Exposure Characteristics of the Analogous β-Carboline Alkaloids Harmaline and Harmine Based

19 Pages·2017·3.48 MB·English

by  Changhong Wang

ORIGINALRESEARCH
published:21August2017
doi:10.3389/fphar.2017.00541
Exposure Characteristics of the
Analogous β-Carboline Alkaloids
Harmaline and Harmine Based on the
Efflux Transporter of Multidrug
Resistance Protein 2
ShupingLi1,YunpengZhang1,GangDeng1,YuwenWang1,ShenglanQi1,
XuemeiCheng1,2,YuemingMa3,YanXie4andChanghongWang1,2*
1TheMOEKeyLaboratoryforStandardizationofChineseMedicines,TheSATCMKeyLaboratoryforNewResourcesand
QualityEvaluationofChinese,InstituteofChineseMateriaMedica,ShanghaiUniversityofTraditionalChineseMedicine,
Shanghai,China,2ShanghaiR&DCentreforStandardizationofChineseMedicines,Shanghai,China,3Laboratoryof
Pharmacokinetics,ShanghaiUniversityofTraditionalChineseMedicine,Shanghai,China,4ResearchCenterforHealthand
Nutrition,ShanghaiUniversityofTraditionalChineseMedicine,Shanghai,China
Harmaline and harmine occur naturally in plants and are distributed endogenously
in human and animal tissues. The two β-carboline alkaloids possess potential for
treatingAlzheimer’sdisease,Parkinson’sdisease,depressionandothercentralnervous
Editedby:
YurongLai, system diseases. However, studies have showed that the two compounds have
Gilead,UnitedStates similar structures but with quite different bioavailability. The aim of this study was to
Reviewedby: elucidate the exposure difference and characterize the in vitro transport, metabolism,
CesareIndiveri,
and pharmacokinetic properties of harmaline and harmine. The results showed that
UniversityofCalabria,Italy
AndreiAdrianTica, the harmaline and harmine transport across the Caco-2 and MDCK cell monolayers
UniversityofMedicineandPharmacy
was varied as the time, concentration, pH and temperature changed. The absorption
ofCraiova,Romania
of harmaline and harmine was significantly decreased when ES (OATPs inhibitor), TEA
*Correspondence:
ChanghongWang (OCTs/OCTNs substrate),NaN3 (adenosine triphosphate inhibitor),orsodium vanadate
[email protected] (ATPase Na+/K+-dependent inhibitor) was added. However, when given MK571 and
probenecid (the typical MRP2 inhibitor), the P of harmine was increased (1.62-
Specialtysection: appAB
Thisarticlewassubmittedto and 1.27-folds), and the efflux ratio was decreased from 1.59 to 0.98 and from 1.59
DrugMetabolismandTransport, to 1.19, respectively. In addition, the uptake ratio of harmine at 1 µM was >2.65 in
asectionofthejournal
the membrane vesicles expressing human MRP2. Furthermore, harmine could slightly
FrontiersinPharmacology
up-regulate the expression of MRP2, which implying harmine might be the substrate
Received:15June2017
Accepted:03August2017 of MRP2. Particularly, the CL -value for harmine was ∼1.49-folds greater than that
int
Published:21August2017
of harmaline in human liver microsomes. It was worth noting that the F-value of
Citation:
harmine was increased 1.96-folds after harmine co-administration with probenecid.
LiS,ZhangY,DengG,WangY,QiS,
ChengX,MaY,XieYandWangC To summarize, comprehensive analysis indicated that harmaline and harmine were
(2017)ExposureCharacteristicsofthe absorbed by transcellular passive diffusion and a pH- and Na+-dependent mechanism
Analogousβ-CarbolineAlkaloids
might be mediated by OATPs and OCTs/OCTNs. MRP2 but MDR1 or BCRP might be
HarmalineandHarmineBasedonthe
EffluxTransporterofMultidrug involvedinthetransportofharmine.Furthermore,harminewasmoreunstableandeasily
ResistanceProtein2.
metabolizedthanharmaline.Allthesefindingssuggestedthatharminenotonlyappears
Front.Pharmacol.8:541.
doi:10.3389/fphar.2017.00541 be an MRP2 substrate, but also possesses weak metabolic stability, and eventually
FrontiersinPharmacology|www.frontiersin.org 1 August2017|Volume8|Article541
Lietal. TransportofHarmalineandHarmine
leads to a low oral bioavailability. Taken together, the elucidated absorption, transport,
metabolismaswellaspharmacokineticcharacteristicsofharmalineandharmineprovide
useful information for designing delivery systems, pharmacological applications and
avoidingdrug-druginteractions.
Keywords:β-carbolinealkaloid,transport,metabolism,pharmacokinetic,MRP2
INTRODUCTION particularly harmine (Li et al., 2016). The drug concentration
(250–500 µM) may exceed the safe dosage in the Caco-2 cells,
The analogous β-carboline alkaloids, harmaline, and harmine whichwillresultindamagetocellsandthenchangetheactivity
(Figure S1), dominant pharmacological ingredients of plants of transporters or the permeability of the cell monolayers.
Peganum harmala L., Passiflora incarnata L., and Banisteriopsis Additionally,itwasfarfromtheillustrationoftheirdifferences
caapi (Spruce ex Griseb.). Morton, are ubiquitously available in the oral bioavailability based on the above results. Hence,
in a variety of medicinal plants (Khan et al., 2013; Ingale a systematic and rigorous study on the transport mechanism
and Kasture, 2014; Stankovic´ et al., 2015; Li et al., 2017). of these alkaloids should be conducted to clarify the exposure
Harmaline and harmine are endogenously produced in human characteristics.
