NZ283139A

NZ283139A - Antimicrobial composition of glucose oxidase and glucose

Info

Publication number: NZ283139A
Application number: NZ283139A
Authority: NZ
Inventors: David Wilson Ashworth; Walter Graham Guthrie; David Vincent Roper
Original assignee: Knoll Ag
Priority date: 1994-03-26
Filing date: 1995-03-18
Publication date: 1998-09-24
Also published as: ZA952363B; WO1995026137A1; AU2110395A; IL113070A0; GB9406075D0; IL113070A; AU697046B2; EP0752815A1; CA2186531A1

Description

<div class="application article clearfix" id="description"> New Zealand No. 283139 International No. PCT/EP95/01037 TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 26.03.1994; Complete Specification Filed: 18.03.1995 Classification:^) A01N63/00; A61K38/44; A61K31/70 Publication date: 24 September 1998 Journal No.: 1432 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Method of killing microorganisms Name, address and nationality of applicant(s) as in international application form: KNOLL AKTIENGESELLSCHAFT, 67061 Ludwigshafen, Federal Republic of Germany I w' -j J - 1 - METHOD OF KILLING MICROORGANISMS The present invention relates to a method of killing microorganisms such as bacteria, protozoa, fungi and viruses and to a packaged chemical composition 5 suitable for use in such a method. International Patent Publication No. W091/11105 (The Boots Company PLC) discloses anti-microbial compositions comprising iodide anions and thiocyanate anions in a weight : weight ratio of from 0.1:1 to 50:1 10 and having a combined anion weight concentration of at least 5 mg/kg, D-glucose in a weight concentration of at least 0.2 g/kg and an effective amount of ' the oxidoreductase enzyme glucose oxidase. The present invention provides a method of killing microorganisms with an improved speed of kill by mixing effective amounts of D-glucose and glucose oxidase, incubating the resulting mixture which comprises both iodine ions and thiocyanate ions wherein the weight ratio of iodide ions to thiocyanate ions is from 0.1:1 to 50:1 and the combined anion weight concentration is at least 5mg/kg wherein the concentration of glucose oxidase in the resulting mixture is at least 150 U/kg, at a temperature of from -10 to 50°C and at a pH of from 1-8 2 0 for a period of from 30 minutes to 48 hours and then applying the incubated mixture to the microorganisms to be killed. Remarkably, the method of the invention provides improved speed of kill. Preferably, the resulting mixture is incubated for at least 1 hour, preferably at least 2 hours, more preferably at least 4 hours. The inventors have found that the fast-kill activity increases rapidly on incubation from 30 minutes to 48 hours. A favoured incubation period is 1 2 to 48 hours. A particularly favoured incubation period is 24 hours, as the mixture can be made up a day in advance and the incubation step carried out during transport to an end user. intellectual property office of n.z. 10 AUG 1998 received The incubated mixture of the invention has been found to retain its fast-kill properties for at least a 2 year test period. Iodide and thiocyanate anions are generally included in the compositions according to the invention in the fcrm of salts. Suitable iodide salts include alkali metal salts such as potassium iodide and sodium iodide and mixtures thereof. Suitable thiocyanate salts include, for example, potassium, sodium, ammonium, ferric and cuprous salts of thiocyanate and mixtures thereof. Preferably the weight concentration of iodide anions is at least 5 mg/kg and the weight concentration of thiocyanate anions is at least 2 mg/kg. The weight-.weight ratio of iodide: thiocyanate anions is preferably in the range 0.2:1 to 2 0.1, more preferably 0.5:1 to 15:1, particularly 1:1 to 5:1. All units (U) of enzyme activity referred to herein relate to International Units of activity defined as the amount of enzyme required to catalyse the transformation of 1.0 micromole of substrate per minute at 25°C under optimal conditions. All concentrations referred to herein relate to amounts per kilogram of the total composition. intellectual property office' of n.z. 1 o AUG 1S98 Received ! - 3 - 10 Oyt-j / < <'< ') X. ' - The method may be used to kill most if not aril types of microorganisms, for example gram negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, gram positive bacteria such as Staphylococcus aureus and Propionibacterium acnes, moulds such as Aspergillus niaer and Penicillium funiculosum, yeasts such as Candida albicans, Saccharomvces cerevisiae and Pitvrosporum ovale, dermatophytic fungi such as Trichophyton rubruro, microalgae such as Chlorella spp. and Sovrocrvra spp. and viruses such as Herpes virus and Picornavirus. <1 The oxidoreductase enzyme, gludose oxidase, catalyses the production of H2O2 by oxidation of D-glucose in the presence of water and oxygen. It is 1 ^ classified as E.C.I.1.3.4. (IUPAC) and is defined herein in International Units (amount of enzyme required to catalyze the oxidation of 1.0 micromole {3-D-glucose per minute at pH 7.0 and 25°C). Glucose oxidase is available commercially from a number of sources, for example from Sturge-ABM under the trade designations "Glucox P200" (2000 U/ml) and "Glucox PS" (75 U/mg) . Glucose oxidase concentrations in excess of 150 U/kg provide excellent protection against bacterial, mould and yeast growth. 