 (Delivered by Kobe (c.) Methylglyoxal manuka honey powder aqueous solu- Gakuin University) was incubated in heart infusion tion broth (50g of beef heart infusion, 1g of pepton and 20 0.5g of sodium chloride per 100ml) at 37°C for about [0175] 14.29% w/v of manuka honey powder aqueous 15 hours until OD 600 reached approximately 0.7. solutionwas prepared by dissolvingmethylglyoxal manu- This culture was 25 fold diluted and used for meas- ka honey powder (Manuka Health Co., Ltd.) with water. urement. [0176] Solutions a. to c. above were sterilized by filtra- (3) Samples prepared using compositions A to F of 25 tionwith 0.45 mm filter and each filtrate was added to heart Examples 1 to 6. infusion broth with the ratio of (1:1).

[0166] Antimicrobial compositions A & B manufactured Measurement of antimicrobial activity as shown in Examples 1 and 2 were each dissolved in 100mL of water so that the concentration could be30 [0177] 5g of Heart infusion broth and 100mL of water 29.09% w/v (the concentration of manuka honey and al- were added to 200mL Erlenmeyer flask and sterilized in pha-cyclodextrin was 13.1% w/v, and 15.9% w/v, respec- an autocrave at 120°C for 20 minutes. To 2ml of the ster- tively) and sterilized by filtration with 0.45mm filter. ilized medium, 2ml of either sample or reference sample [0167] The filtrate was diluted with water (1 in 2, 1 in prepared as above was added. 2ml of distilled water was 4, and 1 in 8). 35 added in place of manuka honey aqueous solution as [0168] After dilution each filtrate was added to heart the control. infusion broth with the ratio of (1: 1). Thus the final con- [0178] 50mL of 25-fold diluted strain solution prepared centration of manuka honey and alpha-cyclodextrin or as above was added to each test tube and incubated at gamma-cyclodextrin in heart infusion broth were as fol- 37°C while shaking. lows: 40 [0179] Optical absorption was measured at 610nm, and the time of incubation and change in bacterial count 2 fold (1 in 2) dilution: manuka honey (3.27% w/v), was determined. α-cyclodextrin or γ-cyclodextrin (4.0% w/v) 4 fold (1 in 4) dilution: manuka honey (1.64% w/v), Results α-cyclodextrin or γ-cyclodextrin (2.0% w/v) 45 8 fold (1 in 8) dilution: manuka honey (0.82% w/v), [0180] As shown in Figure 1, antimicrobial composition α-cyclodextrin or γ-cyclodextrin (1.0% w/v). A containing 7.14% w/v of manuka honey and 8.7%w/v of alpha-cyclodextrin, and the composition containing [0169] Antimicrobial composition A (Example 1) was 3.27% w/v of manuka honey and 4.0%w/v of alpha-cy- also dissolved in water so that the concentration was50 clodextrin each showed higher antimicrobial activity than 31.75% w/v, (the concentration of manuka honey and α- that of manuka honey alone, or alpha-cyclodextrin alone. cyclodextrin was 7.14% w/v, and 8.7% w/v, respectively) [0181] Both antimicrobial composition A containing and sterilized by filtration with 0.45mm filter. 1.64%w/v of manuka honey and 2.0%w/v of alpha-cyclo- [0170] The filtrate was added to heart infusion broth dextrin, and the composition containing 0.82%w/v of with the ratio of (1:1). 55 manuka honey and 2.0%w/v of alpha-cyclodextrin, are [0171] Antimicrobial compositions C, D, & E (Exam- lower in final manuka honey concentration than was the ples 3 to 5, respectively) were each sterilized by filtration reference sample of manuka honey alone. Despite this, with 0.45mm filter. each showed equivalent antimicrobial activity equivalent