and animal tissues as a low molecular weight product of Cell-based assays for evaluation of permeability can glean
secondary metabolism (Li et al., 2016). They could affect the insight into potential issues with intestinal transport. Several
contentofneurotransmittersbystronginhibitionofmonoamine cell culture models for determination of the in vitro intestinal
oxidase(Jiangetal.,2015),acetylcholinesterase(Liuetal.,2014), permeability have been developed and currently gained great
andmyeloperoxidase(Bensalemetal.,2014).Furthermore,they popularity.Amongstvariousmodels,Caco-2andMadin-Darby
possess capability to bind to imidazoline, serotonin, dopamine, caninekidney(MDCK)cellshavebeenextensivelyusedasthein
opiate,andbenzodiazepinereceptors,whichcausephysiological, vitromodelforevaluatingdrugintestinaltransportmechanism
biochemical, and behavioral changes in human and animals (Volpe, 2011; Chen et al., 2014; Shen et al., 2015). In the late
(Wu et al., 2009; Zhao et al., 2011, 2012). They have attracted 1990s,transfectedMDCKcelllineshavebeenreportedtoexpress
muchattentioninrelationtotheirbiologicalactivities,andtheir highlevelsofmultidrugresistancegene1(MDR1),breastcancer
proposedusefortreatmentofneurologicaldisordersonthebasis resistance protein (BCRP) and multidrug resistance-associated
of activity as inhibitors of acetylcholinesterase in vitro (Khan protein isoform 2 (MRP2) on the apical side of the polarized
etal.,2013;Zhaoetal.,2013;Lietal.,2017).Sincethebiological cell monolayer. Furthermore, inverted membrane vesicles with
efficacydependssignificantlyontheoralbioavailabilityofdrugs, over-expressed transporters allow a way to study the transport
it is important to understand the molecular properties such mechanismofdrugs.Thevesicle-basedassaypossessesahigher
as metabolic stability and cell permeability that limit the oral throughputandconveniencecomparedtowholecellassayssince
bioavailability. theycanbepreparedinlargebatchesandcryopreservedforlater
In view of the pharmacokinetic study of harmaline and use.Theinteractiondatageneratedinthevesiclesystemcanbe
harmine, it was interesting to find that significant difference usedtopredicttheinvivotransporter-mediateddispositionand
presented in the absolute bioavailability (F) of the two analogs possible drug-drug interactions (Deng et al., 2016). Therefore,
among different experimental animals, such as rats, dogs and thecombineduseoftheCaco-2,MDCKandtransfectedMDCK
mice.TheF-valueswere63.22and24.99%forharmaline,while cells,aswellasspecificinvertedmembranevesicles,couldyield
only 4.96% and 5.33% for harmine after oral administration clear advantages in the transport study of these β-carboline
in rats and dogs with the total alkaloid extracts (140 mg/kg) alkaloids.
from P. harmala (Zhang, 2013; Shi et al., 2014). According to The purpose of this study was to comprehensively clarify
another study of Guan et al. (2001), the F-value of harmine the exposure difference and characterize the in vitro transport,
was 3% in rats by gastric gavage (20 mg/kg). Following an metabolism, and pharmacokinetic properties of the analogous
intraperitoneallyinjection,theF-valuesofharmalinewere73.7, harmaline and harmine. In the present study, the bidirectional
98.2, 68.6, 60.0% in wild-type and CYP2D6-humanized mice transportofharmalineandharmineacrosstheCaco-2,MDCK,
witheither5or15mg/kg(Wuetal.,2009).Generallyspeaking, MDCK-MRP2 cells in the absence and presence of influx
theoralbioavailabilityofharminewasmuchlowerthanthatof and efflux transporter inhibitors or substrates was investigated.
harmalineindifferentanimals.Furthermore,Khanetal.(2004) Considering the structural features of the two alkaloids and
reported that the intestinal transport of β-carboline alkaloids the transporter expression of the Caco-2 and MDCK cells, the
(harmaline, harmine, harmalol, harmol, and harmane) in the experimentsweretentativelyconductedontheSLCinflux(OATs,
concentration range of 250–500 µM using a human colon OATPs, OCTs, OCTNs, MCTs, SGLT1, PEPT1, etc.) and ABC
carcinoma (Caco-2) cell monolayers. It revealed that the efflux efflux transporters (MDR1, BCRP, MRP2; van Montfoort et al.,
ratios (ER) of harmaline and harmine were <1.0, suggesting 2003; Koepsell and Endou, 2004; Seithel et al., 2006; Koepsell
a passive diffusion mechanism for their transport across the et al., 2007; Volpe, 2011; Koepsell, 2013; Chen et al., 2014;
Caco-2monolayer.However,compositeevidencesindicatethat Arimany-Nardi et al., 2015; Shen et al., 2015). Remarkably,
thesealkaloidspossessastrongcytotoxicitytovariouscelllines, many members are present in the groups of OATs (OAT1,
FrontiersinPharmacology|www.frontiersin.org 2 August2017|Volume8|Article541
Lietal. TransportofHarmalineandHarmine
OAT2, OAT3, OAT4, and OAT5), OATPs (OATP-A, OATP-B, standard environmental conditions (12h light-dark cycles) and
OATP-C, OATP-D, OATP-E, etc.), OCTs (OCT1, OCT2, and givenfreeaccesstorodentchowandtapwaterpriortothestudy.
OCT3), OCTNs (OCTN1, OCTN2, and OCTN3), and MCTs Ratswerefastedfor12handprovidedfreeaccesstowaterpriorto
(MCT1,MCT2,MCT3,MCT4,MCT5,etc.;vanMontfoortetal., theexperiments.Allanimal-useprocedureswereinaccordance
2003;KoepsellandEndou,2004;Shenetal.,2015).Evaluations with the regulations for animal experimentation issued by the
were also carried out using inverted membrane vesicles to State Committee of Science and Technology of the People’s
determine the effect of efflux transporters on the alkaloids Republic of China on 14 November 1988 and approved by the
transport. Moreover, in vitro metabolic stability and in vivo AnimalEthicsCommitteeofShanghaiUniversityofTraditional
pharmacokineticpropertiesofthetwoanalogswereunderwayto Chinese Medicine (No. SUTCM-2011-1107; Approval date: 10
characterize. The results of the current study will be helpful in November,2011).
