Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
101520253035CA 02264286 1999-02-22WO 98/09520 PCT/US97/00276ANTIMICROBIAL COMPOSITIONS____I... Field of the Invention This invention relates to antimicrobialcompositions useful for topical application to an animal'sskin to kill infectious microorganisms and moisturize theanimal's skin.Background of the InventionLotions, salves and the like can be applied to theskin for various restorative purposes. For example,lotions may be used to condition or moisturize the textureof dry skin, or they may have antimicrobial properties toprevent or to treat infections. Thus, a diverse range ofproducts is potentially available for human and veterinaryuse.Of particular importance in the veterinary contextis the prevention of mastitis in cows and the moisturizingof cow teats. Cows are susceptible to mastitis, and cowteat skin is susceptible to drying. Currently, various"teat dips" are available for treatment of cow teats toprevent bacterial colonization. Available teat dips,however, include aqueous carriers (or vehicles) which cancause frostbite and skin damage in cold weather.Consequently, the use of teat dips is often interruptedduring cold weather, increasing the likelihood ofbacterial colonization of cow teats, resulting in bovinemastitis.There is a continuing demand for effective andinexpensive moisturizing or antimicrobial compositionsuseful for treating animal and human skin, and inparticular for treating cow teats to moisturize andprevent mastitis. There is particular need for such acomposition that may be used in cold weather without riskof frostbite.1015202530CA 02264286 1999-02-22WO 98/09520 PCT/US97/00276summarv of the InventionIn a first aspect, the invention involves anantimicrobial ointment including an antimicrobial fattyacid monoester of a polyhydroxy alcohol, an ointmentvehicle composition including petrolatum, and a chelatingagent.The polyhydroxy alcohol used to form theantimicrobial monoester preferably is selected from thegroup consisting of glycerol, propylene glycol andThe fatty acid used to form theantimicrobial monoester preferably is selected from thesaccharides.group consisting of caproic acid, heptanoic acid, caprylicacid, capric acid, undecanoic acid and lauric acid.Particularly preferred monoesters include glycerolmonolaurate and propylene glycol monocaprylate.The antimicrobial ointment preferably includes atleast about 25% by weight of petrolatum and morepreferably at least about 50% by weight of petrolatum.The antimicrobial ointment preferably includes betweenabout 0.1% and about 10% by weight of the fatty acidmonoester of a polyhydroxy alcohol.A preferred antimicrobial ointment of theinvention also includes at least about 0.1% by weight of achelating agent, with lactic acid being a preferredchelating agent.The antimicrobial ointment may include at leastTheantimicrobial ointment may also include at least aboutTheantimicrobial ointment may include at least about 1% byabout 1% by weight of a viscosity modifying agent.0.5% by weight of an inactive emollient.weight of wax. The antimicrobial ointment may similarlyinclude at least about 0.5% by weight of cetyl alcohol. Apreferred antimicrobial ointment of the invention includes1015202530CA 02264286 1999-02-22WO 98/09520 PCT/US97/00276in percent by weight at least about 0.1% glycerylmonolaurate, at least about 0.1% propylene glycolmonolaurate, at least about 0.1% lactic acid, and at leastabout 1% wax.Preferred antimicrobial ointments of the inventionproduce at least about a 105 fold reduction in colonyforming units of bacteria in an in vitro bacterial killtest.invention produce at least about a ten (10) fold reductionSimilarly, preferred antimicrobial ointments of thein Streptococcus uberis contamination eight hoursfollowing application to cow teat skin.In another aspect, the invention involves a methodof producing an antimicrobial composition including thesteps of (a) melting a vehicle composition comprisingpetrolatum, waxes or a mixture thereof; and (b) adding (1)an antimicrobial fatty acid monoester of a polyhydroxyalcohol, and (2) a chelating agent to the melt