PILOTSTUDYOFUPTAKEOFLONGCHAINFATTYACIDSFROMALGALOILCONSUMEDINGELCAPAND

LIPOSOMALBEVERAGEFORM

OVERVIEWOFSTUDYRESULTS

JULY9,2019

OVERVIEWOFSTUDYRESULTS

PILOTSTUDYOFUPTAKEOFLONGCHAINFATTYACIDSFROMALGALOILCONSUMEDINGELCAPANDLIPOSOMALBEVERAGEFORM

Background:

Longchainomega-3fattyacids(EPAandDHA)asnaturaltriglyceridesfromoilyfishareessentiallycompletelybio-available,estimatedat90–95%.Thefatemulsiondropletsfromthisformareaboutonemicronindiameter

Dietarysupplementformscomeasnaturaloiltriglycerideorinare-esterifiedformastheethylesterorastriglyceridesenrichedinEPAandDHA.Severalfactorsaffecttheavailabilityoftheomega-3fromthesesupplements,includingthespecificmolecularform,whetherornotconsumedwithfood(andthefood’sfatcontent),andthemannerandextentofdispersionoftheomegainthesupplementmatrix.Inaddition,thereissignificantvariabilitybetweenindividuals.

StudyDesignforOmega-3TriglyceridesfromAlgalOil(uncontrolled)

HMRI(Pasadena,Dr.MichaelHarringtonPI)recruited31studyparticipantsonconsent#27197foralongchainfattyacidplasmaPKstudy.Allparticipantswerehealthynon-obeseadultswhowerenottakingomega-3supplementsorprescriptionmedications.Withinformedconsent,individualsparticipatedinan8-hourstudyandwererandomlyallocatedtooneofthetreatmentgroups,eachwiththesameoverallquantityofDHA/EPA.Onegroupreceivedaliposomepreparation,theothercommercialGELCAPS.Thetwogroupswerefoundtobematchedbyage,sex,andBMI.Theycommencedthestudyafteranovernight(12h)fastat8am.Afterabaselineblooddraw,theywereallgivenalowfatbreakfastat9am,followedbylunchatnoon.

Thefollowingprocedurewasemployed:

• Intravenouslineinserted• 2mLofvenousbloodatbaseline• Drinkliposomebeverage(nof15)orswallowgelcap(nof15).• Combined(EPA+DHA)dose900mgineachcase• ThealgaloilinbothformswasDSM“Life’sOmega45”• Hourlybloodcollectionsof2mLto8hours• Separateandaliquotplasmasamples,into100ulcontainers,andfreeze(-80C)• Assayfattyacidsbyextraction,derivatizationandGCMS

SummaryofResults

Figures1and2showallthedataforincreaseabovebaselineforEPAandDHAconcentrationsinplasmainug/ml.Theleftportionofeachchartshowstheliposomeformdataandtherightportiontheresultsforgelcaps.Thearrowineachpanelshowsthedivisionbetweengroups.Eachparticipantisrepresentedbynineconsecutivedatapoints(baselineto8hours)startingwithafattyacidconcentrationchangeof

zero;dataareshownconsecutivelywitheach“hump”inthecharteffectivelyshowingresultsforoneindividual.Ofthe31individuals,datawereanalyzedfor28,fourteenineachgroup.Baselineplasmasampleswereinadvertentlypooledforthreesubjectsandplasmalevelchangesrelativetobaselinecouldthusnotbeevaluated.Thechartsshowthesesubjectsasbaseline(zero)valuesforalltimepoints.Astrikinglyanomaloushighearlyvalue(bothDHAandEPA)wasobserved,andconfirmed,foronegelcapsubject.Insomeanalysesthisvaluewasexcluded.

