ClinicalstudyofPCSO-524(ANTINOLä)asneutraceuticalincanineallergicskindisease

Introduction

PCSO-524 is an extract from New Zealand green lipped mussel (Perna canaliculus). Itcontains several classes of lipid. PCSO-524 contains two of the long chain omega-3polyunsaturated fatty acid (PUFAs); eicosapentaenoic acid (EPA) and docosahexaenoic acid(DHA).PCSO-524iscomposeof13%EPA,21%DHAandabout30%cholesterol.1Inaddition,italso contains novel ω-3 PUFAs; 5,9,12,15-octodecatretraenoic acid, 5,9,12,16-nondecatertraenoic acid, 7,11,14,17-eicosatetraenoic acid, and 5,9,12,15,18-heneicsapententaenoicacid.2EPA,DHAandtheo-3PUFA7,11,14,17-eicosatetraenoicacidhavesimilar structure to arachidonic acid which is the precursor of the inflammatory agents,prostaglandinsandleukotrienes.2

TherehavebeenmanyreportsinhumanstudyaboutP.canaliculuseffects.EPAandDHAhavebeen reported to reduce the levelsof inflammatoryprostaglandins and leukotrienesascompetitive substrates for the cyclooxygenase enzyme (COX) and the lipoxygenase enzyme(LOX). IthasbeenshownthatPCSO-524can inhibitPGE2productionandabolish leukotrieneformation.3,4Anotherstudyshowedanti-histamineactivityofP.canaliculus.5

Inanimalstudy,ithasbeenshownthatPCSO-524hasmildanti-inflammatoryactivitytoreduceswellingininducedarthritis.4OtherstudyreportedthatPCSO-524orglucocorticoidalonedidnothaveclinicalbenefitonreducing inflammationbutcombinedtreatmentof these twosubstancescanreducepawswelling.6However,therehavebeennoreportedtocompareclinicaleffects of PCSO-524 and glucocorticoid in standard model or arthritis.2 The effects of P.canaliculushasalosobeendoneinthecollagentype-II-inducedseverepolyarthritis inratandmouse.TheresultsshowedthatP.canaliculuscanreduceincidenceofarthritisfrom58%fromcontrolgroupto17%inP.canaliculustreatedgroup.7ManystudiesofPCSO-524effectsindogswith arthritis have been reported. Bui and Bierer (2001) reported beneficial effects of P.Canaliculusthatcantreatmentgroupshowedanimprovementofosteoarthritiswhencomparedtountreateddogs.8ThisstudyshowedthatPCSO-524hasbeneficialeffectsonArthritisscores(mobility, pain, swelling and crepitus). Another report showed effectiveness of green lippedmuscleextractindogswithgenerativejointdisease.9Thisstudyshowedthatgreenlippedmusselextracthavebeneficialeffectstoimprovedmusculoskeletaldysfunctionby67%ofthe81lamedogs with no side effects throughout the treatment period. This study also showed clinicalresponse was seen at 56 days after treatment. A comparison between PCSO-524 and non-steroidalanti-inflammatorydrug(carprofen)wasdonein45dogswithosteoarthritis.10Dogsinplacebogroupshowed20-40%improvementinpain,mobilityindexandlocomotionafter8-weektreatment period while dogs treated with PCSO-524 and carprofen showed 67% and 86%improvementinpainVAS.SocarprofenwasshowntobemoreeffectivethanPCSO-524butitsadverse effects, especially with long term use, are of concern. Even though many studiesreported anti-inflammatory effects of PCSO-524 but no clinical study in inflammatory skindiseaseshavebeenreported.