to form amelt mixture, the melt mixture forming the antimicrobialointment upon cooling. A preferred antimicrobial ointmentproduced by this method includes at least about 25% byweight of the vehicle composition and between about 0.1%and about 10% by weight of the monoester. The vehiclecomposition preferably includes petrolatum.In another aspect, the invention involves a methodof treating a bovine teat comprising the step of applyingto skin of the bovine teat an antimicrobial compositionincluding (a) an antimicrobial fatty acid monoester of apolyhydroxy alcohol; (b) a chelating agent; and (c) avehicle composition, wherein the vehicle composition isThe treatment methodgenerally is effective at preventing bacterialeffectively water insoluble.colonization of bovine teats as well as at preventingdrying of the skin of the teat. The preferred1015202530CA 02264286 1999-02-22WO 98/09520 PCT/US97/00276antimicrobial composition for use in the treatment methodincludes between about 0.1% and about 10% by weight of themonoester, wherein the polyhydroxy alcohol is selectedfrom the group consisting of glycerol, propylene glycoland saccharides, and wherein the fatty acid is selectedfrom the group consisting of caproic acid, heptanoic acid,caprylic acid, capric acid, undecanoic acid and lauricacid.The antimicrobial compositions of the inventionhave significant advantages with respect to moisturizingand skin conditioning capabilities over aqueous teat dipscommercially available for preventing bovine mastitis.The antimicrobial compositions are effective against boththe products of theinvention can be produced with a wide range ofbacteria and viruses. Furthermore,consistencies to give the consumer a variety of choicesfor applying the product. A significant advantage of thecan be applied to farm animalsOtheradvantages will be apparent from the detailed descriptioncompositions is that theyduring cold weather without risk of frostbite.and claims.Description of the Preferred EmbodimentsThe invention involves antimicrobial compositionsuseful for treating skin to prevent or to cure infectionand/or dryness.In one preferred embodiment, the inventioncombines an antimicrobial agent and a chelating agentwithin an ointment vehicle. The antimicrobial ointmentsinclude at least one antimicrobial monoester as anantimicrobial agent. The chelating agent is included as afurther synergistic component with respect to theAdditionalcompounds may be added to modify the emollient properties,antimicrobial properties of the composition.1015202530CA 02264286 1999-02-22WO 98/09520 PCT/US97/00276the rheology, the aesthetic properties and other aspectsof the ointment.The preferred active antimicrobial agents areTheantimicrobial ointment typically has between 0.01% and 20%fatty acid monoesters of polyhydroxy alcohols.by weight of the antimicrobial monoesters, preferablybetween 0.1% and 10% and more preferably between 0.5% and5% by weight of the antimicrobial monoesters.Appropriate polyhydroxy alcohols (i.e., polyols orpolyhydric alcohols) suitable for use as a componentwithin the antimicrobial monoesters include glycerol,propylene glycol, polyalkylene glycol such as polyethyleneglycol and polypropylene glycol, polyglycerol, andsaccharides. Saccharides include monosaccharides such asglucose, disaccharides such as sucrose and polysaccharidessuch as starch. Preferred polyhydroxy alcohols have lessthan about eight carbon atoms. Glycerol and propyleneglycol are particularly preferred polyhydroxy alcohols forthe formation of antimicrobial esters for use in ointmentsof the invention.Appropriate fatty acids suitable for use as acomponent within the antimicrobial monoesters includestraight and branched carboxylic acids with greater thanabout six carbon atoms. The carbon chains of the fattyacids may be saturated, monounsaturated orpolyunsaturated. Preferred fatty acids have between aboutsix carbon atoms and about 24 carbon atoms, and morepreferably between about 8 carbon atoms and about 16carbon atoms. Generally, preferred fatty acids includestraight chained, saturated fatty acids.It may be desirable to use a combination of morethan one