AnalysisofpercentchangeoverbaselineforthetotalofEPA+DHAisshowninFigure3forthegelcapandliposomegroups.Dataareshownasmeanwithstandarderrorastheerrorbar.Forcomparison,peak(Cmax)dataforLovaza(purifiedethylesterformfromfishoil),dosedat3.6gEPA+DHA,isshownattheappropriatetimepoint(6hours)Harrisetal(2103),asisacalculatedfour-foldlowervaluefor0.9gdose.Withnumerouscaveats,adosecorrected(arithmetic)crossstudycomparisonsuggeststhealgaloilgivesbetteruptake(Cmaxratio)byaboutafactorof3ingelcapsandbetweenfourandfivefortheliposomalpresentation.

Forabsolute(ug/ml)increasesinEPAandDHAinplasmatheaverageddatafortheindividualsineachofthetwogroupsareshowninFigures4and5(meanwithSEMerrorbars).Theliposomalformshowsabouta35%enhancementforEPAandalmost80%enhancementforDHA,whetherassessedbyCmaxorAUCto8hours.

Evenintheaverageddatacharts,itisclearthatthereisamorerapidappearanceoftheEPAandDHAinplasmafromtheliposomalpresentation.ForindividualsubjectsthelinearregressionanalysisofinitialslopeforEPAandDHArisetoCmaxinplasmaareshowninFigures6and7,withaveragesshownasthedashedline.Allratesareshownasug/ml/hour.ForEPAandDHA,respectively,therateofappearanceis1.4andalmost3timesgreater.Inthisanalysis,thenon-responderswereexcludedfromeachgroup–theseindividualsarereadilyapparentinFigures1and2.Notrevealedbythesegraphsisthelagtimeseenforsomeofthegelcaprecipientsforwhomanysignificantriseinplasmadoesnotbeginuntilhour2.Thislagis,however,visibleinplotsofthepercentchangeoverbaselinefor(EPA+DHA)forindividuals.TheseareshowninFigures8and9thatincludeexpandedplotsfortheinitialuptakeperiod.Changeoverbaselineattwohoursislessthan10%for6of14GelCapconsumers(andlessthan20%for10ofthe14).Incontrastfortheliposomegrouponlytwoindividualshadalessthan10%risebytwohours(andthreelessthan20%atthesametimepoint).Whilethe10%and20%thresholdsarearbitrary(butrelatetotheCVofabout10%fortheassaymethod),thefractional“delayed-response”toomega-3dosingisaquantitativeindicatorofthecleardifferenceinlagseenforthedifferentdosageforms.

Overall,thereisnocorrelationofuptakewithage,sex,weightBMIetc.Whilethisstudyonlyhas14individualsineachgroup,thedatahintatadependenceofpeakplasmalevelandAUC(8hrs)onthebaselinetotalfattyacidlevelinindividualsconsumingtheliposomalform.Suchadependenceappearsessentiallyabsentforgelcapconsumers.Figures10-13.Thismayreflectanatural(baseline)digestiveefficiencyfortriglyceridesthatcorrelateswithhigherbaselinefattyacidlevels.Thatefficiencyispotentiallyimmediatelyaccessibleforprocessingthehighlydigestibleliposomepresentationofomega-3triglycerides(inthenaturalmoleculardispersioninphospholipids).Itdoesnotfactorintodigestionof

thegelcapsforwhichtriglyceridesarriveintheduodenuminmuchlargerfatemulsiondropletsinchyme.

AsshowninFigure14,baselinetotalfattyacidlevelswererandomlydistributedbetweenthetwogroups