Allergicdermatitisreferstoanyinflammatoryskindiseasescausedbyanytypesofallergy.Itiscausedbyvariousantigensinducingimmunologicalreactions.Theseverityandprogressionof clinical signs depend on etiologic factors. The clinical presentation of hypersensitivity caneitherbeshort-livedorlifelong.Insmallanimalpractice,severaltypesofallergicdiseasesaredocumentedbut themost common in dermatology practice includes atopic dermatitis, foodhypersensitivity,contacthypersentitivity,andfleaallergicdermatitis.11-13Thecharacteristicsofallergicdermatitisarepruritusandsubsequentinflammation.Therefore,allergicdermatitisisagood candidate to study anti-inflammatory effects of PCSO-524 for the treatment of skindiseases.Objectives

1.TostudythebeneficialeffectsofPCSO-524(ANTINOLä)asaneutraceuticaltoimproveskinandcoatcondition

2. To investigate therapeutic effects of PCSO-524 (ANTINOLä) in canine allergic skindiseases

Materialsandmethods

Animals The31dogsincludedinthisstudywereselectedfromdogswithskindiseasespresentingtotheSmallAnimalHospital,FacultyofVeterinaryScience,ChulalongkornUniversity.Alldogshadclinicalhistoriesconsistentwithchronicpruritis,includingsomedegreeoferythema,focalormultifocalalopecia,excoriation,andrecurrentbacterialoryeastinfection.Thedogsremainedundertheirowner’scarethroughoutthestudyperiod.

InclusioncriteriaThedogsunderwentageneralanddermatologicalexaminationconductedbythesame

veterinarian,toidentifyskinlesions.Hematologicalandbiochemicalanalysiswereperformedtoevaluatehealthstatus.Cytologyexaminationandbacterialcultureandsusceptibilitytestwerealso done to identify the presence of yeast and bacterial infection and select appropriatetreatmentforaffecteddogs.Allergicskindiseasewasdefinedusingacriterionofchronicpruritusandinflammationofskinwithotherskindiseasesexcludedbasedonhistory,clinicalsigns,skinscraping,cytologyexamination,hematology,andbloodchemistryprofiles.13

TreatmentprotocolDogsincludedinthisstudywereclassifiedinto3treatmentgroupsbasedonseverityof

dermatologicalsigns;mild,moderateandsevere.Theclassificationwasmadeonthebasisofdegreeofpruritus,distributionandextensionofthelesionsandskinandcoatcondition.14forethicalreasons,therewasnocontrolgroupwithouttreatment.Group1Milddegree: PCSO-524Group2Moderatedegree: PCSO-524+antihistamine(hydroxyzinehydrochloride)Group3Severedegree: PCSO-524+prednisolone

All dogs were treated with appropriate symptomatic therapy to control secondaryinfection in order to reduce other involving factors apart from allergic reaction. This wasprescribedbeforestartoftheexperimentorduringexperimentalperiodifsecondaryinfectionwas diagnosed up on clinical examination and cytology examination. Adverse effects weremonitoredclinicallybyveterinariansandnotedifanyadverseeffectswereseenbyownersorveterinarians.

Doseprescription

• PCSO-524: All the dogs received PCSO-524 of loading dose for 2 weeks followed bymaintenancethroughouttheexperiment.Loadingdosewasprescribedas1capsule/10kgBWtwiceadayandmaintenancedosewas1capsule/10kgBWonceaday.

• Antihistamine(hydroxyzinehydrochloride)2-4mg/kgbid

• Prednisolone:0.5-1mg/kg/d for7days then taperdown to0.25mg/kg/donceadayeveryotherday

• Cephalexin:25-30mg/kg/dbid

MonitoringofthedogsAlldogswerescheduledtorevisitandbeevaluatedat2weeks,4weeks,8weeks,12

weeksand16weeksaftertreatment.Evaluationofclinicalresponsei.e.improvementofclinicalsigns, response to therapy and skin and coat condition were assessed by owners andveterinarians.

VeterinaryevaluationGrossobservationanddermatologicalexaminationweredonebyveterinarianstoassess

effects of the treatment on each visit. Two veterinarian subjectively assessed 4 parameters;erythema, lichenification,excoriationandself-inducedalopeciausingcanineatopicdermatitisextentandseverityindex03(CADESI-03)atweek0,2,4,8,12and16.Thescoresassignedbyveterinarianweresummedtoformaveterinary-assessedindexscore.