16 29 EP 2 344 170 B1 30 to that of the reference sample of manuka honey alone. antimicrobial composition A with additional manuka hon- [0182] Figure 2 shows that the high antimicrobial ac- ey also showed high antimicrobial activity. tivity of antibacterial composition A, containing 7.14%w/v of manuka honey and 8.7%w/v of alpha-cyclodextrin was INDUSTRIAL APPLICABILITY maintained to almost 100 hours. Figure 2 also shows the 5 antimicrobial activity of antimicrobial composition A, con- [0191] Antimicrobial compositions of this invention taining 3.27%w/v of manuka honey and 4.0%w/v of al- containing methylglyoxal or material with a methylglyoxal pha-cyclodextrin, was maintained for a longer time than presence are capable of maintaining or enhancing the that of manuka honey. original antimicrobial activity present, and can be used [0183] Thus, Figures 1 and 2 shows that antimicrobial 10 in a variety of applications where antimicrobial activity is composition A (with manuka honey and alpha-cyclodex- desired, such as in materials for consumer goods includ- trin) showed higher antimicrobial activity than that of ing foods and beverages,medical devices, medical sup- manuka honey alone at the same manuka honey con- plies, pharmaceuticals, functional foods, drugs, or in in- centration. Antimicrobial activity equivalent to that of dustrial products. Methods of preparing such composi- manuka honey was obtained at a lower manuka honey 15 tions, and methods of using such compositions, for ex- concentration in those compositions comprising alpha- ample in the treatment of microbial disease or in promot- cyclodextrin. ing wound healing, have application in the medical and [0184] Figure 3 shows that antimicrobial composition industrial fields. B diluted to contain 3.27%w /v of manuka honey and 4.0%w/v of gamma-cyclodextrin exhibited higher antimi- 20 crobial activity than that of manuka honey alone. Claims [0185] Figure 3 also shows that antimicrobial compo- sition B diluted to contain 1.64%w /v of manuka honey 1. An antimicrobial composition comprising methylgly- and 2.0%w/v of gamma-cyclodextrin, or containing oxal and cyclodextrin. 0.82%w /v manuka honey and 2.0%w/v of gamma-cy- 25 clodextrin, showed antimicrobial activity equivalent to 2. The antimicrobial composition according to claim 1 that of the manuka honey reference sample at the lower wherein the methylglyoxal is provided in the form of manuka honey concentration. manuka honey. [0186] From the result of Figure 3, the enhancement of antimicrobial activity of antimicrobial composition B 30 3. The antimicrobial composition according to claim 2 comprising manuka honey and gamma cyclodextrin ap- wherein the manuka honey has a methylglyoxal con- pears to be dependent on the concentration. centration of greater than about 30mg/kg, than about [0187] As shown in Figure 4, antimicrobial composition 38mg/kg, than about 40mg/kg, about 50mg/kg, solution E, in which manuka honey and alpha cyclodex- about 60mg/kg, about 70mg/kg, about 80mg/kg, trin were added to the medium at the same concentration 35 about 90mg/kg, about 100mg/kg, about 150mg/kg, of antimicrobial composition A (manuka honey: about 200mg/kg, about 250mg/kg, about 300mg/kg, 7.14%w/v and alpha-cyclodextrin:8.7%w/v), showed about 350mg/kg, about 400mg/kg, about 450mg/kg, high antimicrobial activity. However, the antimicrobial ac- about 500mg/kg, about 550mg/kg, about 600mg/kg, tivity of antimicrobial composition solution E diminished about 650mg/kg, about 700mg/kg, about 750mg/kg, aftermore than 28 hours. This suggests that antimicrobial 40 about 800mg/kg, about 850mg/kg, about 900mg/kg, composition A which was powderized beforehand pos- about 950mg/kg, greater than about 1000mg/kg, sessed longer-lasting enhancement of antimicrobial ac- greater than about 1100mg/kg, greater than about tivity. 1200mg/kg, greater than about 1300mg/kg, greater [0188] Also, antimicrobial composition solution F con- than about 1400mg/kg, greater than about taining 7.14%w/v of methylglyoxal honey powder and 45 1500mg/kg, greater than about 1600mg/kg, greater 8.7%w/v of alpha-cyclodextrin was shown to have high than about 1700mg/kg, greater than about antimicrobial activity, though that activity was lower than 1800mg/kg, greater than about 1900mg/kg, or about that of antimicrobial composition A containing the same 2000mg/kg, or wherein the manuka honey has an concentration of manuka honey and alpha-cyclodextrin. NPA rating greater than 10, than 15, than 20, than [0189] As shown in Figure 5, high antimicrobial activity 50 25, 26, 27, 28, 29, 30, than 31, 32, 33, 34, or greater of antimicrobial composition A containing 7.14%w/v of than 35. manuka honey and 8.7%w/v of alpha-cyclodextrin lasted almost 60 hours. Further, antimicrobial composition so- 4. The antimicrobial composition according to any one lution C which was manufactured in the similar way as of claims 1 to 3 wherein the cyclodextrin is alpha- that of antimicrobial composition A with additional alpha 55 cyclodextrin, gamma-cyclodextrin, or a combination cyclodextrin showed high antimicrobial activity. of alpha-cyclodextrin and gamma-cyclodextrin. [0190] Also, antimicrobial composition solution D which was manufactured in the similar way as that of 5. The antimicrobial composition according to any one