evaluating the pharmacology and toxicology of the β-carboline
alkaloids in further development, and could provide significant Cell Culture
informationforclinicalpractice. Caco-2, MDCK and MDCK-MRP2 cells were donated by
Prof. Yan Xie and Yueming Ma from Shanghai University of
MATERIALS AND METHODS Traditional Chinese Medicine. The cells were grown in culture
dishes(Corning(cid:13)R Costar,Cambridge,MA,USA)usingDMEM
Materials
supplemented with 10% FBS, 1% NEAA and 1% penicillin-
Harmaline, harmine, tacrine hydrochloride (internal standard, streptomycin solution. The cells were cultured in 5% CO and
2
IS), verapamil hydrochloride, quinidine, Ko143, apigenin, 95% O with 90% relative humidity at 37◦C. The medium was
2
MK571, probenecid, sodium vanadate, cimetidine, estrone-3- replaced every other day during incubation. The cells were
sulfate (ES), tetraethylamine (TEA), phloretin, sodium azide passaged every 3–4 days between 70 and 80% confluence at
(NaN3),phloridzin,glycylsarcosine(Gly-Sar),dimethylsulfoxide a 1:5 split ratio using 0.25% trypsin-EDTA. For the transport
(DMSO),ethylenediaminetetraaceticacid(EDTA),andallother experiments, the cells from passages between 30 and 45 were
chemicals used were purchased from Sigma Aldrich Co., Ltd seededat1×105cells/cm2ontopermeablepolycarbonateinserts
(St. Louis, MO, USA). Dulbecco’s modified eagle’s medium (0.45 µm pore size, seeding surface of 0.6 cm2, Millipore, MA,
(DMEM),phosphatebufferedsaline(PBS),Hanks’balancedsalt USA) in 24-well plastic plates. The media in the culture plates
solution(HBSS)withcalciumchlorideandmagnesiumchloride, were changed every other day for the first week after seeding
HBSS without calcium chloride and magnesium chloride, heat and were replaced daily afterward. The integrity of the cell
inactivated fetal bovine serum (FBS), penicillin-streptomycin monolayersandtightjunctions(TJ)weretestedandconfirmed
solution, non-essential amino acids (NEAA), 0.25% trypsin- bymeasuringtransepithelialelectricalresistance(TEER)witha
EDTA, and other reagents were purchased from Gibco Lab Millicell(cid:13)R ERS-2electrode.Thecellswereusedforthetransport
(Grand Island, NY, USA). The cell counting kit-8 (CCK-8), experiments21–28(Caco-2)or5–7(MDCKandMDCK-MRP2)
BCA protein quantification kit, PBST, RIPA lysis buffer, bovine daysafterseeding,andonlymonolayerswithaTEER-valueabove
serum albumin (BSA), 30% acrylic amide, 10% SDS, TEMED, 420(cid:127)cm2wereusedduringthestudies.
and sample loading buffer (4X) were purchased from YEASEN
BiotechnologyCo.,Ltd(Shanghai,China).Proteaseinhibitorand Cytotoxicity Assay
phospholipase inhibitor were purchased from Roche Applied
The cytotoxicity of harmaline and harmine was measured by
Science (Foster City, CA, USA). PVDF membrane, Millicell(cid:13)R
CCK-8assay.Inbrief,thecellswereseededon96-wellplatesat
ERS-2, 24-well cell culture standing inserts, and ImmobilonTM 1×105 cells/mLandculturedin100µLofculturemediumat
WesternchemiluminescentHRPsubstratewerepurchasedfrom
incubatorfor24h.Themediumineachwellwasthenreplaced
Millipore (Billerica, MA, USA). Marker, rabbit anti-MRP2 and with100µLofmediumcontainingharmalineorharmineatthe
anti-glyceraldehyde3-phosphatedehydrogenase(GAPDH),and followingconcentrations:0.5,1,2.5,5,25,and100µM.After12h
HRP-conjugated anti-rabbit IgG antibodies were purchased incubation,10µLofCCK-8dyewasaddedtoeachwell,andcells
from Abcam Technology (Cambridge, MA, USA). Acetonitrile,
wereincubatedforanother2h.Cellviability(%)wascalculated
methanol,andformicacidofHPLCgradewerepurchasedfrom
usingthefollowingEquation(1):
Fisher Scientific Co. (Santa Clara, CA, USA). Deionized water
waspurifiedusingaMilli-QAcademicSystem(MilliporeCorp.,
Cellviability(%) = (OD −OD )/
Billerica,MA,USA).Allotherchemicalswereofanalyticalgrade. sample blank
(OD −OD ) ×100% (1)
control blank
Animals
Fifty-sixpathogen-freeSprague-Dawleyadultratscomprising28 OD referstotheabsorbanceofawellwithtreatedcellsand
sample
malesand28females(weighingwithintherangeof200–250g) CCK-8.OD referstotheabsorbanceofawellwithmedium
blank
wereobtainedfromDrugSafetyEvaluationandResearchCenter andCCK-8butwithoutcells.OD referstotheabsorbance
control
ofShanghaiUniversityofTraditionalChineseMedicine.Animals ofawellwithuntreatedcellsandCCK-8.Theabsorbanceat450
wereraisedunderanenvironmentallycontrolledbreedingroom nm was measured by a microplate reader (Biotek Instrument;
for 7 days before commencing the experiments. Animals were GeneCo.,Ltd.,VT,USA),andtheresultsarepresentedasmean
housedinawell-lightedair-conditionedroom(25±1◦C)under ±SDfromtriplicatewells.
FrontiersinPharmacology|www.frontiersin.org 3 August2017|Volume8|Article541
Lietal. TransportofHarmalineandHarmine
Transport Study compartment. P is the P -value measured in BL to AP
appBA app
Theapical(AP)andbasolateral(BL)sidesaretypicalofpolarized direction and P is the P -value measured in AP to BL
appAB app
Caco-2, MDCK and MDCK-MRP2 epithelial cells. Before the direction. It indicated the involvement of an active transport
experiments, the polarized cell monolayers were washed twice processwhentheER-valueisabove1.5(Hubatschetal.,2007).