OwnerevaluationOwners answered a questionnaire on general history of the dogs including gross

appearanceofthedogs,generalskinandcoatcondition,degreeofpruritus,ageofonsetandprevioustreatmentwererecordedonfirstvisit.Oneachfollowingvisit,observationofownersongeneralskinandcoatconditionanddegreeofpruritus(pruritusvisualanalogscale,PVAS)werenotedtoformanowner-assedindexusedadescriptivescaleof0-10.14

MajoroutcomecriteriaandstatisticalanalysisThemajoroutcomecriterionwastheproportionofdogswithimprovement,stableand

worseningofclinicalsignsperformedateachvisit.Statisticalanalysiswerecarriedoutbyusingthe Statistical Analysis System (SAS) software version 9.0 (SAS Inst. Inc.,Cary, NC, USA).DescriptivestatisticswereobtainedusingMEANSandFREQprocedures.AnalysisofvariancewasappliedusingtheMIXEDandGLMprocedures.Traitsanalyzedwerescoreof improvement inskinandcoatcondition,degreeofpruritusandlesionscores.One-wayANOVAswereusedtoevaluatechangesinCADESI-03andpruritusscoresbetweenthetreatmentgroupsfromweek0to16.Theproportionsofdogsineachtreatmentgroupsthatshowedimprovementatweek0,2, 4, 8, 12 and 16 were assessed. Chi-square test was used to analyze differences in theproportionsofdogsthatshowimprovement.ValueswithP<0.05wereregardedasstatisticallysignificant.

Week02481216

Evaluationbyowner

• Generalcoatandskincondition

• Degreeofpruritus

Evaluationbyveterinarians

• Grossobservation/clinicalsigns

• Hematologyandbloodchemistryprofiles

• Cytologyexamination

• Lesionscore(CADESI-03)

Treatmentprotocol

Loadingdose Maintenancedose

Result

Breeddistribution Therewere31dogsdiagnosedashavingallergicskindiseases.SeveralbreedsincludedinthestudywiththehighestnumberinShihtzu(n=9).Theotherbreedsalsoincludedinthisstudywere Poodle (n=6), Pomeranian (n=6),Mixed breed (n=3),Miniature pincher (n=2),Maltese(n=1),Englishcocker(n=1),Pug(n=1),Bangkaew(n=1)andChihuahua(n=1). Sex Fromtotalof31dogs,thenumbersofmaleandfemaledogwere15(48%)and16(52%).Thenumberofdogsandpercentagewerenotstatisticallydifferent. Comparisonbetween3treatmentgroups

Numberofdogswithimprovementinskinandcoatconditioningroup1,2and3are9of12,3of9and5of10 respectively.Group1showedhigherpercentage (75%)comparedwithgroup2(33.33%)andgroup3(50%).Similarly,numberofdogswithreductioninpruriticscore(PVAS)wasalsohigherinimprovementingroup1(50%)withlesseffectsingroup2(44.44%)and3(40%).Themoreprominenteffectsoflesionscore(CADESI-03)improvementwereobservedwithsignificanthigherpercentageascomparedwiththeothertwogroups.Group1showedtheshortesttimeforimprovementseenwhencomparedwithgroup2and3.Timeat improvementseen inGroup1,2and3were5.33+1.56,7.33+2.70and12+2.09,respectively. Adverseevents One dog in group 3 had diarrhea 2 weeks after treatment. Diarrhea was mild andtransient.Therewasnoevidenceofserioussideeffectsormoderateorseveregastrointestinalevents.Inparticular,nochangesinhematologyandbloodchemistryprofilesindogstreatedwithPCSO-524alone.Somedogs ingroup3 (PCSO-524+prednisolone)had increased liverenzymelevels.