fatty acid monoester of a polyhydroxy alcohol.The combination of two or more monoesters can provide a1015202530CA 02264286 1999-02-22WO 98/09520 PCT/US97/00276broader range of antimicrobial activity relative to asingle monoester. Preferred combinations include two ormore monoesters of straight chained, saturated fattyacids, with between about six carbon atoms and abouttwelve carbon atoms. Particularly preferred saturatedfatty acids for incorporation into the monoesters includecaproic, heptanoic, caprylic, pelargonic, capric,undecanoic, lauric, myristic, and palmitic. Furtherdescription of useful fatty acid monoesters may, forexample, in U.S. Patent 5,378,731 and references therein.The antimicrobial compositions of the inventioncontain a nonaqueous vehicle, or carrier, that ispreferably water insoluble. The nonaqueous vehiclesgenerally may include organic and inorganic, oils andwaxes. Preferred nonaqueous vehicles include ointmentvehicles, such as hydrocarbons, especially petrolatum.Preferred antimicrobial ointments have at least about 25%by weight of petrolatum and more preferably at least about50% by weight of petrolatum. The present inventor hasfound that the antimicrobial monoesters when used in apetrolatum vehicle generally are as effective as currentlyavailable aqueous products.Suitable chelating agents for use in theantimicrobial compositions of the present inventioninclude those selected from the group consisting ofethylenediamine tetraacetic (EDTA) acid and its salts,hydroxyalkanoic acids such as lactic acid and citric acid,polyphosphoric acid and its salts, acidic sodiumhexametaphosphate (commercially available as SPORIX acidicsodium hexametaphosphate, from International Sourcing,Inc., Upper Saddle River, N.J.), and mixtures thereof.Lactic acid is a preferred chelating agent because it mayadvantageously function as both a chelator and a1015202530CA 02264286 1999-02-22W0 98l09520 PCT/US97/00276moisturizing agent in the composition of the presentinvention. The antimicrobial composition generallyincludes an amount greater than about 0.1% by weight ofchelating agent per quantity of antimicrobial ointment,preferably between 0.1% and 5% and more preferably betweenabout 0.5% to about 2% by weight of chelating agent.The antimicrobial compositions of the inventionmay contain emollients or moisturizers in addition to theantimicrobial fatty acid monoesters of polyhydroxyalcohols, which may also act as emollients, and thechelating agents, which may also act as a moisturizers(e-9-,suitable for use in the invention.lactic acid). A variety of typical emollients arePreferred emollientsinclude cetyl alcohol, stearyl alcohol, lanolin, lanolinderivatives, isostearic acid, esters of isostearic acid,isostearyl alcohol, ethylene glycol esters, propyleneAnantimicrobial ointment of the invention, when an emollientglycol esters, emollient glycerides and the like.is used, generally includes greater than about 1% byweight of emollients, preferably between about 2% and 25%and more preferably between about 5% and 15% by weight ofemollients.The antimicrobial compositions of the inventionmay also include viscosity modifiers as inert ingredients.The viscosity modifiers can be used to alter theproperties of the ointment vehicle to be more suitable forsome of the viscosity modifiersTheviscosity modifiers are used to alter the viscosity anda particular application.are also suitable for use as an ointment vehicle.more generally the rheologic properties imparted to theantimicrobial ointment by the ointment vehicle.Organic solvents may be used as viscositymodifiers as well as beeswax, paraffin, carnauba wax,1015202530CA 02264286 1999-02-22WO 98/09520 PCT/US97/00276polyethylene glycol, ethylene glycol monostearates,ethylene glycol distearates, mineral oil, and syntheticspermaceti wax. The antimicrobial compositions, whencontaining viscosity modifiers, generally include at leastabout 1% by weight of viscosity modifier and preferablybetween about 2% and about 20% by weight of viscositymodifier. The viscosity may be adjusted with such agentsto produce a product for application in a particularlydesired way.The