20 25 30 The oxidisable substrate for glucose oxidase, namely D-glucose, is generally included at a concentration (in the resulting mixture, before incubation) of at least 0 2 g/kg, preferably at least 0.5 g/kg, preferably at least 1 g/kg, and more particularly at least 2 g/kg. It will be appreciated by those skilled in the art that D-glucose may be provided per se or may intellectual property office | of n.z. 10 AUG 1923 received WO 95/26137 PCT/EF95/01037 - 4 - be formed in situ from suitable precursors, for example, as a result of the breakdown of an oligomer or polymer containing D-glucose. Suitable precursors such as sucrose or starch may be used alone or in admixture 5 with D-glucose and may advantageously support more sustained anti-microbial activity than obtained with D-glucose alone. The efficiency of iodide and thiocyanate anion oxidation in the presence of H2C>2 may be enhanced by the 10 addition of small amounts of a peroxidase enzyme such as lactoperoxidase, preferably at least 100 U/kg lactoperoxidase. Lactoperoxidase is classified as E.C.I.17.1.7 (IUPAC) and is defined herein in International Units (amount of enzyme required to 15 catalyse the reduction of 1.0 micromole H2O2 per minute at pH 7.0 and 25°C). Lactoperoxidase is available commercially from a number of sources, for example from DMV (De Melkindustrie Veghel bv) (275 U/mg). It may be supplied, for example, in the form of a freeze-dried 20 powder or in an aqueous salt solution e.g. 1.8% NaCl or 12% NaCl. The compositions according to the invention which further comprise lactoperoxidase exhibit effective killing activity against the organisms listed above. The method of the invention may, if desired, 25 incorporate further agents which may supplement or enhance the anti-microbial activity thereof, for example other enzymes such as'" lactoferrin or salts such as calcium chloride. Anti-microbial activity may be enhanced by the addition of agents having antioxidant 30 activity. Typical antioxidants include, for example, butylated hydroxyanisole, butylated hydroxytoluene, a-tocopherol and esters thereof, ascorbic acid, salts and esters thereof, gallic acid, salts and esters thereof e.g. propyl galiate, quinones such as 2,5-35 ditertiary butylhydroquinone, propolis, flavenoic- WO 95/26137 PCT/EP95/01037 - 5 - containing materials such as quercetin, sulphur-containing materials such as dilauryl-3,3-thiodipropionate and distearyl-3,3-thiodipropionate, and mixtures thereof. Preferred antioxidants are selected 5 from butylated hydroxyanisole, butylated hydroxytoluene, a-tocopherol and esters thereof and ascorbic acid, salts and esters thereof, preferably in a weight concentration of at least 1 mg/kg, more preferably at least 50 mg/kg. The use of a-tocopherol and esters thereof as "natural" 10 antioxidants is particularly preferred. There is also provided apparatus in the form of a packaged chemical composition for use in the method defined above, said apparatus comprising: a) a first reservoir comprising a source of D-glucose; 15 b) a second reservoir comprising a source of glucose oxidase; c) an incubation chamber connected to said first and second reservoirs; and d) means for introducing controlled quantities of said 20 D-glucose and glucose oxidase into said incubation chamber to prepare and incubate a biocidal mixture of glucose and glucose oxidase ready for use. Preferably, the first reservoir further comprises a source of thiocyanate and/or iodide anions. • X 25 Preferably, the second reservoir further comprises a source of lactoperoxidase. Suitably, the effective concentrations of D-glucose, glucose oxidase, lactoperoxidase and thiocyanate and iodide are such as to provide a biocidal 30 mixture having the following composition on mixing (but before incubation): WO 95/26137 PCT/EP95/01037 - 6 - (i) 10 to 500 mg/kg iodide anions; (ii) 5 to 200 mg/kg thiocyanate anions; (iii)0.2 to 100 g/kg D-glucose; and (iv) 150 to 20000 U/kg glucose oxidase; 5 wherein the weight : weight ratio of iodide : thiocyanate anions is 0.2:1 to 20:1 and the combined anion weight concentration is at least 25 mg/kg, in combination with a suitable carrier or excipient. One aspect of the invention provides concentrated 10 biphasic compositions in packaged and substantially non-reacting form which may be stored for prolonged periods prior to use. Concentrated compositions according to the invention will maintain physical separation of the glucose oxidase and its substrate, namely D-glucose, 15 such that ^2^2 Pr°duction is substantially prevented during storage. However, it will be understood that prior to storage concentrated compositions may contain a low level of at least one such substrate sufficient to support ar. initial reaction but insufficient to sustain 20 activity under the desired storage conditions. The initial reaction may advantageously provide adequate self-preservation of the concentrated compositions according to the invention. Self-preservation is of particular benefit in aqueous concentrates according to 25 the invention which may otherwise require the use of conventional chemical preservatives to avoid microbial spoilage curing storage! The substantially non-reacting concentrated compositions according to the invention are intended to be mixed or diluted and activated 30 immediately prior to use by bringing the glucose oxidase and substrates thereof into intimate admixture to produce compositions having the desired anti-microbial properties. WO 95/26137 PCT/EP95/01037 - 7 - The concentrated compositions according to the invention optionally further comprise suitable carriers and/or excipients. Advantageously the compositions may incorporate at least one buiifering agent to minimise the 5 fall of pH which may otherwise occur after activation of the concentrated composition. The concentrated compositions may be provided in the form of packs containing one or more discrete units of an appropriate weight or volume for batch or unit dosing. 10 ' Concentrated compositions according to the invention may comprise substantially anhydrous mixtures of each of the essential components mentioned hereinbefore, optionally combined with suitable nonaqueous carriers or excipients. 