Context

Thereisverylimitedinformationaboutthequantitativeextentofuptakeforomega-3supplements,i.e.fractionofthedosegiventhatisabsorbed.ThekeystudiesarebyLawsonandHughes1988(a)whousedlinseedoilasaninternalstandardfor“naturaluptake”.Thoseauthors’workincludedassessmentoftheeffectofahighfatmeal(44gfat)consumedwiththeomega-3productLawsonandHughes1988(b).DatafromthosestudiesarereferencedtotheCmaxforlinseedoilas100%,butthenaturaluptakeofdietaryfatisknowntobebetween90%and95%.ThedatafromLawsonandHughesarepresentedinFigure15below,correctedto92.5%uptakefordietarytriglycerides(illustratedhereforsalmon).TheadditionoffatapproximatelytriplesuptakeforethylesterformsofEPAandDHA.ForthetriglycerideformhigheruptakeforbothEPAandDHAisseeninthelowfatcase.However,foradditionoffattheEPAuptakeapproachesthatseenfordietaryfatswhereastheDHAuptakeislargelyunchanged.Theadditionoffatispresumedtostimulatethedeploymentoflipases,bilesaltsandphospholipidstoboostdigestion.Ethylestersareknowntobelessreadilycleavedthantriglyceridesbypancreaticlipasesandthusshowahigherresponsetothisdigestiveboost.Forthetriglycerides,susceptibilitytoenzymatichydrolysishasbeenpostulatedtorelatetodifferentialpositionaldistributionofEPAandDHAonglycerol,aswellasapotentialeffectoftheadditionaldoublebondclosetotheesterlinkageforDHA.

Thereareotherstudies,someathigherdoses,thatsuggesttheethylestersandtriglyceridesgivesimilarplasmaincreasesfortheselongchainfattyacids.TheassumptionweuseisthatinthemoderatedoserangeweandLawsonandHugheshavestudied(lessthan1.7gramEPA+DHA)thetriglyceridesprovideaboutthreetimestheplasmariseasethylesters.ApplyingtheratioforindividualCmax(EPAandDHAabsoluteincreaseoverbaseline)forliposomes(vsgelcaps)fromFigures4and5providestheprojectedextentofliposomaluptakeforboththesefattyacidsasalmostthatofdietarytriglycerides.ThealternativecalculationusingratiosofpercentincreaseoverbaselineofcombinedEPA+DHAtotheethylesterdataofHarrisetal(2013)(Figure3)givesconsistentEPA+DHAcompositeresultsforgelcapsandliposomesshownbythegreenverticalarrowsinFigure15

Conclusion

Itisnotnecessarytotakehighpotencytriglycerideformgelcapswith300+caloriesoffat(a44gfatmeal

=1.25ozbutter!)fornatural,dietarytriglyceride,uptakeofEPA.Further,theliposomescanimprove

DHAuptakeinawaythatevenahighfatmealcannot.Theliposomalbeveragecanprovideessentially

naturaluptakelevelsofbothlongchainomega-3fattyacidswitharelativelyrapidrisetopeakplasma

levelsafterconsumption.

Figure1:ChangeinEPAconcentrationinplasma–alldata

Figure2:ChangeinDHAconcentrationinplasm

a–alldata

Figure3:Percentchangein(EPA+DHA)inplasm

a

2.9x

4.7x

Figure4:AbsoluteincreaseinEPAinplasma

Figure5:AbsoluteincreaseinDHAinplasm

a

Figure6:EPArateofincreasetoCmaxinplasm

a

Liposom

es:GelCaps1.44x

Figure7:DHArateofincreasetoCm

axinplasmaAllratesareshow

nasug/ml/hour.

Liposomes:GelCaps2.95x

Figure8(a):RateofuptakeGelCap(EPA+DHA)percentincreaseinplasm

aoverbaseline

Figure8(b):expanded-initial3hoursAtt=2hours:6/14show

<10%changeoverbaseline

10/14show<20%

changeoverbaseline

Figure9(a):RateofuptakeLiposomes(EPA+DH

A)forindividuals.

Figure9(b):expandedAtt=2hours:2/14show

<10%changeoverbaseline

3/14show<20%

changeoverbaseline

Figure10:GelCapsAUCvsbaselinetotalfattyacid

Figure11:GelCapsCmaxvsbaselinetotalfattyacid

.

Figure12:LiposomesAUCvsbaselinetotalfattyacid

Figure13:LiposomesCm

axvsbaselinetotalfattyacid.,

Figure14:Baselinetotalfattyaciddistributionbygroup

Figure15:Bioavailability.,