antimicrobial ointment optionally may includefragrance, fillers and other compounds to alter the costand aesthetics of the ointment, including materials suchas FINSOLV TNTâ, which are useful for improvingspreadability and texture.The antimicrobial compositions, includingointments, of the invention have a variety of uses forapplication to skin. For example, the antimicrobialcompositions may be applied to human skin to preventinfections while moisturizing the skin. Similarly, theantimicrobial compositions may be used as a first aidabrasions and the likeproduct to treat cuts, scratches,on animals and humans. The composition may be used as ahoof moisturizer for horses and as a treatment for skininfections on dogs. A preferred use of the antimicrobialcomposition involves application of the antimicrobialcomposition to cow teats to prevent bacterial colonizationof teat skin while moisturizing the teat. Colonization ofteat skin with bacteria is correlated with the developmentof bovine mastitis. An additional preferred use of theantimicrobial composition of the invention involvesapplication to bovine skin to prevent or cure the viralinfection Bovine Herpes Mammallitis.1015202530CA 02264286 1999-02-22wo 98/09520 PCT/US97/00276The invention may be illustrated by way of thefollowing examples.EXAMPLESThe following examples demonstrate theantimicrobial effectiveness and the skin conditioning ofthe products of the invention.A test ointment (nonaqueous teat dip) is preparedfrom the following ingredients given in percents byweight:Ingredient % [ wtLauricidinTM 1 . 0Propylene Glycol Monocaprylate 1.0Lactic Acid 2.0Cetyl Alcohol 3.0Lanolin 10.0White Beeswax 5.0Finsolv TNTM 4.0Ungerer Mask DD 49978TM 0.5White petrolatum 73.5LauricidinTM (glyceryl monolaurate) was purchased fromIL.glycol monocaprylate was purchased from Unichema NorthMed-Chem Laboratories Inc., Galena, The propyleneAmerica, Chicago, IL. The white beeswax was purchasedfrom ACROS Organics, Pittsburgh, PA.mixture of alkyl benzoates with the alkyl groups havingFinsolv TNTâ is achain lengths ranging between C12 to C15 purchased fromFinetex, Inc., Spencer, NC. Ungerer Mask DD 49978TM is afragrance purchased from Ungerer, Inc., Lincoln Park, NJ.The white petrolatum was purchased from PennsylvaniaRefining Co., Butler, PA.To produce the test ointment, the white petrolatumand the lanolin were added to a mixing tank. The mixingtank was heated. Mixing was begun when the white1015202530CA 02264286 1999-02-22WO 98/09520 PCT/US97/00276petrolatum and the lanolin were partially melted. Aftermixing was begun, the other ingredients were addedsequentially in the following order: Finsolv TNTM, cetylalcohol, LauricidinTM, propylene glycol monocaprylate,white beeswax and lactic acid. Heating and mixing wascontinued while these other ingredients were added. Themixture was heated to 160°F i 10°F (71°Ct6°C) to produce aclear liquid melt. Heating was stopped and mixing wascontinued while the mixture cooled. When the mixture hadcooled to 120°F - 130°F (48.9°C - 54.4°C), the fragrance(Ungerer Mask DD 49978TM) was added while mixingcontinued. when the batch had cooled to about 115°F(46.1°C), the mixture was poured into ointment jars.To evaluate the antimicrobial effectiveness of thetest ointment described above, both in vitro tests andtests on cow teats were performed. These tests aredescribed below.Example 1 and Comparative Examples 1-3These examples involve in vitro tests of theantimicrobial effectiveness of the test ointment of theexample.of bacteria, Staphylococcus aureus (gram positive, ATCC#25923) and Escherichia coli (gram negative, ATCC #25922).The test was performed with two different typesFor comparison with the test ointment of the invention,three commercially available aqueous udder balms were alsotested. Dr. NaylorTM is distributed by H.W. Naylor Co.,Inc., Morris, NY, Ken AgTM is distributed by Ness & Clark,Inc., Ashland, OH,Mills Fleet Farm, Appleton, WI.antimicrobial activity involve a bacterial count afterand 4 Seasons TM is distributed byThe comparisons ofexposure to the antimicrobial ointment for 0 minutes andfor 20 minutes at 37°C.