15 Concentrated water-containing compositions, optionally combined with suitable carriers or excipients, may be packaged and maintained prior to use. They may be in the form of, for example, solutions, suspensions, pastes or gels. 20 Compositions useful in the present invention may take the form of two or more powders, liquids, pastes or gels which prevent the glucose oxidase and D-glucose from reacting until the two are combined prior to use. For example, the glucose and D-glucose might be 2 5 formulated as dry granules to be activated by addition of liquid prior to use/ allowing the reagents to react. Examples of such-products include : a) deodorants e.g. for topical administration in the form of roll-on or stick formulations; 3 0 b) antibacterial skin washes e.g. in the form of lotions; c) anti-acne preparations e.g. in the form of lotions cr creams; WO 95/26137 PCT/EP95/01037 - 8 - d) anti-athlete's foot preparations e.g. in the form of lotions; e) anti-dandruff preparations e.g. in the form of shampoos or lotions; 5 f) dental preparations e.g. mouth washes suitable for general oral hygiene and in particular having anti-plaque properties, and dentifrices such as toothpastes, toothpowders, chewing gums and lozenges; 10 g) impregnated materials e.g. wound dressings, sutures and dental floss; h) pharmaceuticals e.g. wound irrigants and burn treatments, anti-diarrhoeal agents and medicaments suitable for the treatment of infections such as 15 Candida and Tinea infections; i) ophthalmic preparations e.g. eye washes and solutions for rinsing and/or sterilising contact lenses; j) sterilants e.g. for bciby bottles and surgical or 20 dental instruments. k) sterilants for use in healthcare/manufacturing environments to decontaminate for example surgical instruments or food processing equipment; and 1) surface cleaning agents for use in food 25 preparation/handling or healthcare environments to effectively decontaminate working surfaces. In another aspect, it is preferred that the incubated mixture is' provided as a concentrate for dilution to _produce a solution for killing 3 0 microorganisms. In this aspect, the incubation step to activate the mixture for fast-kill activity is carried out during manufacture and/or distribution to an end user. A range of oral hygiene preparations may be 35 envisaged which incorporate the anti-microbial WO 95/26137 PCT/EP95/01037 - 9 - compositions of the invention into conventional dental preparations such as mouthwashes, gargles and dentifrices as an anti-plaque agent and/or as a general antiseptic agent, for example in denture cleansing 5 tablets or solutions. The oral hygiene compositions of the present invention may, if desired, contain one or more active ingredients conventionally used in the art. These include, for example, other anti-plaque agents such as bromochlorophene, triclosan, cetylpyridinium 10 chloride and chlorhexidine salts; fluoride ion sources such as sodium fluoride, sodium monofluorophosphate and amine fluorides; anti-tartar agents such as zinc salts, preferably zinc citrate, and water soluble pyrophosphate salts, preferably alkali metal pyrophosphates; and 15 agents which reduce tooth sensitivity including potassium salts such as potassium nitrate and potassium chloride and strontium salts such as strontium chloride and strontium acetate. It will be appreciated that the compositions and 20 methods of the invention are suitable for a whole host of anti-microbial applications in areas such as agriculture, horticulture, veterinary medicine, and aquaculture, such as trout farming. The invention will be understood with reference to 25 the following non-limiting Tests and Examples: - i Comparative Test A Two phases of an antimicrobial composition were prepared as follows: WO 95/26137 PCT/EP95/01037 Phase A 10 - Component Concentration (w/v%) D-Glucose Sodium Thiocyanate 5 Potassium Iodide 45 to 55 0.42 to 0.52 0.66 to 0.80 Phase B Component Concentration Lactoperoxidase Glucose Oxidase 5500 Units/ml 2250 Units/ml 10 Both phases were adjusted to between pK 5.5 and 6.5 with buffer solutions. 15 A test solution was prepared by mixing Phases A and B together with water to concentrations of 0.9% and 0.05% respectively. An aliquot of the resulting solution was removed immediately and inoculated with Pseudomonas aeruginosa. The inoculated solution was incubated at room temperature. Samples were taken at regular intervals (viz at 0, 5, 10, 15, 30 and 60 minutes after mixing) 2 0 and subjected to serial dilution and agar plating in known manner (normal aseptic technique being used throughout) to determine the numbers of any surviving' organisms. At each serial dilution point a parallel sample was 25 taken, inoculated into a nutrient broth and incubated and then checked at an appropriate time for visual signs of growth. These parallel samples acted as so-called "broth controls" to check that the inoculation had been WO 95/26137 PCT/EP95/01037 - 11 - performed correctly and to check whether viable organisms were present which might be missed by the serial dilution process. These controls show up as counts of "less than 10M (<10) on the agar plates. 5 The original mixture of Phases A and B and water was stored at room temperature for three months. Aliquots were taken at regular intervals {viz at 4 hours, 24 hours, 48 hours, 7 days, 13 days, 21 days, 27 da^s, 2 months and 3 months) and inoculated with 10 Pseudomonas aeruginosa as described above to determine any change in the speed of kill. Results are set out below in Table 1. In each case the "broth controls" are recorded as "+ve" or M-ve" above the corresponding plate counts. 15 Plate counts are given in "colony forming units per ml" (c.f.u. ml~^) . In each case "T" denotes the test solution and "C" a control (the buffer solution without Phases A and B above). Comparative Test B 20 The procedure of Comparative Test A above was repeated with the modifications that the test and control solutions were inoculated with Staphylococcus aureus rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 25 2. Comi arative Test C The procedure of Comparative Test A above was repeated with the modifications that the test and control solutions were inoculated with Escherichia coli WO 95/26137 PCI7EP95/01037 - 12 - rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out. in Table 3. Comparative Test D . The procedure of Comparative Test A above was 5 repeated with the modifications that the test and control solutions were inoculated with Enterobacter cloacae rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 4. 