Table1Subjectcharacteristics

Subject

Age(year)

Sex

Breed

Ageofonset

(year)Group1 1

23456789101112

74123889

10.559510

FMMMMFsMMFMMF

PoodleShihtzuShihtzuChihuahuaShihtzuShihtzuPomeranianPomeranianPomeranianMaltesePomeranianShihtzu

4-71-31-31-34-74-74-74-74-74-71-34-7

Group2 123456789

54887.5144919

FFFsMFMFMM

ShihtzuPugMixedPomeranianPooleBangkaewMiniaturepincherShihtzuMiniaturepincher

1-31-3<14-74-71-3<14-7>7

Group3 12345678910

810881312613149

MFMFFFFMFF

MixedPoodlePomeranianMixedPoodlePoodleShihtzuPoodleEnglishcockerShihtzu

4-71-3>74-74-74-74-7>7>74-7

M=male;F=female;Fs=spayedfemale

Figure 1 Diagram shows breed distribution of 31 dogs included in the study; Shih tzu (n=9),Poodle (n=6), Pomeranian (n=6),Mixedbreed (n=3),Miniaturepincher (n=2),Maltese (n=1),Englishcocker(n=1),Pug(n=1),Bangkaew(n=1)andChihuahua(n=1).

29%

20% 19%

10%

7%

3% 3%

3% 3% 3%

Breed

Shihtzu Poodle Pomeranian MixedbreedMiniaturepincher Maltese Englishcocker PugBangkaew Chihuahua

Figure2Diagramshowssexof31dogsdiagnosedashavingallergicskindiseaseinthisstudy;male(n=15)andfemale(n=16).

48%

52%

Sex

Male

Female

Figure3Ageofonsetnotedinallsubjects(31dogs)wereclassifiedinto4ageranges;<1years(n=2),1-3years(n=8),4-7years(n=17)and>7years(n=4).

6%

26%

55%

13%

Ageofonset

<1year

1-3years

4-7years

>7years

Figure4Clinicalresponse(skin&coatconditionanddegreeofpruritus)inallergicdogstreatedwithPCSO-524alone(n=12).

0

1

2

3

4

5

6

7

8

9

Improved Stable Worseskin&coatcondition 9 3 0

Degreeofpruritus 6 5 1

Num

bero

fdog

sGroup1:PCSO-524

Figure5Clinicalresponse(skin&coatconditionanddegreeofpruritus)inallergicdogstreatedwithcombinationtherapyofPCSO-524andantihistamine(n=9).

0

1

2

3

4

5

6

Improved Stable Worseskin&coatcondition 3 6 0

Degreeofpruritus 3 6 0

Num

bero

fdog

sGroup2:PCSO-524 +Antihistamine

Figure6Clinicalresponse(skin&coatconditionanddegreeofpruritus)inallergicdogstreatedwithcombinationtherapyofPCSO-524andprednisolone(n=10).

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Improved Stable Worseskin&coatcondition 5 4 1

Degreeofpruritus 4 5 1

Num

bero

fdog

sGroup3:PCSO-524 +Prednisolone

Figure7Canineatopicdermatitisextensionandseverityindex(CADESI-03)scoreofallergicdogstreatedwithPCSO-524alone(n=12),combinationtherapyofPCSO-524andantihistamine(n=9)andPCSO-524andprednisolone(n=10).

0

2

4

6

8

10

12

Improved Stable Worse

PCSO-524 11 1 0

PCSO-524+AH 3 5 1

PCSO-524+Pred 4 4 2

Num

bero

fdog

s

CADESI-03score

Table2Percentageofimproveddogsandtimeatimprovementseenforevaluatedvariables,per

group

Numberofdogswithimprovement(%)

Timeatimprovementseen

(weeks)

Group1PCSO-524(n=12)

Group2

PCSO-524+AH(n=9)

Group3

PCSO-524+Pred(n=10)

Group1PCSO-524(n=12)

Group2

PCSO-524+AH(n=9)

Group3

PCSO-524+Pred(n=10)

Skin&coatcondition

9/12(75%)

3/9

(33.33%)

5/10b,c(50%)

5.33+1.56c

7.33+2.70

12+2.09a

Degreeofpruritus(PVAS)

6/12(50%)

4/9

(44.44%)