withwarmHBSSsolutionandsubsequentlypreincubated(37◦C,
30 min). Afterward, HBSS solution on both sides of the cell Vesicular Transport
monolayers was removed using a mini desktop vacuum pump. The vesicular transport assay was implemented as described
To measure the AP to BL permeability (absorptive transport), in the GenoMembraneTM vesicular transport kit protocol
400 µL HBSS containing harmaline or harmine was added to (GenoMembrane,Kanagawa,Japan)withminorchanges.Briefly,
theAPside,while600µLblankHBSSwasaddedtotheBLside. vesiclestockswerethawedanddilutedwithassaybuffer(50mM
TomeasuretheBLtoAPpermeability(secretivetransport),600 MOPS-Tris, 70mM KCl, and 7.5mM MgCl2). Ten microliters
µLHBSScontainingharmalineorharminewasaddedtotheBL harmaline or harmine at the concentration of 1 or 10 µM was
side and 400 µL blank HBSS was added to the AP side. The mixedwith5µLassaybufferand20µL10mMMgATPsolution;
harmaline or harmine solution was freshly prepared in DMSO. 10 µL vesicle was mixed with 5µL assay buffer; after 5min
The final DMSO concentration in the HBSS or drug mixture preincubation,thetwomixtureswerethenmixedandincubated
was below 0.5%. The monolayers were incubated at 37◦C, and for another 5min. MgAMP instead of MgATP was used as a
100µLsamplesweretakenat30,60,90,and120minfromthe control. Apart from these, 4mM glutathione was also included
acceptorcompartmentandimmediatelyreplacedwiththesame in BCRP and MRP2 assays. Transport was terminated with ice
volume of prewarmed fresh blank HBSS. TEER measurements coldassaybuffer,andthensampleswerequicklytransferredtoa
forassessingthemembraneintegritytookplacebeforeandafter filterplate.Wellswereimmediatelywashed5timeswith200µL
theexperiments. assaybufferanddried.Thevesiclesontheplateweredissolvedin
Harmaline and harmine transport was assessed in both 50µLof80%methanolandcentrifugedat2,000rpmfor2min
directions at different concentrations (1, 2, and 5 µM). The aftercollection,andrepeatingtheaboveprocedures.Mixedthe
effect of pH on harmaline or harmine transport in both twofiltratestogetherandaddedthepre-cooledmethanol.After
directions was studied using the following pH in the acceptor centrifugationat12,000rpmfor5minat4◦C,a5µLaliquotof
or donor compartment: 5.5/5.5 and 7.4/7.4. The harmaline supernatantwasinjectedintotheUPLC-ESI-MS/MSsystemfor
or harmine transport at 4 and 37◦C in both directions was analysis.Furthermore,theuptakeofNMQ(MDR1),LY(BCRP),
evaluated to investigate the temperature effect. To reveal the andE217βG(MRP2)wasusedtoevaluatetheactivityofspecific
paracellularharmalineorharminetransport,thecellmonolayer vesicles.
waspreincubatedwith5mMEDTAinHBSSwithoutCa2+ and
Mg2+ for30min.Subsequently,theharmalineorharminewith Western Blot Analysis
5mMEDTAinHBSSwithoutCa2+andMg2+wasaddedtothe The Caco-2 cells were seeded into 6-well plates at a density of
APorBLcompartment. 1 × 105 cells/mL and cultured for 24h. After treatment with
The transport specificity experiments were performed by harmaline or harmine for 48h with their concentrations of 1,
testinginhibitionoftransportbyselectiveinhibitorsorsubstrates 2, and 5 µM, cells were washed 3 times with ice-cold PBS
ofthechosentransporters.Toinvestigatetherolesofinfluxand and lysed on ice with RIPA lysis buffer containing protease
efflux transporters in harmaline or harmine transport, 50 µM inhibitorandphosphataseinhibitor.Proteinlysatewascollected
sodium vanadate (Na+/K+-ATPase), 10mM NaN (adenosine bycentrifugationat12,000rpmat4◦Cfor10min,andthetotal
3
triphosphate, ATP), 50 µM cimetidine (OATs), 50 µM ES protein content was determined using BCA protein assay kit.
(OATPs),5mMTEA(OCTs/OCTNs),0.3mMphloretin(MCTs), Proteinswerethenmixedwithaquartervolumeofloadingbuffer
0.5mM phloridzin (SGLT1), 10mM Gly-Sar (PEPT1), 100 µM and heated at 100◦C for 5 min. Approximately 20µg of the
verapamil and quinidine (MDR1), 50µM MK571 and 200 µM total protein content was separated by SDS-PAGE through an
probenecid(MRP2),10µMKo143,and25µMapigenin(BCRP) 8% acrylamide gel and transferred to PVDF membranes. After
wereaddedatbothAPandBLsidesofmonolayers.After30min washing with PBST, the membrane was blocked with 5% fat-
preincubation,thesecompounds withharmalineorharmine(2 free milk in PBST for 1 h at room temperature (RT) and then
µM) were added to either the AP or BL side and blank HBSS incubated with anti-MRP2 (1:500) and anti-GAPDH (1:5,000)
bufferwasaddedtotheotherside,respectively.Asacontrol,in overnight at 4◦C. Afterwards, the membranes were rinsed with
eachinhibitionexperimentharmalineorharminetransportwas PBSTandincubatedwithHRP-conjugatedanti-rabbit(1:5,000)
alsoassessedintheabsenceofanyinhibitororsubstrate. secondaryantibodyfor2hatRT.Aftercompletewashingwith
The apparent permeability coefficients (P ) and ER were PBST,theproteinbandswerevisualizedwithECLprimekit(GE
app
calculatedasEquations(2)and(3): Healthcare,NA,UK).
P = (dQ/dt)/(A×C ) (2) Drug Analysis
app 0
ER = P /P (3) ExtractionProcedure
appBA appAB
Aconvenientandrapidprecipitationmethodwasusedtoprepare
wheredQ/dt isthesteady-stateflux,Aisthemembranesurface the transport and following plasma samples. A 100 µL aliquot
area, and C is the initial drug concentration in the donor of sample was added with 400 µL acetonitrile containing IS
0
FrontiersinPharmacology|www.frontiersin.org 4 August2017|Volume8|Article541
Lietal. TransportofHarmalineandHarmine
in a 1.5mL centrifuge tube and then vortex-mixed for 1.0min; heparinized tubes at 0 (predose), 0.03, 0.08, 0.25, 0.5, 0.75, 1.0,
subsequently, the mixture was centrifuged at 12,000rpm for 2.0,4.0,8.0,12.0,and24.0hafteradministration.Ratshadfree
10min at 4◦C. Up to 400µL of supernatant was transferred accesstowaterafter4.0hofbloodsamplecollection.Serialblood
to another clean tube and evaporated to dry at 37◦C under a samples were immediately centrifuged at 3,000rpm for 15min,
slight stream of nitrogen. The dried residue was reconstituted and100µLofthesupernatantplasmalayerwastransferredinto
with 80 µL of 9% acetonitrile and vortexed for 1.0min. After another new 1.5mL centrifuge tube and stored at −80◦C until
centrifugationat12,000rpmfor10minat4◦C,a5µLaliquotof analysis.