-10-1015202530CA 02264286 1999-02-22WO 98/09520 PCT/US97/00276The bacterial cultures were prepared by suspendingthe bacteria in tryptic soy broth and incubating thesuspensions at 37°C for 24 hours. The suspensions weredesigned to have an initial inoculum count of between 107to 108 CFUs/ml, which was later confirmed with plateThen, a 0.1 mlaliquot of the 24 hour bacterial culture suspension wascounts. (CFU = colony forming unit).added to a centrifuge tube containing 10 mls ofantimicrobial ointment to form an ointment bacterialsuspension (OBS). Prior to the addition of the bacterialculture suspension, the antimicrobial ointment wassoftened in a water bath at 37°C to assist with the mixingof the CBS.for 20 seconds and returned to the water bath afterThe OBS in the centrifuge tube was vortexedaliquots were removed.A 1.0 ml aliquot of OBS was removed from thecentrifuge tube both immediately after vortexing and 20minutes after vortexing. Serial dilutions were performedwith each 1.0 ml aliquot. To perform the first dilution,the 1.0 ml aliquot was placed in a test tube containing9.0 mls of Letheen BrothTM to form a 1:10 dilution. Twoadditional serial dilutions were made by first placing a 1ml aliquot of the 1:10 dilution into 9.0 mls of LetheenBrothTM to form a 1:100 dilution and subsequently placinga 1 ml aliquot of the 1:100 dilution into 9.0 mls ofLetheen BrothTM to form the 1:1000 dilution. LetheenBrothTâ is a neutralizing agent to prevent furthermultiplication of bacteria, sold by Difco Laboratories.The letheen broth was warmed prior to mixing to 37°C tomaintain optimum mixing. The tubes were vortexed.Following vortexing, 0.1 ml of solution from eachof the serial diluted tubes separately was spread ontryptic soy agar plate. The inoculated plates were-11-10152025303540WO 98/09520incubated at 37°C for fortyâeight hours.CA 02264286 1999-02-22FollPCT/US97l00276owingincubation, the bacterial colonies on the plates werecounted and compared to the initial inoculum count.results of the tests are shown in Table 1.ProductMinutesDr. NaylorX 10x 105Ken Ag10<1OCFU/gm4 SeasonsTNTC<l0CFU/gmTest<10CFU/gmOintment<10CFU/gmTABLE 1TheIN-VITRO BACTERIAL KILL ASSAY RESULTSActiveIngredient(s)Bacteria8âHydroxy- S. Aureuaquinoline E. ColiMethyl Paraben, S. AureusPropyl Paraben, E. ColiBronopolChloroxylenol S. AureusE. ColiGlyceryl S. AureusMonolaurate,Propylene E. ColiGlycol Mono-caprylate,Lactic Acid* TNTC=Too numerous to count1.2 X 10InoculantM Count5.0 x 10886.0 x 1081.2)(1o86.0 X 101.2 X 106.2 x 101°1.9 x 101°Zero 20Minutes82.7 X 10 7.681.9 X 10 7.4TNTC* 5.2 x4.4)z1o5TNTCTNTCe.4)<1o5<10CFU/gmThe test ointment had superior antimicrobial propertiescompared with the aqueous udder balms.1015202530CA 02264286 1999-02-22WO 98/09520 PCT/US97/00276Example 2 and Comparative Example 4These examples were designed to test therelatively long term (eight hour) antimicrobialeffectiveness of the test ointment in use on cow teatsspecifically against Streptococcus uberis. For comparison(Comparative Example 4), the test was also performed withan aqueous, commercial teat dip, LauricareTM from 3M,Saint Paul, MN.Cows were selected for normal teat size andstructure. At the start of the test teats were washed andrinsed with alcohol. All teats then were dipped to about3/4 length in a bacterial solution of S. uberis at aconcentration of 1.0xlO7 bacteria/ml in milk suspension.Then,ointment vehicle without germicide (monoesters) was addedThe teats were allowed to dry for ten minutes.to control teats (left side) and test ointment was addedto test teats (right side).TMSimilarly, other cows weretreated with Lauricare teat dip, an aqueous basedproduct, on test teats (right side) with no treatment onthe control teats (left side).Eight hours after treating the teats, the numberof colony forming units (CFU's) recovered from the testand control teats was determined by first scraping theteats separately with a spatula with the residue placedNext,16 mls of letheen broth, which was placed into a separateinto a centrifuge tube. each teat was rinsed withcentrifuge tube. Then, each teat was scrapped again withany residue being placed into the same