17 31 EP 2 344 170 B1 32

of claims 2 to 4 wherein the manuka honey content the composition consists of manuka honey and cy- of the composition is within the range of about 10.0 clodextrin. to about 99.0 %wt.

6. The antimicrobial composition according to any one 5 Patentansprüche of claims 1 to 5 wherein the methylglyoxal content of the antimicrobial composition is within the range 1. Antimikrobielle Zusammensetzung, umfassend Me- of about 0.003 %wt to about 0.15 %wt. thylglyoxal und Cyclodextrin.

7. The antimicrobial composition according to any one 10 2. Antimikrobielle Zusammensetzung nach Anspruch of claims 1 to 6 wherein the composition is a con- 1, worin das Methylglyoxal in der Form von Manuka- sumer good, an industrial product, a food, drink, food Honig bereitgestellt ist. additive, drink additive, dietary supplement, nutri- tional product, medical food, medical device, medi- 3. Antimikrobielle Zusammensetzung nach Anspruch cal supply, nutraceutical, medicament or a pharma- 15 2, worin der Manuka-Honig eine Methylglyoxal-Kon- ceutical composition. zentration von mehr als etwa 30 mg/kg, als etwa 38 mg/kg, als etwa 40 mg/kg, etwa 50 mg/kg, etwa 60 8. The antimicrobial composition according to claim 7 mg/kg, etwa 70 mg/kg, etwa 80 mg/kg, etwa 90 wherein the composition is a pharmaceutical com- mg/kg, etwa 100 mg/kg, etwa 150 mg/kg, etwa 200 position formulated for oral, topical, or parenteral ad- 20 mg/kg, etwa 250 mg/kg, etwa 300 mg/kg, etwa 350 ministration. mg/kg, etwa 400 mg/kg, etwa 450 mg/kg, etwa 500 mg/ kg, etwa 550 mg/kg, etwa 600 mg/kg, etwa 650 9. The antimicrobial composition according to any one mg/kg, etwa 700 mg/kg, etwa 750 mg/kg, etwa 800 of claims 1 to 8 wherein the composition comprises mg/kg, etwa 850 mg/kg, etwa 900 mg/kg, etwa 950 one or more additional antimicrobial agents. 25 mg/kg, mehr als etwa 1000 mg/kg, mehr als etwa 1100 mg/kg, mehr als etwa 1200 mg/kg, mehr als 10. A method of preparing a composition comprising etwa 1300 mg/kg, mehr als etwa 1400 mg/kg, mehr methylglyoxaland cyclodextrin, the methodcompris- als etwa 1500 mg/kg, mehr als etwa 1600 mg/kg, ing admixing methylglyoxal, or a material comprising mehr als etwa 1700 mg/kg, mehr als etwa 1800 Methylglyoxal, or both methylglyoxal and a material 30 mg/kg, mehr als etwa 1900 mg/kg oder etwa 2000 comprising methylglyoxal, with cyclodextrin or a ma- mg/kg aufweist oder worin der Manuka-Honig eine terial comprising cyclodextrin, or both cyclodextrin NPA-Bewertung von mehr als 10, als 15, als 20, als and a material comprising cyclodextrin. 25, 26, 27, 28, 29, 30, als 31, 32, 33, 34 oder mehr als 35 aufweist. 11. The method according to claim 10 wherein the ad- 35 mixing is of powderized manuka honey with cyclo- 4. Antimikrobielle Zusammensetzung nach einem der dextrin, optionally comprising the preliminary step of Ansprüche 1 bis 3, worin das Cyclodextrin Alpha- drying manuka honey prior to or during powderising Cyclodextrin, Gamma-Cyclodextrin oder eine Kom- the manuka honey. bination von Alpha-Cyclodextrin und Gamma-Cyc- 40 lodextrin ist. 12. An in vitro method for controlling one or more mi- crobes, the method comprising contacting the one 5. Antimikrobielle Zusammensetzung nach einem der or more microbes with a composition comprising Ansprüche 2 bis 4, worin der Manuka-Honig-Gehalt methylglyoxal and cyclodextrin. der Zusammensetzung innerhalb des Bereichs von 45 etwa 10,0 bis etwa 99,0 Gew.-% liegt. 13. A composition comprising methylglyoxal and cyclo- dextrin for use as a medicament. 6. Antimikrobielle Zusammensetzung nach einem der Ansprüche 1 bis 5, worin der Methylglyoxal-Gehalt 14. The composition for use according to claim 13 der antimikrobiellen Zusammensetzung im Bereich wherein the methylglyoxal is provided in the form of 50 von etwa 0,003 Gew.-% bis etwa 0,15 Gew.-% liegt. manuka honey. 7. Antimikrobielle Zusammensetzung nach einem der 15. The composition of claim 13 or 14 for use in the treat- Ansprüche 1 bis 6, worin die Zusammensetzung ein ment of a microbial disease or disorder, for control- Konsumgut, ein industrielles Produkt, ein Nahrungs- ling a microbial infection, or for promoting wound 55 mittel, ein Getränk, ein Nahrungsmittelzusatz, ein healing, in a subject in need thereof. Getränkezusatz, ein Nahrungsergänzungsmittel, ein Lebensmittelprodukt, ein medizinisches Nah- 16. The composition of any one of claims 2 to 8, wherein rungsmittel, eine medizinische Vorrichtung, ein me-