4/10(40%)

4+0.95

4.67+1.40

5+1.17

Lesionscore(CADESI-03)

11/12b,c

(91.67%)

3/9a

(33.33%)

4/10a

(40%)

4.55+1.28c

4.67+2.45

10.50+2.12a

asignificantdifferencebetweenwhencomparewithgroup1bsignificantdifferencebetweenwhencomparewithgroup2csignificantdifferencebetweenwhencomparewithgroup3

Table3Acomparisonof3parameter(mean+SEM);skinandcoatcondition,degreeofpruritus

andlesionscorein3treatmentgroupsatweek0,2,4,8,12and16

Week

Mean+SEM

0 2 4 8 12 16

Skin&coa

tcond

ition

Group1PCSO-524(n=12)Group2PCSO-524+AH(n=9)Group3PCSO-524+Pred(n=10)

6+0.67

7+0.77

6+0.73

5.7+0.61

6.13+0.68

6.00+0.68

3.91+0.64

5.22+0.74

5.1+0.71

2.67+0.56c

4.29+0.73

5.30+0.61a

2.89+0.66b

5.40+0.88a

4.50+0.69

1.88+0.63

5.50+0.72

4+0.67

Degreeof

pruritu

s(PVA

S)

Group1PCSO-524(n=12)Group2PCSO-524+AH(n=9)Group3PCSO-524+Pred(n=10)

5+0.72

6.89+0.83

6.1+0.79

4.4+0.59

6.38+0.67

5.75+0.67

3.5+0.63

5.33+0.73

5.1+0.69

2.33+0.6b,c

5.00+0.85a

4.9+0.66a

2+0.58b,c

5.6+0.73a

5.13+0.58a

2+0.78b

4.67+0.90a

3.85+0.84

Lesion

scores

(CAD

ESI-0

3)

Group1PCSO-524(n=12)Group2PCSO-524+AH(n=9)Group3PCSO-524+Pred(n=10)

250+52.51

254+60.63

359+57.51

196+53.17

326.67+68.64

255+59.44

89.75+36.77b,c

211.56+42.46a

211.30+40.28a

94.22+26.75b,c

124.57+30.34a

231.30+25.38a

75.89+32.18b,c

206.14+36.49a

232.50+34.13a

37.25+34.31b,c

177.57+36.67a

240.71+36.67a

asignificantdifferencebetweenwhencomparewithgroup1bsignificantdifferencebetweenwhencomparewithgroup2csignificantdifferencebetweenwhencomparewithgroup3

Discussion

Allergicdermatitisisdefinedasanyinflammatoryskindiseasescausedbyanytypesofallergy.Itcanbecausedbyavarietyofallergeninducingimmunologicalreactionofanimals.Inthisstudy,allergicdogsreferstodogswithclinicalsignsofatopicdermatitisandfoodallergyasother diseaseswere ruled out by history, physical examination, cytological examination andbloodtests.Thediagnosisfollowed2010clinicalpracticeguidelinesfromtheinternationaltaskforceoncanineatopicdermatitis.14

Eicosapentaenoicacid(EPAanddocosahexaenoicacid(DHA)arethemainactiveportionsof fish oil. their anti-inflammatory effects are possibly due to inhibition of eicosanoids andcytokinesproduction.15AnextractfromNewZealandgreenlippedmussel(Pernacanaliculus),PCSO-524 also contains polyunsaturated fatty acid (PUFAs); eicosapentaenoic acid (EPA) anddocosahexaenoicacid(DHA)includingnovelω-3PUFAs(7,11,14,17-eicosatetraenoicacid).1,2Manystudiesshowedanti-inflammatoryactivityofthesePUFAsandalsoanti-histamineactivity.4Fishoilhasbeenshown tohavehigher inhibitoryeffectsonCOX-1andCOX-2productionascompared to PCSO-524. However, hydrolysed form of PCSO-524 showed similar inhibitoryactivity on COX enzyzmes production.16 PCSO-524 has beneficial effects on reducinginflammation in animal study. It hasbeen shown to reduce swelling in arthritis.3 In addition,PCSO-524hasalsobeenshowntohaveanti-inflammatoryeffectswhenusedincombinationwithglucocorticoid.6 The anti-inflammatory effects was absent when animals were treated withPCSO-524orglucocorticoidalone.5AnotherstudyshowedtheeffectivenessofcombinationofPCSO-524,EPAandDHAinthetreatmentofrheumatoidarthritis.17However,thestudyofPCSO-524effectsonskindiseaseshasneverbeeninvestigated.