centrifuge tubewith the first scrapings.The materials in the tubes were appropriatelydiluted, and the diluent was plated onto sheep blood agar.The inoculated plates were incubated at 35â37°C for 48hours. The colonies were counted on plates having between-13..101520253035WO 98/09520CA 02264286 1999-02-22PCT/US97/0027630 and 300 colonies per plate to determine the CFU's perml. The log reduction in bacteria from the test teatsrelative to bacteria from the control teats was calculatedfor each set of teats. The results from the rinse testsand the scrapping tests were averaged together. For eachcow the results for the two left (control) teats and tworight (test) teats also were averaged together. Theresults of the experiments, presented as percentreductions and log reductions in contamination, with thetest ointment and with LauricareTM are presented in Table 20TABLE 2LONG TERM (8 HR.) RESIDUAL GERMICIDAL EFFECTS OFAGAINST STREP. UBERIS ON TEAT SKINProduct Percent Reduction Log ReductionCow#1 Cow#2 Cow#3 Cow#1 Cow#2 Cow#3Test Ointment 98.12 93.96 96.66 1.73 1.22 1.47LauricareTMTeat Dip 81.40 96.13 97.70 0.73 1.41 1.63The test ointment had comparable effectiveness as theaqueous teat dip against these bacteria under realisticconditions.Example 3This example demonstrates effectiveness of thetest ointment against the Bovine Herpes Mammillitis virus.The Bovine Herpes Mammillitis virus (strain NewYork 1, VR-845) was obtained from the American Tissue TypeCollection, Rockville, MD. TMminimal essential medium (sold by Gibco-BRL, Grand Island,The test medium was EaglesNY) supplemented with 2% (v/v) fetal bovine serum heatinactivated by heating to 56°C for 30 minutes. The medium-14..1015202530WO 98/09520CA 02264286 1999-02-22PCT/US97/00276was also supplemented with 100 units/ml penicillin (soldby USB, Cleveland, OH), 10 pg/ml gentamicin and 2.5 pg/ml(Amphotericin B sold by Sigma, St. Louis, MO).Then,the cell debris was removed, and aliquots of the virusFungizoneTMThe virus was grown to a sufficiently high titer.were stored at -70°C until use.Viruses were exposed to the test ointment insuspension for exposure times of 1 minute (23°C) or 20minutes (40°C). A 1.0g quantity of test ointment wasplaced in a test tube along with a 0.5ml aliquot of virussuspension. For the 1 minute exposure, the mixture wasvortexed for the entire one minute. For the 20 minuteexposure, the mixture was vortexed for one minute andevery four minutes thereafter. The mixture was held at40°C during exposure except during vortexing. Immediatelyfollowing the exposure period, the mixture was titredusing a series of 10-fold dilutions using Eagle'sTMminimal essential medium. The series of dilutions wereassayed for infectivity.Several controls were used. A virus controlincluded a 0.5 ml aliquot of virus suspension mixed with1.0g of test media. cytotoxicity controls included 1.0gof test ointment mixed with 0.5 ml of Eagle'sTM minimalessential medium. A series of dilutions of thecytotoxicity control mixture was challenged with stockvirus and assayed for infectivity in order to determinethe dilution of the test ointment which has virucidalactivity. The experiments with the cytotoxicity controlmixture were performed in the same way as the testointments experiments.Infectivity was determined using bovine kidneycell lines obtained from ViroMed Laboratories, Inc., Cell-15.-10152025CA 02264286 1999-02-22WO 98/09520 PCT/US97/00276Culture Division. Cultures were grown and used asmonolayers in disposable tissue culture labware.Dilutions of test mixtures and controls were inoculatedinto bovine kidney cell cultures in quadruplicate. Thecells were inoculated with 100 pl of each dilution andincubated at 36-37°C in 5.1â6.5% C02. The cells wereobserved for seven days for the presence of virus and/orcytotoxic effects. Bovine Herpes Mammillitis virusproduces a typical cytopathic effect on bovine kidneycells. The method of Karber was used to calculate 50percent end points, TCID5o (TCID=Tissue CultureInfectivity Dose), which is the dose that results ininfection of 50% of the cells.are obtained as follows:1 â (TCID5o/TCID5o virus control) 100(ASTM Standards on Materials and EnvironmentalPercent reduction values%ReductionMicrobiology, 1994, E1052-85; Diagnostic Procedures forViral, Rickettsial, and Chlamydial Infections, E.H.Lennette and N.J. Schmidt, editors, Fifth Edition, 1979, p32-35).The results of the one minute and 20 minutesThe titre of thevirus control in both tests was 6.75 loglo.exposure tests are displayed in Table 3.Infectivitywas not detected in either test for the test ointmentsamples. This puts a lower limit on the titre of $1.5loglo and an upper limit on the percent reduction of299.999%.