18 33 EP 2 344 170 B1 34

dizinisches Hilfsgut, ein Nahrungstherapeutikum, Revendications ein Medikament oder eine pharmazeutische Zusam- mensetzung ist. 1. Composition antimicrobienne comprenant du mé- thylglyoxal et de la cyclodextrine. 8. Antimikrobielle Zusammensetzung nach Anspruch 5 7, worin die Zusammensetzung eine pharmazeuti- 2. Composition antimicrobienne selon la revendication sche Zusammensetzung ist, die zur oralen, topi- 1 dans laquelle le méthylglyoxal se présente sous la schen oder parenteralen Verabreichung formuliert forme de miel de manuka. ist. 10 3. Composition antimicrobienne selon la revendication 9. Antimikrobielle Zusammensetzung nach einem der 2 dans laquelle le miel de manuka a une concentra- Ansprüche 1 bis 8, worin die Zusammensetzung ei- tion en méthylglyoxal supérieure à environ 30 mg/kg, nes oder mehrere zusätzliche antimikrobielle Mittel à environ 38 mg/kg, à environ 40 mg/kg, environ 50 umfasst. mg/kg, environ 60 mg/kg, environ 70 mg/kg, environ 15 80 mg/kg, environ 90 mg/kg, environ 100 mg/kg, en- 10. Verfahren zum Herstellen einer Zusammensetzung, viron 150 mg/kg, environ 200 mg/kg, environ 250 die Methylglyoxal und Cyclodextrin umfasst, wobei mg/kg, environ 300 mg/kg, environ 350 mg/kg, en- das Verfahren das Beimischen von Methylglyoxal viron 400 mg/kg, environ 450 mg/kg, environ 500 oder einem Material, das Methylglyoxal umfasst, mg/kg, environ 550 mg/kg, environ 600 mg/kg, en- oder von sowohl Methylglyoxal als auch einem Ma- 20 viron 650 mg/kg, environ 700 mg/kg, environ 750 terial, das Methylglyoxal umfasst, mit Cyclodextrin mg/kg, environ 800 mg/kg, environ 850 mg/kg, en- oder einem Material, das Cyclodextrin umfasst, oder viron 900 mg/kg, environ 950 mg/kg, supérieure à von sowohl Cyclodextrin als auch einem Material, environ 1 000 mg/kg, supérieure à environ 1 100 das Cyclodextrin umfasst, umfasst. mg/kg, supérieure à environ 1 200 mg/kg, supérieure 25 à environ 1 300 mg/kg, supérieure à environ 1 400 11. Verfahren nach Anspruch 10, worin das Beimischen mg/kg, supérieure à environ 1 500 mg/kg, supérieure ein Beimischen von pulverisiertem Manuka-Honig à environ 1 600 mg/kg, supérieure à environ 1 700 mit Cyclodextrin ist, gegebenenfalls umfassend den mg/kg, supérieure à environ 1 800 mg/kg, supérieure vorbereitenden Schritt des Trocknens von Manuka- à environ 1 900 mg/kg ou à environ 2 000 mg/kg, ou Honig vor oder während der Pulverisierung des Ma- 30 dans laquelle le miel de manuka a une activité non nuka-Honigs. peroxyde supérieure à 10, à 15, à 20, à 25, 26, 27, 28, 29, 30, à 31, 32, 33, 34, ou supérieure à 35. 