10 Comparative Test E The procedure of Comparative Test A above was repeated with the modification that the test and control solutions were inoculated with Corynebacterium xerosis rather than Pseudomonas aeruginosa and that the storage 15 times were varied. Results are set out in Table 5. Comparative Test F The procedure of Comparative Test A above was repeated with the modifications that the test and control solutions were inoculated with Candida albicans 20 rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 6. Comparative Test G The procedure of Comparative Test A above was repeated with the modifications that the test and 25 control solutions were inoculated with Aspergillus niger rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 7. WO 95/26137 PCT/EP95/01037 - 13 - Comparative Test H The procedure of Comparative Test A above was repeated with the modifications that the test and control solutions were inoculated with Micrococcus 5 luteus rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 8. Comparative Test I The procedure of Comparative Test A above was 10 repeated with the modifications that the test and control solutions were inoculated with Staphylococcus albus rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 9 . 15 Comparative Test J The procedure of Comparative Test A above was repeated with the modifications that the test and control solutions were inoculated with Pityrosporum orale rather than Pseudomonas aeruginosa and that the 2 0 storage times were varied. Results are set out in Table 10 Comparative Test K - t The procedure of Comparative Test A above was • repeated with th» modifications that the test and 25 control solutions were inoculated with Streptococcus mutans rather than Pseudomonas aeruginosa and that the storage times were varied. Results are set out in Table 11. • • • • Table 1 Pseudomonas aeruginosa c.f.u. ml--*" Sampling time (minutes) Storage 0 5 10 15 30 60 time i 0 hours T +ve +ve h ve -ve -ve -ve 2.1xl06 l.lxlO6 5.8xl04 <10 <10 <10 f 1,7xl06 4 hours T +ve +ve -ve -ve -ve -ve 1.5xl05 <10 <10 <10 <10 <10 C 2.0xl06 24 T +ve -ve -ve -ve -ve -ve hours <10 <10 <10 <10 <10 <10 C 5.9xl05 48 T +ve -ve -ve -ve -ve -ve hours <10 <10 <10 <10 <10 <10 C 2.3xl06 Table 1 continued Sampling time (minutes) Storage time 0 5 10 15 30 60 7 days T 1 +ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 1.3xl06 2.2x10® 13 days T +ve <10 -ve <10 -ve <10 -ve <10 - ve <10 C 2.6xl06 2.0x10® 21 days T +ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 3.6xl06 3.2xl06 27 days T +ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 3.1xl06 3.0x10® Table 1 continued Sampling time (minutes) Storage time 0 5 10 15 30 60 2 months i T +ve <10 -ve <10 -ve <10 -ve <10 C Z.3xl06 7.8xl06 3 months T +ve <10 -ve <10 -ve <10 -ve <10 C 6.0x10® 7.0x10® Table 2 S ©\ W Staphylococcus aureus c.f.u. ml 1 5 10 Sampling time (minutes) Storage time 0 5 10 15 30 60 C hours T 2-. 7x10® * 2.7x10® +ve 2.2x10® +ve 2.2x10® +ve 2.5xl05 +ve <10 C 2.9x10® 4 hours T 2.6x10® 2.5xl05 +ve 2.4x104 +ve 2.OxlO1 +ve <10 -ve <10 C 4.7x10® 24 hours T +ve 2.8x10® +ve 2.4xl03 +ve 2.OxlO1 +ve 2.OxlO1 -ve <10 C 2.8x10® 48 hours T +ve 2.8xl04 -ve <10 -ve <10 -ve <10 -ve <10 C 4.1x10® ft 3 (ii S o Ul Table 2 continued Sampling time (minutes) Storage time 0 5 10 15 30 60 5 days T +ve 2.7xl06 +ve 3.2xl03 +ve <10 -ve <10 -ve <10 C 3.2x10^ 7 days T +ve 6.3xl05 +ve 5.OxlO1 +ve 1.OxlO1 -ve 7.OxlO1 -ve <10 C 3.9x106 14 days T +ve 1.2xl04 +ve <10 -ve <10 -ve <10 -ve <10 C 3.2xl06 28 days T +ve 6.6xl02 -ve <10 -ve <10 -ve <10 -ve <10 C 4.1x106 Table 2 continued Sampling time (minutes) Storage time 0 5 10 15 30 60 2Vt months T +ve 1.7xl04 -ve <10 -ve <10 -ve <10 -ve <10 C 2.6xl06 4 months T +ve 4.8xl05 +ve <10 +ve <10 -ve <10 -ve <10 C 4.4xl06 1.6xl06 6 months T +ve 1.3xl06 +ve 3.4xl03 +ve <10 +ve <10 C 2.3xl06 2.OxlO6 Table 3 S Escherichia coli c.f.u. ml-1 to 5 10 Sampling time (minutes) Storage 0 5 10 15 30 60 time 0 hours r ^ +ve +ve +ve +ve -ve 5„. 6x10 6 5.2xl06 4.8xl06 5.5xl06 8.4xl04 <10 C 4.2xl08 4.6xl06 4 hours T +ve -ve -ve -ve -ve -ve 2.7xl06 <10 <10 <10 <10 <10 C 2.6xl06 6.8xl06 24 T +ve -ve -ve -ve -ve -ve hours <10 <10 <10 <10 <10 <10 C 4.7x10 ^ 4.5xl06 48 T +ve +ve -ve -ve -ve -ve hours 3.3x105 <10 <10 <10 <10 <10 C 4.9xl06 3.4xl06 N3 o 13 1 3 t/i O L--J Table 3 continued Sampling time (minutes) Storage time 0 5 10 15 30 60 6 days T ' +ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 1.8xl06 4.OxlO6 9 days T +ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 3.2xl06 5.2xl06 14 days T +ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 3.lxlO6 3.5x106 23 days T +ve <10 -ve <10 -ve <10 -ve <10 C 3.3xl06 3.6xl06 Table 3 continued Sampling time (minutes) Storage time 0 5 10 15 30 60 28 days T +ve <10 -ve <10 -ve <10 -ve <10 C 2.3x106 3.OxlO6 2 months T +ve 3.7xl04 -ve <10 -ve <10 -ve <10 C 4.9x106 2.6x106 3 months T +ve <10 -ve <10 -ve <10 -ve <10 C 3.2xl06 2.5xl06 Table 4 g hJ C\ N* 1 W Enterobacter cloacae c.f.u. ml-1 ^ 5 10 Sampling time (minutes) Storage 0 5 10 15 30 60 time 0 hours T +ve +ve +ve -ve -ve 2.4xl06 3.5xl06 l.lxlO6 3.9xl06 <10 <10 C 5.7x106 4 hours T +ve -ve -ve -ve -ve -ve 4.4xl05 <10 <10 <10 <10 <10 C 4.6xl06 24 T -ve -ve -ve -ve -ve -ve hours <10 <10 <10 <10 <10 <10 C 2.4xl06 48 T +ve +ve +ve -ve -ve -ve hours 2.7xl04 <10 <10 <10 <10 <10 C 2.5xl05 ro CO *■3 n M *a Ln © o U) -4 Table 4 continued S KJ Os U -J Sampling time (minutes) Storage time 0 5 10 15 30 60 3 days T +ve 6.8xl05 +ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C *2.8xl06 8 days T +ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 3.1xl06 2.6xl06 14 days T +ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 4.7xl06 2.lxlO6 23 days T +ve -ve <10 -ve <10 -ve <10 -ve <10 C 5.OxlO6 5.7xl06 ro 4^ 3? O Table 4 continued £ hJ 0\ Ik) -4 Sampling time (minutes) Storage time 0 5 10 15 30 60 28 days T +ve 1.OxlO1 -ve <10 -ve <10 -ve <10 -ve <10 C 5:8xl06 4.2x106 2 months T +ve -ve <10 -ve <10 -ve <10 C 3.2xl06 3.4xl06 3 months T +ve -ve <10 -ve <10 -ve <10 C 6.7xl06 4.2xl06 ro on i 3 l/t © o w •o '» • Table 5 Corynebacterium xerosis c.f.u. ml-1 Sampling time (minutes) Storage 0 5 10 15 30 60 time 1 0 hours T +ve +ve +ve +ve +ve +ve 1.4xl06 9.3xl04 1.2xl04 5.1xl03 <10 <10 C 1.3xl06 1.1x10s 4 hours T +ve +ve +ve +ve -ve -ve 3.4xl04 <10 <10 <10 <10 <10 C 8.7xl05 3.5xl05 24 T +ve -ve -ve -ve -ve -ve hours <10 <10 <10 <10 <10 <10 C 7.3xl05 7.5xl05 48 T +ve -ve -ve -ve -ve -ve hours 2.7xl03 <10 <10 <10 <10 <10 C 6.3xl05 8.2xl05 Table 5 continued ^ IA h> 0\ u> -0 Sampling time (minutes) Storage time 0 5 10 15 30 60 6 days i T +ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C *:.6xl05 9.4xl05 9 days T +ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 1.2xl05 9.5xl05 14 days T +ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 6.1xl06 4.2xl06 23 days T +ve <10 -ve <10 -ve <10 -ve <10 C 1.4xl06 1.8xl06 rsa vj n s VO ty» © o u -J Table 5 continued Sampling time (minutes) Storage time 1 0 5 10 15 30 60 28 days T +ve <10 -ve <10 -ve <10 -ve <10 C 8.5x10s 9.3xl05 2 months T +ve <10 -ve <10 -ve <10 -ve <10 C 1.9xl06 8.2xl05 3 months T +ve <10 -ve <10 -ve <10 -ve <10 C 6.9xl05 6.lxlO5 J Table 6 ^ yj o\ . — *1 ''' Candida albicans c.f.u. ml 5 10 Sampling time (minutes) Storage time 0 5 10 15 30 60 0 hours T +ve 2.8x106 +ve 3.lxlO6 +ve 3.2xl06 +ve 3.2xl06 +ve 2.4xl06 +ve 1.OxlO6 C 2.6xl06 - 2.3x106 4 hours T +ve 2.lxlO6 +ve 3.lxlO6 +ve 1.2xl06 +ve 2.5xl05 +ve <10 -ve <10 C 2.5xl06 2.2xl06 24 hours T +ve 2.OxlO2 +ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 8.2xl06 2.7xl06 48 hours T +ve 1.6xl06 -ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 1.8xl06 2.2xl06 ro io 1 3 Ul & N* o w -J • J * i Table 6 continued Sampling time (minutes) Storage time 0 5 10 15 30 60 6 days T +ve -ve <10 -ve <10 -ve <10 -ve <10 C 2 ?6xl06 2.2xl06 15 days T +ve 8.6xl04 -ve <10 -ve <10 -ve <10 -ve <10 C 1.5xl06 1.3xl06 2 3 days T +ve 5.5xl05 -ve <10 -ve <10 -ve <10 -ve <10 C 1.2xl06 1.5xl06 27 days T +ve 4.9xl05 -ve <10 -ve <10 -ve <10 -ve <10 C 3.3xl06 3.1xl06 Table 6 continued Sampling time (minutes) Storage time 0 t 5 10 15 30 60 2 months T +ve 6.OxlO4 -ve <10 -ve <10 -ve <10 -ve <10 C 5.9x106 6.6xl06 3 months T +ve 2.5x10s -ve <10 -ve <10 -ve <10 -ve <10 C 1.8xl06 2.OxlO6 Table 7 Aspergillus niger c.f.u. ml-1 Sampling time (minutes) Storage time i 0 5 10 15 30 60 75 0 hours T +ve 1,2xl06 +ve 5.0x10s +ve 1.6xl06 +ve 1.4x106 +ve l.OxlO6 +ve 1.2x106 C 2.2xl07 1.3x10* ; 5 hours T +ve 1.2xl06 +ve 1.2xl06 +ve 1.3xl06 +ve 5.OxlO5 +ve 9.OxlO5 +ve 4.OxlO1 C 1.3xl06 1.OxlO6 24 hours T +ve 5.OxlO6 +ve 1.OxlO1 tve <10 -ve <10 -ve <10 -ve <10 C 3.7xl06 1.2xl06 4 days T 1.2xl05 -ve <10 -ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 2.4xl06 1.4xl06 Table 7 continued Sampling time (minutes) Storage 0 5 1C 15 30 60 75 time 7 days T -ve -ve -ve -ve -ve -ve 4.OxlO6 <10 <10 <10 <10 <10 <10 C 1.2xl06 * 9.OxlO5 14 days T -ve -ve -ve -ve -ve l.lxlO6 <10 <10 <10 <10 <10 C 2.3xl06 1.9xl06 22 days T -ve -ve -ve -ve -ve 1.OxlO5 <10 <10 <10 <10 <10 C 4.OxlO6 2.9xl06 26 days T -ve -ve -ve -ve -ve 1.OxlO6 <10 <10 <10 <10 <10 C 3.OxlO6 2.6xl06 Table 7 continued KJ C\ W Sampling time (minutes) Storage time 0 5 10 15 30 60 75 2 months T 3.OxlO6 -ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 4 .'OxlO6 6.OxlO6 3 months T 9.OxlO5 -ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 3.OxlO6 2.OxlO6 CO I 3 Cil o u -J Table 8 ^ . C\ N* — 1 ^ Micrococcus luteus c.f.u. ml ^ 5 10 Sampling time (minutes) Storage time 0 i 5 10 15 30 60 0 hours T +ve 1.7xl05 +ve 1.8xl05 +ve 1.7xl05 +ve 1.3xl05 +ve 1.2xl05 +ve 2.2xl05 C 2.OxlO5 - 1.4xl05 4 hours T +ve 1.5xl05 +ve 8.OxlO5 +ve 2.5xl04 +ve 7.5xl02 +ve 4.OxlO1 +ve <10 C 1.2xl05 8.7xl04 24 hours T +ve 1.5xl05 +ve 1.4xl04 +ve 3.8xl02 -ve <10 -ve <10 -ve <10 C 1.7xl05 1.7xl05 48 hours T +ve 4.7xl05 +ve 1.4xl02 +ve <10 -ve <10 -ve <10 -ve <10 C 4.7xl05 4.5xl05 OJ on ►a 1 ? t/I S N* o u •>4 Table 8 continued Sampling time (minutes) Storage time 0 5 10 15 30 60 6 days T , +ve 1.2xl05 +ve 3.3xl04 +ve 1.OxlO2 +ve <10 -ve <10 C 1.6xl05 1.3xl05 13 days T +ve 1.8xl05 +ve <10 +ve <10 +ve <10 +ve <10 C 2.2xl05 2.OxlO5 2 0 days T +ve 2.5xl04 +ve <10 -ve <10 +ve <10 -ve <10 C 7.9xl04 8.4xl04 27 days T +ve 1.3xl05 +ve <10 -ve <10 -ve <10 -ve <10 C 1.8xl05 2.0xl05 Table 9 Staphylococcus albus c.f.u. ml-1 5 10 Sampling time (minutes) Storage time 0 5 10 15 30 60 0 hours T +ve 2\3x106 +ve 1.1x106 +ve 2.OxlO6 +ve 2.0x106 +ve 1.6x106 +ve 9.4x106 n v 1.9xl06 - 2.5xl06 4 hours T +ve 2.OxlO6 +ve 8.2xl05 +ve 6.7xl04 +ve 1.5xl04 -ve 2.OxlO1 -ve <10 C 2.4xl06 2.0x106 24 hours T +ve 2.OxlO6 +ve 2.OxlO5 +ve 1.8xl04 +ve <10 -ve <10 -ve <10 C 2.3x106 1.5xl06 48 hours T +ve 1.3xl06 -ve 1.5xl02 -ve <10 -ve <10 -ve <10 -ve <10 C 1.4x106 1.6xl06 Table 9 continued o LA bJ 0\ N* >1 5 Sampling time (minutes) Storage time 0 1 5 10 15 30 60 6 days T +ve 2.lxlO7 +ve l.lxlO5 +ve 8.7xl02 -ve <10 -ve <10 C 3^6xl06 1.8xl06 13 days T +ve 2.8x105 -ve 7.OxlO1 -ve <10 -ve <10 -ve <10 C 1.9xl06 2.3xl06 20 days T +ve 2.7xl05 -ve <10 -ve <10 +ve <10 -ve <10 c 2.3xl06 2.5xl06 27 days T +ve 1.8xl05 -ve <10 -ve <10 -ve <10 -ve <10 C 9.6xl05 l.lxlO6 CjJ 00 I tjt O I-* o U s] Table 10 vo est Si ©\ P-+ -1 w Pityrosporum ovale c.f.u. ml A ^ 5 10 Sampling time (minutes) Storage time i 0 5 10 15 30 60 0 hours T 6.1xl04 4.8x104 5.5x104 +ve 3.7xl04 +ve 4.3xl04 +ve 9.OxlO1 C 4.7xl04 6.4xl04 4 hours T +ve 5.lxlO3 -ve 4.OxlO1 -ve <10 -ve <10 -ve <10 -ve <10 C 6.2xl04 2.8xl04 24 hours T +ve 9.8xl03 -ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 1.3xl04 1.2xl04 48 hours T -ve <10 -ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 8.OxlO4 5.4xl04 oo to £ 3 C/» © -J Table 10 continued Sampling time (minutes) Storage time 0 5 10 15 30 60 8 days T 1 +ve l.lxlO4 -ve <10 sampling interrupted -ve <10 -ve <10 C 1.8x105 A 1.6xl05 15 days T -ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 2.lxlO3 2.OxlO3 22 days T +ve 6.OxlO1 -ve <10 -ve <10 -ve <10 -ve <10 C 2.9x105 2.2x105 29 days T 9.OxlO1 -ve <10 -ve <10 -ve <10 -ve <10 C 4.OxlO5 3.9xl05 Table 11 Streptococcus mutans c.f.u. ml"1 Sampling time (minutes) Storage time 0 i 5 10 15 30 60 0 hours T +ve 3.5x105 +ve 4.7xl05 +ve 3.6xl05 +ve 3.4xl05 +ve 3.4xl05 +ve 3.9xl04 C 3.8xl05 2.0x105 4 hours T +ve 2.2xl05 +ve l.lxlO5 +ve 3.lxlO4 -ve 1.3xl04 +ve <10 -ve <10 C 3.3x105 2.lxlO5 24 hours T +ve 2.1x105 +ve 7.4xl04 -.