Azole Antifungal Inhibition of Buprenorphine, Methadone and Oxycodone In Vitro Metabolism

13 Pages·2015·0.83 MB·English

JournalofAnalyticalToxicology2015;39:374–386
doi:10.1093/jat/bkv030 AdvanceAccesspublicationApril12,2015 Article
Azole Antifungal Inhibition of Buprenorphine, Methadone and Oxycodone
In Vitro Metabolism
DavidE.Moody*,FenyunLiuandWenfangB.Fang
CenterforHumanToxicology,DepartmentofPharmacologyandToxicology,UniversityofUtah,30E2000S,Rm105,
SaltLakeCity,UT84112,USA
*Authortowhomcorrespondenceshouldbeaddressed.Email:[email protected]
Opioid-relatedmortalityrateshaveescalated.Druginteractionsmay oxycodone (1,2, 9). Amajor site ofdruginteraction isthe en-
increase blood concentrations of the opioid. We therefore used zyme involved in the metabolism of the drug (10, 11). While
D
humanlivermicrosomes(HLMs)andcDNA-expressed humancyto- buprenorphine,methadoneandoxycodoneallsharemetabolism o
w
chrome P450s (rCYPs) to studyin vitro inhibition of buprenorphine bycytochromeP450(CYP)3A4,theydifferintheotherenzymes nlo
metabolismtonorbuprenorphine(CYP3A4and2C8),oxycodoneme- involvedinthemetabolismandinthepharmacodynamicactivity ad
e
tabolism to noroxycodone (CYP3A4 and 2C18) and oxymorphone oftheirmetabolites. d
(CYP2D6),andmethadonemetabolismtoR-andS-2-ethylidene-1,5- Buprenorphine is N-dealkylated to norbuprenorphine by fro
m
dimethyl-3,3-diphenylpyrrolidine (EDDP; CYP3A4 and 2B6). In this CYP3A4(12,13)andCYP2C8(14).Otherpathwaysofringand h
study,wehaveexaminedtheinhibitoryeffectof12(mostlyantifun- sidechainhydroxylationhavebeenidentified;theyarealsocat- ttp
s
gal)azoles.Thesecompoundshaveawiderangeofsolubility;tokeep alyzedbyCYP3A4and2C8(15,16),butappeartobeofminor ://a
organic solvent (cid:2)1%, therewas an equally wide range of highest clinicalrelevance(17).Whilenorbuprenorphinehasinvitroac- ca
d
concentration tested (e.g., itraconazole 5mM to fluconazole tivityatthemu-opioidreceptor,itscentralactivityislimiteddue e
m
1000mM).Inhibitorswerefirst incubatedwithHLMs at threecon- toeffluxattheblood–brainbarrierbyP-glycoprotein(3,18,19). ic
centrationswithorwithoutpreincubationofinhibitorwithreducing Methadone is a racemic drug. The R-enantiomer is more .ou
p
equivalents to also screen for time-dependent inhibition (TDI). potent as a mu-opioid receptor agonist (20), whereas both .c
o
PosaconazoledisplayedevidenceofTDI;metronidazoleandalbenda- R- and S-enantiomers are NMDA receptor antagonists (21). m
zolehadnosignificanteffect.Azoleswerenextscreenedatthehighest S-Methadone isthe more potent blocker ((cid:3)2.5–3.5(cid:4)) of the /ja
awcehrieevdaebtleermcoinnecdenfotrramtioonstfoCrYnPo3nA-C4YpPa3thAw4apyasth(wraanygse.sI)Ca5n0dvaoltuheesr(pmaMth)- hasusmocainateetdhewr-iat-hgoa-gmoe-rtehlaatdeodngee-innedu(hcEeRdGp)roKlþoncgheadnnQeTlsitnhtaetravrael t/article
waysasdictatedbyscreenresults:clotrimazole(0.30 – 0.35;others (22).R-andS-methadoneareN-demethylated,withanensuing -a
b
>30mM); econazole (2.2 – 4.9; 2B6 R-EDDP – 9.5, S-EDDP – 6.8; spontaneous cyclization,toR-and S-2-ethylidene-1,5-dimethyl- stra
2C8 – 6.0; 2C18 – 1.0; 2D6 – 1.2); fluconazole (7.7 – 66; 2B6 – 3,3-diphenylpyrrolidine (EDDP), respectively. EDDP is further c
313,361;2C8 – 1240;2C18 – 17;2D6 – 1000);itraconazole(2.5to N-demethylated to 2-ethyl-5-methyl-3,3-diphenylpyrroline t/39