supernatantwasinjectedintotheUPLC-ESI-MS/MSsystemfor The plasma concentrations of harmaline and harmine were
analysis. directly calculated by their calibration curves accordingly. The
plasma concentration vs. time curves were plotted, and all
UPLC-ESI-MS/MSAnalysis the pharmacokinetic parameters of harmaline and harmine,
Harmaline and harmine concentrations were simultaneous such as absorption rate constant (K ), distribution rate
a
quantitativedeterminedonaWaters-ACQUITYTMUPLCsystem constant(K ),eliminationrateconstant(K ),absorptionhalf-life
d e
(Waters Corp., Milford, MA, USA) using an ACQUITY UPLC (T1/2a),distributionhalf-life(T1/2d),eliminationhalf-life(T1/2e),
BEH C18 column (50 × 2.1mm, 1.7 µm particle size). Mass apparent volume of distribution (Vd), clearance rate (CL), area
spectrometricdetectionwasperformedusingatriplequadrupole undertheplasmaconcentration-timecurvefromzerototimet
massspectrometer(WatersCorp.,Milford,MA,USA)equipped (AUC0−t),areaundertheplasmaconcentration-timecurvefrom
withelectrosprayionizationinpositiveionizationmode,andall zero to infinity (AUC0−∞), and mean residence time (MRT),
otherinstrumentalparametersweresetaccordingtoourprevious were processed using the non-compartmental pharmacokinetic
study (Li et al., 2016). The UPLC-ESI-MS/MS method was data analysis software program of PK solution 2.0TM (Summit
well-validated (data not shown) and the analytical method was Research Services, USA). The maximum peak concentration
successfully applied to determine the concentration of harmine (C )andtimeofmaximumplasmaconcentration(T )were
max max
and harmaline in the HBSS buffers. Figure S2 presents the obtaineddirectlyfromtheobservedconcentrationvs.timedata.
representative of typical MRM chromatograms of blank HBSS, TheF-valueofharmalineorharminewascalculatedbytheratios
blank HBSS spiked with harmine, harmaline and IS, and IS- ofdose-normalizedAUC0−∞afteroralandintravenousdosingas
spikedHBSSsamplecollectedat30minafteradministrationof thefollowingEquation(5):
harmineandharmaline.
F=(AUC0−∞,oral×Doseiv)/(AUC0−∞,iv×Doseoral)×100%
MetabolicStabilityofHarmalineandHarminein
(5)
HumanLiverMicrosomes
Harmalineorharmine(2µM)wasincubatedwithhumanliver
Excretion in Rats
microsomes (0.5mg protein/mL) for 0, 5, 10, 20, 30, 45, 60, 90
Sixteen rats with eight males and eight females were put into
min using our previous method (Li et al., 2016). The residual
metabolic cages individually and then fasted overnight till 2h
percentageofharmalineorharminewasplottedvusincubation
afteroraladministrationofharmalineandharmineatadoseof
time. The depletion half-life (T1/2) of harmaline or harmine
40.0mg/kg.Accesstowaterwasmaintainedallthetime.Samples
was calculated by regression analysis of semilogarithmic plots.
including blank urine and feces before dosing, urine and feces
Intrinsicclearance(CL )ofharmalineorharminewasestimated
int
from 0 to 24h, 24 to 48h, and 48 to 72h after dosing were
accordingtotheequation(4)fromtheinvitroT1/2,incubation collectedandstoredat−80◦Cuntilanalysis.
volume(V),andmassofmicrosomalproteinsintheincubation
Urine samples were thawed and an aliquot of 100 µL was
mixture(P)(Liuetal.,2010).
processed for detection as described in Section Drug Analysis.
Feces were homogenized and then weighed, added 6-folds of
CLint =(0.693/T1/2)×(V/P) (4) acetonitrile, extracted by ultrasonic wave for 1 h and then
centrifuged. An aliquot of 100 µL was also processed for
Pharmacokinetics
detectionasdescribedinSectionDrugAnalysis.
Thevalidatedmethodwasappliedtothepharmacokineticstudy
of harmaline and harmine in rats after single intravenous and Data Analysis
intragastric administration of harmaline, harmine, or harmine Data analysis was carried out with SPSS version 17.0 software,
co-administration with probenecid. Forty rats with 20 males andthedatawereexpressedasthemean±SD.Tocomparethe
and 20 females were randomly divided into five groups of twogroups,atwotailedunpairedStudent’st-testwasemployed.
eight rats in each. Harmaline or harmine was administered to P <0.05,P <0.01,andP <0.001wereconsideredstatistically
eight rats by gavage at a dose of 40.0mg/kg, and eight rats significant.
were administered via the caudal vein at a dose of 3.3mg/kg,
which was dissolved in physiological saline with germicidal RESULTS
treatment;theeightotherratswereorallyadministeredwiththe
mixture of harmine (40.0mg/kg) and probenecid (20.0mg/kg). Cytotoxicity Assay
Approximately 0.25mL of blood sample was collected from Harmaline and harmine were tested with CCK-8 assay for
the angular vein of each conscious rat and transferred into possible cytotoxic effects in the Caco-2 and MDCK cell lines.
FrontiersinPharmacology|www.frontiersin.org 5 August2017|Volume8|Article541
Lietal. TransportofHarmalineandHarmine
Over 90% of the Caco-2 and MDCK cells were viable when up harminetransportwaspH-dependent,whichmightberelatedto
to25µMforharmalineor5µMforharminewasusedduring thephysicochemicalpropertiesofdrugs.
the experiments (Figure S3). According to these results, it was
confirmed that none of harmaline and harmine at the tested
concentrations(1,2,and5µM)showedtoxicityordecreasedcell
EffectofTemperature
viability.
As depicted in Figure2, the harmaline and harmine transport
across the Caco-2 cell monolayers at the concentration of 2
Transport Study µM was markedly reduced because P was changed from
appAB
TransportCharacteristics (27.72 ± 1.06) × 10−6cm/s to (7.59 ± 0.33) × 10−6cm/s for
The transcellular harmaline and harmine transport across harmalineandfrom(18.78±1.26)×10−6cm/sto(1.58±0.57)
the Caco-2 and MDCK cell monolayers was investigated as ×10−6cm/sforharminewhenloweringthetemperaturefrom37
time, concentration, pH, temperature changed, and the role of to4◦C(P<0.001).Simultaneously,theharmalineandharmine
paracellularpathway. transportacrosstheMDCKcellmonolayerswasalsoobviously
reduced, the P -values of harmaline and harmine at 37◦C
appAB
EffectofTimeandConcentration weresignificantlyhigher(3.77-and2.49-folds)thanthoseat4◦C
(P<0.001;Table2),sodidtheoppositedirection.Thedecreased
A total of three concentrations (1, 2, and 5µM) were used to
P andP at4◦Cmightindicatethatthetransportwas
examine harmaline and harmine transport across the Caco-2 appAB appBA
and MDCK cell monolayers at 37◦C with the incubation time energy-dependent because decreasing the temperature would
slow down cellular metabolism (Duan et al., 2014). However,
from 0 to 120min on either the AP or BL side. No obvious
furtherstudiesarenecessarytoconfirmtheenergydependence
change in TEER-value was observed among the concentration
oftheCaco-2andMDCKcellsforloweringthetemperaturemay
groups of harmaline and harmine during the 2h experimental
alsoslowdownpassivediffusion.
exposure, suggesting the integrity of the cell monolayers. As
shown in Figure1, the transport of harmaline and harmine in
bothdirectionsincreasedgraduallywithtimeandconcentration.