■"e 8.4xl02 +ve 5.OxlO1 +ve <10 -ve <10 C 2.9x105 2.lxlO5 48 hours T +ve 1.OxlO6 +ve <10 -ve <10 -ve <10 -ve <10 -ve <10 C 1.6x106 1.OxlO6 Table 11 continued Sampling time (minutes) Storage time 0 5 10 15 30 60 1 7 days T I +ve 1.4x105 +ve 2.OxlO3 +ve <10 +ve <10 -ve <10 C 1.. 7x10 5 * 1.3xl05 13 days T +ve 3.9xl04 -ve <10 -ve <10 -ve <10 -ve <10 C 4.lxlO5 3.lxlO5 WO 95/26137 PCT/EP95/01037 - 43 - EFFECT OF PH The following tests were carried out to assess the effect of pH on anti-microbial activity. Test Procedure 5 Two phases of an antimicrobial composition were prepared as follows: Phase A Component D-glucose 10 Sodium thiocyanate Potassium iodide Concentration (w/v %) 45 to 55 0.42 to 0.52 0.66 to 0.80 Phase B 15 Component Lactoperoxidase Glucose Oxidase Concentration 5,500 Units/ml 2,250 Units/ml Both phases were adjusted to between pH 5.5 and 6.5 with buffer solutions. Test solutions were prepared by mixing Phases A and B together with citrate phosphate buffer at pH 3, 4, 5, 20 6, 7, 8 to concentrations of 0.9% and 0.05% ' i respectively. -The test solutions were then stored at room temperature" for 48 hours. 25 Aliquots of the stored test solutions were removed and inoculated with a single organism from the following organisms: E.coli, S.aureus, C.albicans, A.niger. The inoculated solution was incubated at room temperature for 15 minutes serially diluted and agar WO 95/26137 PCT/EP95/01037 - 44 - plated to determine the numbers of any surviving organisms. 5 10 Organism cfu ml-1 E.coli S.aureus C.albicans A.niger Initial Inoculum count 8.2xl05 2.4x106 1..2xl06 1.OxlO5 pH 3 <10 <10 1.9x106 <10 pH 4 <10 <10 <10 <10 pH 5 <10 5.OxlO3 <10 <10 pH 6 <10 5.OxlO5 <10 <10 pH 7 1.2xl02 6.4xl05 <10 3.OxlO1 pH 8 <10 2.1x106 1.7xl05 1.7xl03 The above results demonstrate that pH can be chosen so as to kill a particular micro-organism selectively. For example, at pH 8 only E.coli is killed. Broad 15 spectrum anti-microbial activity is obtained when the pH is around 4 in the above cests. WO 95/26137 PCT/EP95/01037 - 45 - Example 1 Glycol paint for athlete's foot or acne A biphasic glycol paint is prepared to the following composition: 5 Component A Propylene glycol D-Glucose Sodium thiocyanate Potassium iodide 10 Water Component B Glucose oxidase (available under the Trade Designation "Glucox P200") 112U 15 15ppm) Lactoperoxidase 275U (lOppm) Water to 50g Using a suitably designed dispenser 1 part of Component 20 A is combined with 1 part of Component B and allowed to stand for the required activation time. After this period the mixed components can be applied to control two strains of -inter alia Staphylococcus aureus and also Propionibacter "acnes, Candida albicans, Trichoderma 25 rubrum, Trichoderma mentagrophytes and Trichoderma interdigitale. Amount/50a 30g lg 8.4mg 13. 2mg to 50g Amount/50q WO 95/26137 PCT/EP95/01037 - 46 - Example 2 Roll-on Deodorant A biphasic roll-on ifodorant is prepared to the following composition: 5 Component A Amount/8Qq Tetrasodium EDTA 0.125g Mixture of stearates (available under the Trade Designation "Cithrol GMS A/Su) 3.75g 10 Ethoxylated fatty alcohol (available under the Trade Designation "Cromul EM 0685") Light liquid paraffin D-Glucose Sodium thiocyanate 15 Potassium iodide Water Component B Glucose oxidase (available under the Trade Designation "Glucox P200") 20 Lactoperoxidase Water Using a suitably designed dispenser, 4 parts of 25 Component A are combined with 1 part of Component B in a mixing chamber and allowed to stand for the required activation time. After this period the mixed components can be applied to provide the necessary killing activity against inter alia two strains of Staphylococcus aureus. 3.125g 3.75g 0.625g 5.2 5mg 8.2 5mg to 80g Amount/2Qq 280U (37.5ppm) 687.5U (25ppm) to 20g WO 95/26137 PCT/EP95/01037 - 47 - Example 3 Anti-dandruff shampoo A biphasic antibacterial anti-dandruff shampoo is prepared to the following composition: 5 Component A Amount/8Ocrms Sodium laureth-2-sulphate (23% soln) 68.75g Zinc sulphate 0.125g Mixture of diethanolamides (available under the Trade Designation "Empilan CDE") 6.25g 10 Stearic acid toilet 1.25g Mixture of mono and distearates (available under the Trade Designation "Empilan EGMS") 3.75g D-Glucose 0.625g Sodium thiocyanate 5.25mg 15 Potassium iodide 8.25mg Water to lOOg Component B Amount/2Ocrms Glucose oxidase (available under the Trade Designation "Glucox P200") 280U 20 (37.5ppm) Lactoperoxidase 687.5U (25ppm) Water to 20g Using a suitably- designed dispenser 4 parts of Component 25 A are combined with 1 part of Component B and allowed to stand for the required activation time. After this period the mixed components can be applied to achieve the desired killing activity against inter alia S. aureus and Pityrosporum ovale. WO 95/26137 PCT/EP95/01037 - 48 - CONCENTRATED FORMULATION Example Sterilising Concentrate for Dilution Concentration 10 Glucose Sodium Thiocyanate Potassium Iodide Potassium Dihydrogen Phosphate Sodium Hydroxide Glucose Oxidase Lactoperoxidase Water q.s to 45% w/v 0.42% w/v 0.66% w/v 0.62% w/v 0.02% w/v 5600 U 13750 U 100% volume In the above concentrated formulation the components are pre-activated by incubation during 15 manufacture and distribution. The concentration is then diluted 1 part in 100 parts of