12. In-vitro-Verfahren zum Bekämpfen einer oder meh- rerer Mikroben, wobei das Verfahren das Kontaktie- 4. Composition antimicrobienne selon l’une quelcon- ren der einen oder mehreren Mikroben mit einer Zu- 35 que des revendications 1 à 3 dans laquelle la cyclo- sammensetzung umfasst, die Methylglyoxal und Cy- dextrine est une alpha-cyclodextrine, une gamma- clodextrin umfasst, umfasst. cyclodextrine ou une combinaison d’alpha-cyclo- dextrine et de gamma-cyclodextrine. 13. Zusammensetzung, die Methylglyoxal und Cyclod- extrin umfasst, zur Verwendung als Medikament. 40 5. Composition antimicrobienne selon l’une quelcon- que des revendications 2 à 4 dans laquelle la teneur 14. Zusammensetzung zur Verwendung nach Anspruch en miel de manuka de la composition se situe dans 13, worin das Methylglyoxal in der Form von Manu- la plage d’environ 10,0 à environ 99,0 % en poids. ka-Honig bereitgestellt ist. 45 6. Composition antimicrobienne selon l’une quelcon- 15. Zusammensetzung nach Anspruch 13 oder 14 zur que des revendications 1 à 5 dans laquelle la teneur Verwendung bei der Behandlung einer mikrobiellen en méthylglyoxal de la composition antimicrobienne Erkrankung oder Störung, zum Bekämpfen einer mi- se situe dans la plage d’environ 0,003 % en poids à krobiellen Infektion oder zum Fördern der Wundhei- environ 0,15 % en poids. lung bei einem Individuum, das dies benötigt. 50 7. Composition antimicrobienne selon l’une quelcon- 16. Zusammensetzung nach einem der Ansprüche 2 bis que des revendications 1 à 6, laquelle composition 8, worin die Zusammensetzung aus Manuka-Honig est un bien de consommation, un produit industriel, und Cyclodextrin besteht. un aliment, une boisson, un additif alimentaire, un 55 additif pour boisson, un supplément diététique, un produitnutritionnel, un aliment thérapeutique, un dis- positif médical, des fournitures médicales, un nutra- ceutique, un médicament ou une composition phar-