-16-10152025303540CA 02264286 1999-02-22WO 98/09520 PCT/US97/00276T E 3one Minute Exposure Period 20 Minute ExposurePeriodDilution Virus Control Test samples Virus Control Testsamples(CELL CONTROL) 0000 0000 000000001o"2 ++++ oooo ++++00001o'3 ++++ 0000 ++++000010'4 ++++ 0000 ++++000010-5 ++++ 0000 ++++000010-6 ++++ 0000 ++++00001oâ7 o+oo oooo +0000000TCID5g 1o6'75 s1o1'5 1o°â755101'Percent Reduction NA 299.999% NA 299.999%Key:(+) = positive for the presence of test virus(0) = no test virus recovered and/or no cytotoxicitypresent(T) = Cytototoxicity presentThe results of the cytotoxicity control tests aredisplayed in Table 4.Again the samples containing thediluted test ointment eliminated all signs of virusactivity.10152025303540CA 02264286 1999-02-22WO 98/09520 PCT/US97/00276TABLE 4Dilution Cytotoxicity Control: NeutralizationControl:Test Substance + BovineHerpesMammillitis(CELL CONTROL) oooo oooo1o'2 oooo ++++1oâ3 oooo ++++1oâ4 oooo ++++TCD§og0.1ml S101â5 Neutralized at510 °Key:(+) = positive for the presence of test virus(0) = no test virus recovered and/or no cytotoxicitypresent(T) = Cytototoxicity presentExample 4This example demonstrates the effectiveness of thetest ointment for improving the skin condition of a cow'steat.Cows in the study were selected for having normalteats and udders. Teat on a cow's right side were treatedwith the test ointment while the adjacent teats on theFive cows were included in theInleft side were untreated.study each with two test teats and two control teats.other words, ten test teats and ten control teats wereinvolved in the study. The test ointment is applied twicedaily following milking. The tests were performed duringa relatively cold period. The temperature of the barn wasabout 40°Fâ50°F (4.4°C-10.0°C) with low humidity. Therewas a tendency for drying under these conditions.-18-WO 98/09520101520253035CA 02264286 1999-02-22PCT/US97/00276Barrel Dryness and Teat End Score were determinedfor both the treated and the control cow teats. In theevaluation, each teat is given a numerical score, and thescores are added to produce an overall result. Theis unaware of which teats received the productIn the Teat Barrelevaluatorand which teats are the control teats.Test, the teat barrel is evaluated by palpation todetermine the degree of dryness and roughness, and thenumerical score is assigned as follows:1 = normal soft skin2 = mild amount of dry feeling and some roughness3 = moderate loss of moisture and ability to feeledges of skin flakes4 = skin feels very dry, possibly some crackingand serum exudationThe Teat End Keratin Evaluation involves a numericalevaluation of each teat based on the following numericalscale:= none or small amount of soft keratin present2 = small increase in amount and hardness ofkeratin3 = significant increase in amount and hardeningof keratin4 = large amount of very hard keratin presentThe results of the tests are presented in Tables 5 and 6.TABLE 5BARREL DRYNEBSstart 1 Week 2 Weeks 3WeeksRight side treatment 22 18 12 12Left side control 21 19 19 18-19-1015CA 02264286 1999-02-22WO 98/09520 PCT/US97/00276TABLE 6THAT END SCOREstart 1 Week 2 Weeks 3weeksRight side treatment 18 14 20 17Left side control 20 16 13 17The dryness test (Table 5) demonstratessignificant improvement over time for the treated teatcompared with little improvement shown for the controlteats. The teat end scores showed approximately similarresults for the treated and the control teats within thevariability of the results.Other embodiments of the invention are within thescope of the appended claims.-20-