Interestingly,thecumulativetransportfluxesofharmineamong EffectofEDTA
the concentration groups were much higher in the BL to AP Inordertoinvestigatethepotentialparacellularpermeabilityof
direction than those in the AP to BL direction. The Papp and harmaline and harmine, EDTA (5mM) was used to transiently
ER-values of harmaline and harmine in both directions are open the functional barrier of TJ. The cellular TJ was modified
summarizedinTable1.Astheconcentrationincreased,thePapp- byEDTAtoremoveCa2+ ionsfromthemediumviachelation.
valuesofharmalineandharmineincreasedinbothdirections.In The TEER-values decreased, indicating the cell junctions had
theAPtoBLdirection,theharmalineandharminepermeabilities opened, and the TEER-values were below 100 (cid:127) cm2 after
were all lower than that of propranolol, a transcellular flux EDTA treatment. Table2 listed the P -values of harmaline
appAB
markerwithPappAB-valueof(41.90±3.30)×10−6 cm/s(Shen and harmine in both directions across the Caco-2 and MDCK
et al., 2015). The ER-values of harmaline were <1.5 at the monolayerswithorwithoutpretreatmentwithEDTA.Opening
tested concentrations, and it can be confirmed that harmaline these junctions, the P and P -values of harmaline and
appAB appBA
was transported mainly by passive diffusion. However, the BL harmineacrosstheCaco-2cellmonolayerswereincreased(P<
to AP harmine transport was 1.5- to 1.6-folds higher than the 0.05; Table2). After exposure of the MDCK cell monolayers
transport in the opposite direction. The ER-values at different to 5 mM EDTA at both sides, the harmaline and harmine
concentrationswereall>1.5,suggestingthatanactivetransport transport was also increased because P was changed from
appAB
processwasinvolved(Hubatschetal.,2007).Harminemightbe (20.36 ± 1.82) × 10−6 to (29.43 ± 1.45) × 10−6 cm/s for
a substrate for efflux transporters, such as MDR1, BCRP, and harmaline and from (4.60 ± 0.25) × 10−6 to (7.47 ± 1.31) ×
MRP2, which can participate in the efflux process, resulting in 10−6cm/sforharmine(P<0.05),andtheP wasincreased
appBA
lowerconcentrationsofharmineinthereceptorcompartment. from (21.03 ± 2.11) × 10−6 to (26.34 ± 0.93) × 10−6cm/s
for harmaline (P < 0.05) and from (7.32 ± 0.19) × 10−6 to
EffectofpH (8.98±0.90)×10−6 cm/sforharmine(Table2).Interestingly,
TheeffectofpHonharmalineandharmine(2µM)bidirectional the significant increase in harmaline flux was observed when
transport in the two cell monolayers were determined. As the bidirectional experiment was conducted in the presence
presented in Figure2, the cumulative transport fluxes of of EDTA, while for harmine, which mainly increased in the
harmaline and harmine at pH 5.5 were much lower than those AP to BL direction (Table2). This is indicative of paracellular
at pH 7.4 in both directions. The P -values of harmaline and passive diffusion as the primary pathway for the permeability
app
harmineinbidirectionarelistedinTable2.TheP -valuesof of the tested drugs (especially for harmaline) across the cell
appAB
harmalineandharmineatpH7.4weresignificantlyhigher(3.65- monolayers (Zhang et al., 2014). Besides, P of harmine
appBA
and3.56-folds)thanthoseatpH5.5(P<0.001)acrosstheCaco-2 remainedalmostconstantirrespectiveinthepresenceorabsence
cellmonolayers,andwhichwerealsoobviouslyhigher(1.56-and of EDTA, suggesting that harmine permeates the Caco-2 and
2.05-folds)thanthoseatpH5.5(P<0.05)acrosstheMDCKcell MDCK cell monolayers via transcellular route in the AP to BL
monolayers.Consequently,thedataindicatedthatharmalineand direction.
FrontiersinPharmacology|www.frontiersin.org 6 August2017|Volume8|Article541
Lietal. TransportofHarmalineandHarmine
FIGURE1|ThecumulativetransportfluxesofharmalineandharmineintheCaco-2(A)andMDCK(B)cellmonolayersinbothdirections(APtoBLandBLtoAP)at
differenttimesandconcentrations.Here,theAPtoBLdirection(absorptivetransport):harmalineorharminewasaddedtotheAPsideandsampleswerecollected
fromtheBLside;theBLtoAPdirection(secretivetransport):harmalineorharminewasaddedtotheBLsideandsampleswerecollectedfromtheAPside.Data
representthemean±SDfromthreereplicates.
FrontiersinPharmacology|www.frontiersin.org 7 August2017|Volume8|Article541
Lietal. TransportofHarmalineandHarmine
TABLE1|ThebidirectionalPapp-valuesofharmalineandharmineacrossthe harmine, and the permeability in the AP to BL direction was
Caco-2andMDCKcellmonolayers(mean±SD,n=3). obviously decreased (Figure3 and Table3, P < 0.01), while
ER was increased. To investigate the involvement of MCTs,
Cell Drug Initial PappAB PappBA ER
concentration (1×10−6cm/s) (1×10−6cm/s) SGLT1 and PEPT1, the model inhibitors phloretin, phloridzin,
(µM) and glycylsarcosine were used, respectively. However, 0.3 mM
phloretin, 0.5 mM phloridzin and 10 mM glycylsarcosine, the
Caco-2 Harmaline 1 27.45±1.44 25.57±1.95 0.93 concentration sufficient to inhibit MCTs, SGLT1, and PEPT1,
2 27.72±1.06 26.42±0.49 0.95 hadnoeffectonharmalineandharminetransport(Table3).For
5 30.10±3.01 30.98±1.40 1.03
theMDCKmonolayers,thesimilarresultswereobtainedexcept
Harmine 1 15.13±0.73 22.84±0.25 1.51
OATPs(Table4),whichmightduetothequitelowexpressionof
2 18.78±1.26 29.34±1.21 1.56
OATPsintheMDCKcells(Volpe,2011).Insummary,harmaline
5 31.15±1.42 48.29±2.81 1.55
andharminemightbethesubstrateofOATPsandOCTs/OCTNs
MDCK Harmaline 1 15.81±1.76 19.47±2.25 1.23 influxtransporters.