water to yield a steriling solution with fast-kill activity. The concentrated formulation is advantageous because the weight of product is greatly reduced 2 0 compared to the liquid solution, making the concentrated product relatively cheap to distribute. The concentrate formulation is also convenient for the end user because it is easier to transport and takes up less storage space than the equivalent liquid solution. WO 95/26137 PCT/EP95/01037 - 49 - Example 4 Sterilant solution (eg for contact lenses) A biphasic sterilant solution is prepared to the following composition: 5 Component A Amount/5Qq D-Glucose lg Sodium thiocyante 8.4mg Potassium iodide 13.2mg Water to 50g 10 Component B Amount/5Qg Glucose oxidase (available under the Trade Designation "Glucox P200") 112U (15ppm) Lactoperoxidase 275U 15 (lOppm) Water to 50g Using a suitably designed dispenser 1 part of Component A is combined with 1 part of Component B and allowed to stand for the required activation time. After this 20 period the mixed components can be applied to give effective control of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus ,taureus, Streptococcus mutans, Enterobacter cloacae and Candida albicans. An example of packaging suitable for use in the 25 method of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: WO 95/26137 PCT/EP95/01037 - 50 - Figure 1 shows a side section view of a packaged chemical composition according to the present invention, along the line I-I of Figure 2; and Figure 2 shows a plan section view of the packaged 5 chemical composition of Figure 1 along the line II- II of Figure 1. The packaged chemical composition, generally indicated as 1, comprises a first reservoir 2 and a second reservoir 3. The reservoirs are of approximately 10 equal size and are generally tubular in shape. The reservoir walls 4,5 are made from resilient polyethylene but it will be appreciated that other resilient materials, such as polyvinylchloride (PVC) or polyethyleneterephthalate (PET) may also be used if 15 desired. The walls are strong enough to avoid the risk of breakage but flexible enough to permit "squeezing" of the contents in use. One of the reservoirs, 2, houses Component A from Example 1 above and the other reservoir, 3, houses 20 Component B therefrom. The reservoirs 2,3 are substantially sealed so that no leakage of contents can occur from one to the other. For convenience they are welded together at one point by a polythene web 6 which is continuous with the reservoir walls 4,5. The walls 25 of the reservoirs are independently deformable such that it is possible to df.'form one reservoir, for example by "squeezing", without deforming the other, if desired. The lower end of the reservoirs provide a base 7 to allow the packaging to be rested in an upright position. 3 0 The upper ends of the reservoirs 2,3 are constricted to form a neck 8 which is blocked by a neck-piece 9 made of stiff non-resilient polyethylene. The neck 8 and neckpiece 9 support a bulbous incubation chamber 10. The incubation chamber 10 is constricted at its upper end to WO 95/26137 PCT/EP95/01037 - 51 - provide a second neck 11 which is threaded in the manner of a screw-top bottle neck to connect with a screw-cap 12 which may be used to close off the packaging when not in use. 5 Polythene tubes 13,14 extend substantially from the bases of the two reservoirs 2,3 respectively, through holes in the neck-piece 9, with which they form an interference fit, to the base of the incubation chamber 10. The tubes 13,14 are narrow enough to prevent 10 significant leakage of the contents of the reservoirs 2,3 into the incubation chamber 10 during normal storage. Such leakage as may occur is contained by the cap 12. However, when the cap 12 is removed and one or both of the reservoir walls is deformed, for example by 15 "squeezing", a portion of the reservoir contents is forced up the tube 13 or 14 by the pressure created and into the incubation chamber 10. In use, controlled mixing of the reservoir contents (Components A and B of Example 1 above) is achieved by 20 squeezing each of the reservoirs in turn and/or simultaneously by roughly equal amounts to introduce equal quantities of the reservoir solutions into the incubation chamber 10. Indicia 15 are provided on the walls of the incubation chamber 10 to assist in 25 measuring the quanitities to be mixed. The walls of the incubation chamber are made from transport/translucent polyethylene to assist in the measuring process but this is not essential and it will be appreciated that opaque plastics materials may be used if desired. The cap 12 30 may then be replaced and the apparatus 1 may be shaken gently to complete the mixing process. The resulting mixture is left to incubate for the desired period, typically from 5 to 10 minutes. After incubation the cap 12 is removed and the 35 incubated anti-microbial mixture is used in the desired WO 95/26137 PCT/EP95/01037 - 52 - manner, by application to the feet or other areas affected by so-called "athlete's foot". It will be appreciated that the packaging could house any of the other Examples given above and/or t 5 numerous variants thereof. Where the components are not mixed in equal proportions (eg Examples 2 and 3 above) the reservoirs may be made of correspondingly unequal size and corresponding modifications may be made to the other parts of the apparatus as appropriate. Thus, if 10 four parts of component A are needed to each part of component B, then the reservoir housing component A may be squeezed four times as often as the reservoir housing component B (as in Example 3 above), until the desired quantity of mixture has been generated in the incubation 15 chamber 10. Alternatively or additionally, the indicia 15 may be used to measure the required proportions. Instructions for mixing the components in the appropriate proportions will normally be supplied with the apparatus, preferably by printing onto the packaging 20 itself. </div>