>5; others >5); ketoconazole (0.032 – 0.094; 2B6 – 12, 31; 2C8 – (EMDP). We (23, 24), and others (25–27), have shown that /5/3
78;2C18 – 0.98;2D6 – 182);miconazole(2.3 – 7.6;2B6 – 2.8,2.8; CYP3A4and2B6arethemainenzymesinvolvedintheinvitro 74
2C8–5.3;2C18–3.1;2D6–5.9);posaconazole(3.4–20;2C18–3.8; N-demethylation of methadone. CYP2B6 has a higher affinity /76
0
others >30);terconazole(0.48to >10;2C18 – 8.1;others >10)and for S-methadone, whereas 3A4 is non-specific. CYP2C19 (R- 6
9
voriconazole(0.40 – 15;2B6 – 2.4,2.5;2C8 – 170;2C18 – 13;2D6 methadone preferred) and 2D6 (non-specific) also carry out 2 b
>300).ModelingbasedonestimatedKivaluesandplasmaconcentra- the reaction, but appear to have minorroles. EDDPand EMDP y g
tionsfromtheliteraturesuggestthattheorallyadministeredazoles, areessentiallyvoidofopioidactivity(20). ue
s
particularlyketoconazoleandvoriconazole,havethegreatestpotential Oxycodone is N-demethylated to noroxycodone and O- t o
forinhibitingCYP3A4pathways,asdoesvoriconazolefortheCYP2B6 demethylatedtooxymorphone;combinedreactionsformnoroxy- n 0
pathways.Azolesusedformucosalandtopicalapplicationsdidnot morphone.Lalovicetal.(28)haveshownthattheN-demethylation 6 M
exceedthemodelingthreshold. iscarriedoutbyCYP3A4andtheO-demethylationbyCYP2D6. a
y
We recently found that CYP2C18 can also perform the N- 2
0
1
demethylation(29). Oxymorphone andnoroxymorphonewith 9
Introduction theopen3-hydroxylgroupareglucuronidatedwhileoxycodone
Overthepastdecadeandahalf,therehasbeenanotableincrease andnoroxycodoneareeithernot,oronlyslightlyglucuronidated.
inmortalities arising fromopioid use. This is related to the in- Oxymorphoneandnoroxymorphonesharemu-opioid receptor
creased use of opioids in pain management and the increased activitywithoxycodone,whereasnoroxycodonedoesnot.The
abuseoftheseprescribedmedications(1–4).Theforensictoxi- impactofnoroxymorphoneislimitedtotheperipheryasitpoorly
cologycommunityistaskedwithassistingintheinterpretation penetratestheblood–brainbarrier(28).Oxymorphoneishighly
of the cause of these deaths, be they intentional oraccidental, glucuronidated,andthemu-opioidreceptoractivityofoxymor-
self-inflicted, aresultof amedical misadventureor someother phone-glucuronide has not been determined, but this might
reason.Aconfoundingfactoriswhetheruse ofco-medications limititscontributiontotheoverallmu-opioidreceptoractivity.
mayhavecontributedtotheresultantdeath(5–8).Suchdrugin- Studiesondruginteractionswithbuprenorphinehavefocused
teractions mayhave an impact oncausation. Drug interactions primarily on antiretrovirals. Some instances of inhibition and
maychangethepharmacokineticsofthedrug.Threecommonly induction of metabolism have been described, but few have
used (andabused)opioids arebuprenorphine, methadone and hadadverseeffectsundertheconditionsofthestudies(6,30).
#TheAuthor2015.PublishedbyOxfordUniversityPress.Allrightsreserved.ForPermissions,pleaseemail:[email protected]
Drug interactions with methadone have been studied since compoundsstudied arestructurally distinguishedas imidazoles
Kreek’s initialreportin1976thatrifampininduces methadone andtriazoles.Thechemicalstructuresoftheserespectiveazole
metabolism (31). Subsequent studies were sporadic until the compoundsarepresentedinSupplementaryFigures1and2.