19 35 EP 2 344 170 B1 36

maceutique.

8. Composition antimicrobienne selon la revendication 7, laquelle composition est une composition phar- maceutique formulée pour une administration orale, 5 topique ou parentérale.

9. Composition antimicrobienne selon l’une quelcon- que des revendications 1 à 8, laquelle composition comprend un ou plusieurs agents antimicrobiens ad- 10 ditionnels.

10. Procédé de préparation d’une composition compre- nant du méthylglyoxal et de la cyclodextrine, le pro- cédé comprenant le mélange de méthylglyoxal, ou 15 d’un produit comprenant du méthylglyoxal, ou à la fois de méthylglyoxal et d’un produit comprenant du méthylglyoxal, avec de la cyclodextrine ou un produit comprenant de la cyclodextrine, ou à la fois de la cyclodextrine et un produit comprenant de la cyclo- 20 dextrine.

11. Procédé selon la revendication 10 dans lequel le mé- lange est constitué de miel de manuka en poudre avec de la cyclodextrine, comprenant facultative- 25 ment l’étape préliminaire consistant à sécher le miel de manuka avant ou pendant la mise en poudre du miel de manuka.

12. Procédé in vitro permettant de lutter contre un ou 30 plusieurs microbes, le procédé comprenant la mise en contact du ou des microbes avec une composition comprenant du méthylglyoxal et de la cyclodextrine.

13. Composition comprenant du méthylglyoxal et de la 35 cyclodextrine pour utilisation en tant que médica- ment.

14. Composition pour utilisation selon la revendication 13 dans laquelle le méthylglyoxal se présente sous 40 la forme de miel de manuka.

15. Composition selon la revendication 13 ou 14 pour utilisation dans le traitement d’une maladie ou d’un trouble microbien, pour lutter contre une infection 45 microbienne, ou pour favoriser la guérison d’une plaie, chez un sujet qui le nécessite.

16. Composition selon l’une quelconque des revendica- tions 2 à 8, laquelle composition est constituée de 50 miel de manuka et de cyclodextrine.

55

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description

• JP 2008007408 A [0004] • WO 05120250 A [0092] • JP H8239693 B [0004] • US 3773919 A [0127]

Non-patent literature cited in the description

• J. Roy. Soc. Med., 1994, vol. 87, 9-12 [0002] • PORTER N. G. disclosed the chemical, physical and • Mol. Nutr. Food Res., April 2008, vol. 52 (4), 483-489 antimicrobial properties of essential oils of Leptosper- [0003] mum scoparium and Kunzea ericoides. Phytochem- • LIS BALCHIN M. reported the pharmacological and istry, 10 February 1998, vol. 50 (3), 407-415 [0006] antimicrobial studies on different tea-tree oils (mela- • FILOCHE S K et al. reported the antimicrobial effects leuca alternifolia, leptospermum scoparium or manu- of essential oils in combination with chlorhexidine ka and kunzea ericoides or kanuka), originating in digluconate. Oral Microbiology and Immunology, 01 Australia and new Zealand. Phytotherapy Research, August 2005, vol. 20 (4), 221-225 [0006] 01 January 2000, vol. 14 (8), 623-629 [0006] • Scientific Tables, Geigy Pharmaceuticals. Ardley, New York, 1970, 537 [0057] • REMINGTON’S. Pharmaceutical Sciences. Mack Publishing Co, 1980 [0122]

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