Claims

<div class="application article clearfix printTableText" id="claims">



CLAIMS 

1 A method of killing microorganisms with an improved speed of kill by mixing effective amounts of D-glucose and glucose oxidase, incubating the resulting mixture 

5 which comprises both iodide ions and thiocyanate Ions wherein the weight ratio of iodide ions to thiocyanate ions is from 0.1:1 to 50:1 and the combined anion weight concentration is at least 5 mg/kg wherein the concentration of glucose oxidase in the resulting mixture is at least 150 U/kg, at a temperature of from -10 to 50°C and at a pH of from 1-8 for a period of from 30 minutes to 48 hours and then applying the 10 incubated mixture to the microorganisms to be killed.
2 A method as claimed in Claim 1 wherein the resulting mixture Is incubated for 12 to 48 hours. 

15
3 A method as claimed in either Claim 1 or Claim 2 wherein the resulting mixture further comprises a peroxidase enzyme.
4 A method as claimed in Claim 3 wherein the peroxidase enzyme is lactoperoxidase. 

20
5 A method as claimed in any preceding claim wherein the incubated mixture is provided as a concentrate for dilution to produce a solution for killing microorganisms.
6 A method as claimed in any of Claims 1 to 5 wherein the components are 25 provided as a concentrated biphasic composition in packaged and substantially non- 

reacting form.
7 Apparatus in the form of a packaged chemical composition for use in the method defined in Claim 1 said apparatus comprising: 

30 

a) a first reservoir comprising a source of D-glucose; 

b) a second reservoir comprising a source of glucose oxidase; 

c) an incubation chamber connected to said first and second reservoirs; and d) means for introducing controlled quantities of said D-glucose and glucose 35 oxidase into said incubation chamber to prepan mixture of glucose and glucose oxidase ready for us e. N-2' 

1 o AUG 1998 RECEIVPn 

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- 54 - W'
8. A method of killing microorangisms as claimed in claim 1 substantially as herein described with reference to any one of the Examples.
9. An apparatus as claimed in claim 7 substantially as herein described with reference to Figures 1 and 2 of the accompanying drawings. 

EHD Of ClAlWS jflawn JO 0^3 

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intellectual property office of n.z.
10 AUG 1998 RECEIVED 



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