mid-1990s, at which time a number of studies with selective
serotoninreceptor inhibitorswerepublished,andstudieswith
antiretroviralswerejuststartingtoappear(32).Sincethattime Experimentalmethods
a focus on the antiretrovirals has been apparent with only a Materials
fewotherdrugclassesstudied(6).Mostoftheseadverseeffects, Racemic methadone, racemic EDDP perchlorate, oxycodone,
particularlyopioidwithdrawal,havearisenfromCYP-relatedin- noroxycodone, oxymorphone, buprenorphine, norbuprenor-
ductionofmethadonemetabolismthatisassociatedwithwith- phine and their deuterated internal standards were purchased
drawal. Inhibition of metabolism was also seen, but under the fromCerilliant Corporation(RoundRock,TX, USA).Insectcell
controlled clinicalconditionsofthestudieswasnotassociated rCYPs 2B6, 2C8, 2C18, 2D6 and 3A4 (Supersomes) were pur-
withadverseeffects.This,however,showsthatmanydrugscan chased from BD Biosciences (Franklin Lakes, NJ, USA); all had
inhibitmethadonemetabolismandcausehighercirculatingcon- co-expressed NADPH CYP reductase; rCYP2B6 and 3A4 also D
o
centrationsthatunderconditions ofundeveloped tolerance or hadco-expressedcytochromeb .Albendazole,albendazolesulf- w
5 n
ingestion of higher than intended doses could have severe oxide,clotrimazole,econazole,ketoconazole,itraconazole,met- lo
a
adverseeffects. ronidazole, miconazole, posaconazole, terconazole,paroxetine, de
beDenrupguibnltieshraecdtiomnosrteuodfiteesnwiinthreocxeynctodyeoanres;aarefeliwmietxeadm,bpuletshaavree Nm,yNci0n,N, 0D0--tgrliuetchoysleen6e-tphhioopshpohsaptehomraomniodseo(dtihuiomTEsPalAt),,gtrluocleoasned-6o-- d from
providedhere.Manyinvolvethetestingofprototypicalinhibitors phosphatedehydrogenase,b-NADPsodiumsalt,EDTAdisodium h
ozyfmCYesPi2nDt6heantwd3oAm4a(i3n3p)attohwcoanysfiromfmtheetaibnovloislvme.mAefnetwooftthheersecleans-- sCaoltrpa.n(dStMLogCuils2, wMeOr,eUoSbAt)a.iFnleudcofnroamzolSeigamnda-vAolrdirciocnhazCohleemwiecrael ttps://a
c
sicdruginteractantsare:rifampin(34),St.John’swort (35)and obtained from Eurasia Chemicals (Mumbai, India). Gemfibrozil ad
grapefruitjuice(36);andmorerecently,theantiretroviralsrito- glucuronidewas purchased from Toronto Research Chemicals em
navir and lopinavir/ritonavir (37). Inducers of metabolism (Toronto, Canada). Concentrated formic acid (88%), glacial ic.o
decreasetheeffectivenessofoxycodone,andinhibitorsthatin- acetic acid and ammonium hydroxide were purchased from up
creasesystemicexposuretooxycodonecanincreasetheeffect; Fisher Scientific (Fair Lawn, NJ, USA). All other solvents were .co
thisisevenmoresoifsystemicexposuretooxymorphoneisalso HPLC-grade. Water usedinthepreparationofreagents; extrac- m/ja
increased.Thesestudiesshowthatthereisadangerfromdrug tion and liquid chromatography (LC) mobile phasewas drawn t/a
interactionsincreasingexposuretooxycodone,andtherebyin- from a Milli-Q filter apparatus (Millipore Corp., Billerica, MA, rtic
creasingriskofoverdose. USA).OutdatedhumanplasmawasfromtheUniversityofUtah le-a
We have now initiated a series of studies on the in vitro in- bloodbank.Silanizedtubeswerepreparedbyvapor-phasesilani- b
s
hibition of buprenorphine, methadone and oxycodone metab- zationusinghexamethyldisilazane(Pierce,Rockford,IL,USA)in tra
c
olisminhumanlivermicrosomes(HLMs)andcDNA-expressed anovenundervacuumat2508Cfor2h. t/3
CYP450s (rCYPs). We initiated our studies with two groups of 9
/5
widely used compounds with varying histories of drug inter- /3
7
actions, i.e. the H -receptor antagonists and the proton pump Incubations 4
2 /7
inhibitors (38). While the in vitro inhibitory action of many Incubations of buprenorphine, oxycodone or racemic metha- 6
0
6
of the compounds we study has been addressed previously donewithHLMsandrCYPswereperformedusingamodification 9
2
intheliterature,fewpreviousstudiescomparedlargenumbers ofourpreviouslydescribedincubationconditions(16,43).HLMs b
y
of potential inhibitors. Differences in inhibition potency with- were prepared using methods described by Nelson et al. (44), g
u
in CYP pathways are possible. This can be particularly true withthefirstcentrifugationat9,000g,thehomogenizationbuff- e
s
for CYP3A4, and some other CYP, substrates, as the large ercontaining0.25Msucroseand10strokesofhomogenization. t o
n
substrate-binding domain of this enzyme allows various align- The protein content was determined by the method of Lowry 0
6
ments, and thereby various potencies for inhibitor substrate et al. (45). HLMs were pooled from seven sources. Since M
a
interactions (39). this pooldoes not reflect anyone donor, donor demographics y
2
Wenowreportontheeffectofanumberofazoles,mostlyused arenotprovided.Incubationconditionswereoptimizedforen- 0
1
forantifungal indications, but afew with otheranti-protozoal zymeproteinandincubationtimetoassurelinearityofproduct 9
indications. Manyof these compounds are formulated fororal formation and ,10% substrate loss with substrate at 20mM.