2 20.36±1.82 21.03±2.11 1.03
5 21.21±1.32 24.53±2.54 1.16 EffectsofEffluxTransporters
Harmine 1 3.70±0.14 5.81±0.19 1.57 As illustrated in Tables3, 4, the results from transport studies
2 4.60±0.25 7.32±0.19 1.59 performedusingvariousselectiveeffluxtransporterinhibitorsor
5 7.11±0.89 11.58±0.15 1.63 substrates.Verapamilandquinidine(100µM,MDR1inhibitors)
were chosen because they are more selective for MDR1 than
other efflux transporter inhibitors. The transport amount of
harmaline and harmine at 120 min was not changed (Tables3,
EffectsofInfluxTransporters 4, P > 0.05), implying that harmaline and harmine were not
Interestingly, the majority of P -values in the AP to BL theMDR1substrateunderourexperimentalconditions.Similar
appAB
directionshowedastatisticaldifferencecomparedtothecontrol, experiments with verapamil and quinidine, the harmaline and
butnosignificantdifferencewasfoundintheBLtoAPdirection harmine efflux was unaffected by BCRP inhibitors Ko143 (10
in the experiments with inhibitors or substrates. Thus, the µM) and apigenin (25 µM). As displayed in Tables3, 4, the
resultswerefocusedontheeffectsofinhibitorsorsubstrateson transportamountofthetesteddrugsat120minandpermeability
harmalineandharminetransportintheAPtoBLdirectioninthe werealmostunchangedbyKo143andapigenintreatment(P>
twocellmonolayers. 0.05).ItsuggestedthatbothMDR1andBCRPtransporterswere
Figure3 and Tables3, 4 presented the data from transport notinvolvedinharmalineandharminesecretion.
experimentsusingvariousselectiveinfluxtransporterinhibitors However,whenMK571(atypicalMRP2inhibitor)wasadded
or substrates. The investigations of harmaline and harmine to the AP side in the Caco-2 cell monolayers, the PappAB of
influxmechanismutilizedcimetidineandES(OATsandOATPs harmineat120minwasincreased(1.62-folds),andtheER-value
inhibitors, respectively) on both sides. As Figure3 and Table3 was decreased from 1.59 to 0.98 (Figure4A and Table3, P <
indicated, no significantly decrease of harmaline and harmine 0.01), implying that MRP2 transporter governed the secretion
transport amount at each sampling points, as well as Papp and of harmine. This result was validated by the values of PappAB
ER, occurred after cimetidine addition (P > 0.05) during the andERwhentheeffluxofharminewasinhibitedbyprobenecid.
experiment,implyingthatOATsdidnotcausetheharmalineand Probenecid is an MRP2 inhibitor that markedly increased
harmineinflux.However,theharmalineandharmineabsorption harmine transport from AP to BL direction with the PappAB-
was significantly decreased when 50 µM (Figure3, P < 0.01) value increased (1.27-folds; Figure4A and Table3, P < 0.05).
ESwasaddedontheAPside;theP decreasedfrom(27.48 However, both inhibitors of MRP2 (MK571 and probenecid)
appAB
± 0.18) × 10−6 to (24.33 ± 0.49) × 10−6 cm/s for harmaline hadnoeffectonharmalinetransport,andthePappAB andERin
(P < 0.01) and from (18.52 ± 2.06) × 10−6 to (14.64 ± 1.31) harmalinetransportweresimilartothecontrolintheAPtoBL
× 10−6 cm/s for harmine (P < 0.05), respectively, while the direction(Tables3,4,P>0.05).Therefore,theinhibitoryeffect
ER increased from 0.95 to 1.12 for harmaline and from 1.59 to of MK571 and probenecid implied that MRP2 was primarily
1.94 for harmine (Table3). Therefore, harmaline and harmine responsiblefortheharmineeffluxintheAPtoBLdirection.
mightbetransportedintotheCaco-2cellsviaOATPs.Moreover, Insummary, among thethreeselected ATP-binding cassette
TEA is the OCTs/OCTNs substrate behaves also as inhibitor, (ABC) transporter inhibitors (MDR1, BCRP, MRP2), especially
and it also inhibited the absorption of harmaline and harmine, MRP2, revealed a high affinity for harmine during harmine
and the permeability in the AP to BL direction was obviously secretion because MK571 and probenecid enhanced the
decreased (Figure3 and Table3, P < 0.01), while ER was permeabilityofharmineintheAPtoBLdirection.Additionally,
increasedfrom0.95to1.13forharmalineandfrom1.59to1.88 the same results have been verified in other cells (MDCK and
forharmine.TofurtherinvestigatetheATP-mediatedtransport MDCK-MRP2;Table4andFigures4B,C,P<0.05).
of harmaline and harmine, different substrates or inhibitors
were added at both sides. NaN (10mM, an ATP inhibitor) Vesicular Transport
3
and sodium vanadate (50µM, ATPase Na+/K+-dependent To validate the transport properties of the commercial Sf9
inhibitor) dramatically reduced the transport of harmaline and membranevesiclesexpressinghumanMDR1,BCRP,andMRP2,
FrontiersinPharmacology|www.frontiersin.org 8 August2017|Volume8|Article541
Lietal. TransportofHarmalineandHarmine
FIGURE2|ThecumulativetransportfluxesofharmalineandharmineintheCaco-2(A)andMDCK(B)cellmonolayersinbothdirections(APtoBLandBLtoAP)at
differentconditions(pH7.4or5.5,37or4◦C,withorwithoutEDTA).Datarepresentthemean±SDfromthreereplicates.