From total of 31 dogs, the proportion ofmale and female dogs diagnosed of havingallergicdermatitiswasnotsignificant.Therewere15maledogs(48%)and16femaledogs(52%).Theresultscorrelatedwithpreviousstudythatallergicdermatitishasnogenderpredisposition.14

Dogsingroup1,treatedwithPCSO-524only,showedsignificantimprovementofskinandcoatconditioncomparedwithgroup2and3.Thiswasevaluatedbytheowners,withscorerangefrom0to10basedonskinandcoatgrossappearance.Similarly,numberofdogswithreductioninpruriticscore(PVAS)wasalsohigheringroup1(50%)withlesseffectsingroup2(44.44%)and3 (40%).Theresults indicatedthatdogswithmilddegreeofallergicdermatitisseemtohavebenefitfromPCSO-524byimprovingskinandcoatconditionandreducingdegreeofpruritus.Dogswithmoderatedegree(group2)andseveredegreeofallergicdermatitis(group3)didnothavebeneficialeffectsbyownerevaluationresults.Theimprovementobservedingroup1waspossibly due to anti-inflammatory and anti-histamine activity of PCSO-524.2-4 Failure oftreatmentsingroup2and3couldbeacombinationofdegreeofseverity,presenceofrecurrentbacterialandyeastinfectionandchronicityofthedisease.Olivryetal.(2010)14suggestedthatclinical signsofallergicdogs,e.g.urticariaorpruritus,mightbepresentwithorwithout skinlesion.ItwasreasonableforDogsingroup1withmilddegreeofallergytohaveimprovedclinicalappearance.Dogsingroup2and3withmoderateandseveredegreeofallergymighthavehigherdegreeof inflammationorhypersenstitivity, inwhich, combination therapywas incapableoftreatingthedogs.Allallergicdogsinthisstudymightrepresentatopicdermatitisorfoodallergy

orboth.Therelationshipbetweenatopicdermatitisandfoodallergyhasbeencontroversialbutitissuggestedfoodcomponentsmightcausetheflareofatopicdermatitis.Thisincludesurticariaorprurituswithorwithoutskinlesion(9ofguideline).Therefore,dietaryfactorscouldbeonefactoroftreatmentfailure.Moreover,dogsingroup2and3withhigherdegreeofseverityhadmorefrequentrecurrenceofbacterialoryeastinfection.Thisisamajorflarefactorsinallergicdogs.14

EvaluationbyveterinarianalsoshowedconsistentresultsofPCSO-524benefitonreductionin lesionscore (CADESI-03).The improvementof lesionsscoreswasobservedwithsignificanthigherpercentageingroup1comparedwiththeothertwogroups.Thetimeat improvementseenwasalsoobservedto investigatepropertreatmentperiodforeffectiveresults.Averagetimetohaveimprovementingroup1,2and3were5.33+1.56,7.33+2.70and12+2.09weeks,respectively.Theresultswereconsistentwithpreviousstudy.18Conclusion PCSO-524mighthavebeneficialeffectsforthetreatmentofcanineallergicskindisease.Theimprovementinskinandcoatcondition,degreeofpruritusandlesionscoreswasobservedindogswithmilddegreeofallergicskindiseasebutnotindogswithmoderateorsevereallergicdermatitis.Othercontributingfactors,i.e.secondarybacterialoryeastinfection,skinandcoathygieneandcausingallergensshouldbeofconcernandwell-controlledinmoreseverecases.References

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