orintravenous(iv)administrationtocombatsystemicinfections. The optimization experiments and incubation conditions are
Somearelimitedforuseinthemucosaloralandvaginalcavities. discussed under results and presented in Supplementary data.
Others arelimitedtotopical applications.Assuchplasmacon- Incubations were carried out in an incubation buffer (0.1M
centrationsarehighestafterivororaladministration,withmuch phosphate buffer, pH 7.4 with 1.0mM EDTA and 5.0mM
lowerconcentrationsaftermucosalandevenloweraftertopical MgCl ) with an NADPH-generating system (NADPH-GS) of
2
administration (40–42). We have focused on seven drugs used 10mM glucose-6-phosphate, 1.2mM NADP and 1.2 units of
fororalorivtherapy(albendazole,fluconazole,itraconazole,keto- glucose-6-phosphate dehydrogenase. The total volume was
conazole, metronidazole, posaconazole and voriconazole), the 0.2mL.Forallincubations,protein(HLMorrCYP)and incuba-
active metabolite of albendazole, albendazole sulfoxide, three tionbufferwereaddedfirst.Forincubationswithoutpreincuba-
examplesofmucosaldrugs(clotrimazole,miconazoleandterco- tion, substrateand inhibitorwere thenadded andthereaction
nazole) and a drug used solely topically (econizole). The azole was initiated by adding the NADPH-GS. For incubations with
AzoleInhibitionofOpioidMetabolism 375
preincubation,inhibitorandtheNADPH-GSwereadded,samples accurate and precise quantification in HLM was demonstrated
wereincubated at378C for 15min in awater bath shakerand during method validations (14, 29, 48). Calibrators and QCs
then substrate was added. Time-dependent inhibition (TDI)- wererunatthebeginningandendofthebatch.QCswererun
positive controls were included with HLM incubations. atlow,mediumandhighconcentrationswithanN(cid:5)2percon-
Troleandomycin at 5mM was used with all three substrates as centration.Overthecourseoftheseanalyses,theaccuracyand
the CYP3A4-positivecontrol; 10mM thioTEPA,0.5mM paroxe- precision expressedas%target/%CVfor theQCs wereas fol-
tine and 5mM gemfibrozil glucuronide were included as the lows: buprenorphine and norbuprenorphine low (0.250ng/
CYP2B6 (methadone), CYP2D6 (oxycodone) and CYP2C8 mL) 98.0%/8.2%, 106%/6.0%; medium (20ng/mL) 101%/3.4%,
(buprenorphine)-positive controls, respectively (46, 47). 104%/2.9%; high (70ng/mL) 101%/3.1%, 95.1%/2.4%; R- and
Incubationswerethenstartedorcontinuedforspecifiedtimes, S-methadone, R-EDDP and S-EDDP low (5.0ng/mL) 95.6%/
andwereterminatedbyadditionof100mLofice-coldmethanol 11.7%, 96.0%/11.0%, 104%/14.2%, 104%/13.5%; medium
andtransferofsamplestoanicebath.Allsampleswereprepared (100ng/mL) 101%/5.9%, 101%/5.9%, 102%/13.7%, 103%/
induplicate. 12.6%;high (425ng/mL)99.3%/4.3%, 98.8%/4.0%,101%/6.7%,
102%/7.4%; oxycodone, noroxycodone and oxymorphone low D
o
(0.60ng/mL) 100%/7.5%, 100%/11.2%, 95.8%/6.6%; medium w
n
SamplepreparationandLC–electrospray (10.0ng/mL) 96.9%/2.9%, 96.9%/3.7%, 93.8%/3.5%; high lo
a
ionization-tandemmassspectrometry(LC–ESI-MS-MS) (200ng/mL)96.5%/4.1%,99.5%/4.0%,97.5%/4.1%. d
e
Tthheemeentahnotdiofmorero-xsypceocdifiocnemaentdhomdetfaobrolmiteesth(a2d9o)nweer(e48p)erafonrd- d from
med as previously described; theyareonly briefly described at h