FrontiersinPharmacology|www.frontiersin.org 9 August2017|Volume8|Article541
Lietal. TransportofHarmalineandHarmine
TABLE2|ThebidirectionalPapp-valuesofharmalineandharmineintheCaco-2 64.16 and 54.25%, respectively (Figure7). The T1/2-value was
andMDCKcellmonolayersatdifferentconditions(pH7.4or5.5,37or4◦C,with 147.45minforharmalineand99.00minforharmineinhuman
orwithoutEDTA;mean±SD,n=3).
liver microsomes. The CL -value was 2.8mL/min/mg for
int
harmine,whichwas∼1.49-foldsgreaterthanthatofharmaline
Cell Drug pH/ PappAB PappBA ER
Temperature (1×10−6cm/s) (1×10−6cm/s) (1.88mL/min/mg), suggesting that harmaline is more stable in
humanlivermicrosomesthanharmine.
Caco-2 Harmaline Control 27.72±1.06 26.42±0.49 0.95
5.5 7.60±0.74*** 6.72±0.76*** 0.88
Pharmacokinetics
4◦C 7.59±0.33*** 8.71±1.83*** 1.15
Thepharmacokineticparametersofharmalineandharminewere
withEDTA 35.23±2.68* 39.57±2.10* 1.12
allobtainedafteroralandintravenousadministrationatthedose
Harmine Control 18.78±1.26 29.34±1.21 1.56
of 40.0 and 3.3mg/kg, respectively. Figure8 presents the mean
5.5 5.27±0.29*** 4.17±1.03*** 0.79
plasma concentration-time curves of harmaline and harmine
4◦C 1.58±0.57*** 2.41±0.85*** 1.52
after administration in rats. Table5 gives the corresponding
withEDTA 24.11±2.53* 29.67±2.22 1.23
pharmacokineticparametersofharmalineandharminefollowing
administrationinrats.
MDCK Harmaline Control 20.36±1.82 21.03±2.11 1.03
Following administration by gastric gavage, harmaline, and
5.5 13.03±2.54* 15.40±0.91* 1.18
4◦C 5.40±0.99*** 6.65±0.64*** 1.23 harmine can be absorbed into blood circulation with a short
T of0.56±0.13handlowC of67.05±34.29ng/mLfor
withEDTA 29.43±1.45* 26.34±0.93* 0.90 max max
harmine, and a relative long T of 1.76 ± 1.11 h and high
Harmine Control 4.60±0.25 7.32±0.19 1.59 max
C of 117.80 ± 59.01ng/mL for harmaline after a single oral
5.5 2.24±0.29*** 3.42±0.48*** 1.53 max
dose of 40.0 mg/kg in rats (Figure8 and Table5). The plasma
4◦C 1.85±0.55*** 3.02±0.45*** 1.63
concentrationvs.timecurveofharmalineandharmineproduced
withEDTA 7.47±1.31* 8.98±0.90 1.20
a slow phase of decrease with T1/2e of 5.13 ± 1.52h and 4.73
*P<0.05;**P<0.01;***P<0.001(comparedwithcontrol). ± 0.71h, respectively after dosing, which presented the same
trend with intravenous dosage. To ascertain whether harmine
isthesubstrateofMRP2,harminewasorallyco-administration
thethreeprobesN-methylquinidine(NMQ),luciferyellow(LY) withprobenecid(anMRP2inhibitor).AspresentedinFigure8
and estradiol-17 β-glucuronide (E217βG) were used. As shown andTable5,theCmax,Tmax,T1/2e,AUC(0−t),AUC(0−∞),MRT-
inFigure5,theuptakeratios(UR)ofthethreeprobeswere2.05, values of harmine in probenecid co-administration group were
19.66,and7.71respectively,whichindicatedthatthemembrane significantlyhigherthanthoseofharmineadministrationgroup
vesiclescouldbeusedinfurtherstudies(allUR-valueswereabove (Table5,P<0.05,P<0.01,orP<0.001).Onthecontrary,the
2.00). It also showed that the UR-values of harmaline at 1 and Vd-andCL-valueswereobviouslylowerthanthoseofharmine
10µMwere<2.00inallmembranevesiclesexpressinghuman administration group (Table5, P < 0.01 or P < 0.001). It
MDR1,BCRP,andMRP2.Particularly,theUR-valueofharmine was worth noting that the F-value of harmine was increased
at1µMwas>2.65inthemembranevesiclesexpressinghuman 1.96-folds after probenecid co-administration, which further
MRP2,whichimplyingharminemightbethesubstrateofMRP2 demonstratedthatharminemightactasasubstrateofMRP2.
(Figure5C).
Excretion in Rats
Western Blot Analysis
The cumulative excretion of harmaline and harmine in urine
The protein expression of MRP2 in the Caco-2, MDCK, and
andfecesafteroraladministrationof40.0mg/kgwaspresented
MDCK-MRP2cellsweredeterminedbywesternblot.Asdepicted
inTable6.Harmalineandharmineinraturineweredetectable
in Figure6A, MRP2 significantly expressed, low expressed
withlowconcentrationwithin72hafteroraldosing,whilenone
and over-expressed in the Caco-2, MDCK, and MDCK-MRP2
of harmaline and harmine could be found in feces within 48–
cells, respectively. Furthermore, it can be seen from Figure6B,
72h.Harmalineandharminewerepoorlyexcretedviaurineand
harmine(2and5µM)couldslightlyup-regulatetheexpression
feces, and four metabolites for harmaline (harmine, harmalol,
of MRP2 compared with the control group, indicating that
harmol,M279)andtwometabolitesforharmine(harmol,M279)
harmine might be the substrate of MRP2 and could probably
were found in rats. The cumulative excretion was 5.05 ±
inhibittheabsorptionofthosecomponentsmediatedbyMRP2.
4.12% for harmaline and 0.69 ± 0.36% for harmine in urine
However,harmalineatthetestedconcentrations(1,2,and5µM)
and feces within 72h after intragastric dosing. Considering the
had no effect on the expression of MRP2, demonstrating that
combination of intact drugs and their metabolites in urine and
MRP2wasnotresponsibleforharmalinetransport.
feces, about 16.04 ± 5.45% for harmaline and 28.18 ± 8.24%
forharminewererecovered,whichsuggestedthatharminewas
Metabolic Stability of Harmaline and
easily metabolized than harmaline in rats in vivo after oral
Harmine in Human Liver Microsomes administration.Thelowrecoveryrateofharmalineandharmine
After incubating of harmaline or harmine at 2 µM, the was probably due to they transformed into other undetected
residual percentage values of harmaline and harmine were metabolites,whichneedtobefurtherconfirmed.
FrontiersinPharmacology|www.frontiersin.org 10 August2017|Volume8|Article541