thistime. Results ttps
R- and S-methadone and R- and S-EDDP determination used Enzymeassayoptimization ://a
c
deuteratedinternalstandardsandliquid:liquidextractionwith Assayswere optimizedtomeet threecriteria recommendedby a
d
methyl t-butyl ether. The m/z 310 (MHþ) to 265, m/z 313 theUnitedStatesFoodandDrugAdministrationforinvitroin- em
(MHþ) to 268, m/z 278 (MHþ) to 234 and m/z 281 (MHþ) to hibition studies: substrates were kept below K (to the extent ic
m .o
234 selected ion transition monitoring were used to analyze possible), substrate loss did not exceed 10% and product for- u
p
methadone, methadone-d , EDDP and EDDP-d , respectively. mation was linear over the time of incubation. In addition, ex- .c
3 3 o
Enantiomer separation was achieved with a chiral column perimentswereperformedtotestforTDI(49).Inourprevious m
/ja
(ChiralAGP50(cid:4)2mm,5mm,currentlyfromChiralTechnologies, study, oxycodone and methadone metabolism were optimized t/a
Inc.,WestChester,PA,USA)withamobilephasegradientofmetha- at 2mM using 0.5mg/mL of HLMs (38). We found that bupre- rtic
nol:10mMammoniumacetate(80:20%to60:40%). norphine at this concentration was metabolized rapidly; a con- le
-a
Oxycodone,noroxycodoneandoxymorphonedetermination centration of 20mM with HLM concentration reduced to b
s
useddeuteratedinternalstandardsandliquid:liquidextraction 0.05mg/mLwas requiredto achieve optimalincubationcondi- tra
with4:1n-butylchloride:acetonitrile.Them/z316(MHþ)to tionswitha10-minincubation;proteincontentofCYP3A4and ct/3
298and322to304transitionswereusedtoanalyzeoxycodone 2C8 were likewise optimized to 2.5 and 10pmol/mL with 9
/5
anditsinternalstandardoxycodone-d6.Them/z302(MHþ)to 10min incubations (Supplementary Figure 3). Methadone and /37
284and305to287transitionswereusedtoanalyzenoroxyco- oxycodone incubation conditions were therefore also adjusted 4
/7
done,oxymorphoneandtheirinternalstandardsnoroxycodone- to use of substrate at 20mM with appropriate reduction in en- 6
0
6
d3andoxymorphone-d3.Noroxycodoneandoxymorphonehave zyme protein and selection of incubation time (Supplementary 92
thesametransition;theiridentificationswereachievedbyHPLC Figure 4). Methadone incubations were for 15min with HLM, b
y
columnseparation. CYP3A4 and CYP2B6 at 0.2mg/mL, 5pmol/mL and 10pmol/ g
u
Themethodfordeterminationofbuprenorphine andnorbu- mL. Oxycodone incubations with HLM were for 60min at e
s
prenorphine was modified from our previously described 0.3mg/mL; those with CYP3A4, 2D6 and 2C18 were for t o
n
method (14). To permit a larger analytical range, the upper 30min with 5, 5 and 25pmol/mL (see also Supplementary 0
6
limit of quantitation was increased from 10 to 100ng/mL. Table IV). M
Calibrators were changed to 0.1, 0.25, 1.0, 10, 25, 50, 75 and Theuseofsubstrateat20mM(i.e.,6,200to9,360ng/mL)had ay
2
100ng/mL. Quality control samples (QCs) were changed to animpactonthemassspectrometricconditions.Withsubstrate 0
1
0.25, 20and70ng/mL.Accuracyandprecisionforthe calibra- at such high concentration, it was not possible to keep both 9
tion curves over the first six runs, the curve parameters for substrate and metabolite within the linear range of the assay.
theserunsandaccuracyandprecisionoftheQCsareillustrated Only the metabolite was quantitated. The substrate peak area
inSupplementaryTablesI–III,respectively.Buprenorphineand ratio was evaluated to assure no large fluctuations. The linear
norbuprenorphinedeterminationuseddeuteratedinternalstan- rangeforthemetabolitewasdictatedinpartbyincubationvol-
dards and liquid:liquid extraction with 4:1 n-butyl chloride: ume (e.g., for norbuprenorphine, the 0.2-mL incubate was cal-
acetonitrile. The m/z 468 (MHþ) to 396, m/z 472 (MHþ) to culated as a 5-fold dilution and the analytical range thereby
400,m/z414(MHþ)to101andm/z417(MHþ)to104selected adjusted from 0.1–100 to 0.5–500). This also resulted in back-
iontransitionmonitoringswereusedtoanalyzebuprenorphine, grounddetectionofmetabolites(SupplementaryFigure5)rang-
buprenorphine-d , norbuprenorphine and norbuprenorphine- ingfrom0.006to0.48%ofsubstrateadded.Backgroundsamples
4
d ,respectively. thatcontainedonlysubstrateat20mMwereroutinelyincluded
3
For all three assays, calibrators and QCs were prepared in with all batches, and the amounts detected subtracted from
human plasma; the ability of plasma calibrations to provide studysamples.
376 Moodyetal.
Impactofazolepolarity notmissed.AsthemagnitudeofinhibitionofCYP2B6,2C8and
It is also highly recommended that in vitro inhibition studies 2D6maynotbeaccuratelyexpressedinHLMduetotheirlesser
keep thefinalconcentration of organicsolvent (cid:2)1%(49). The percentageoftotalCYPprotein,inhibitionofthesepathwayswas
azolecompoundshaveawiderangeofpolarity/aqueoussolubil- firsttestedat a maximumconcentrationof azole todetermine
ity,whichlimitedthefinalconcentrationsthatcouldbetested which would requireIC50determinations (Figure 4). Based on
forsomecompounds(e.g.,itraconazole)aslowas5mM.Acorre- these studies, we decided that CYP3A4 IC50 determinations
lation(R2¼0.473,P¼0.0194)wasfoundbetweenthepartition wouldincludealbendazolesulfoxidewithmethadone;CYP2B6,
coefficient (XLogP3)andthehighestfinalconcentrationtested 2C8, 2C18and2D6 IC50 determinations would include econa-
(Figure1). zole, fluconazole, ketoconazole, miconazole, and voriconazole
(except2D6);andCYP2C18IC determinationswouldalsoin-
50
cludeterconazoleandposaconazole(Figure4).
AzolesinHLMwithorwithoutpreincubation
The11azoledrugs,albendazole,clotrimazole,econazole,flucon-
azole, itraconazole, ketoconazole, metronidazole, miconazole, IC50determinationsforazolesinrCYPs D
posaconazole,terconazoleandvoriconazole,andthemetabolite IC50 determinations for selected azoles were carried out as ow
of albendazole, albendazole sulfoxide, were first incubated in described above. rCYPswereincubated with theselected sub- nlo
HLMwitheitherbuprenorphine,racemicmethadoneoroxyco- strates and azoles at six concentrations (each in duplicate) ad
e
doofnineh,iwbiittohroarndwiNthAoDuPtHa-G15S-(mFiignuprere2in)c.uCboanttiroonlsinintchluedperdestehnocsee bofasseodluobnilittyheoffitnhdeincogsmpwoituhndt.hFeosrcmreoesnt caonmdptohuenudps,ptehrelIiCm5i0t d from
withnoinhibitor,thosewithnoinhibitorplus1%DMSO(solvent wasdeterminedusingnonlinearregression(seeSupplementary h
control)(SupplementaryFigure6)andpositivecontrolsforTDI Figure 7 for examples). For a few azoles, extrapolation from ttp
s
(Figure 3). Preincubation always lead to significantly lower where the curve intercepted the 50% inhibition point was ://a
control activity; addition of 1% DMSO had variable effects used. A summary of the IC50 determinations for each CYP- ca
d
(Supplementary Figure 6). These controls were not pooled. dependent pathwayis presented inTableI.Comparisonof the e
m
Mnoorspthaiznoel,eRc-oamndpoSu-nEDdsDcPauassewde.ll5a0s%noinrohxibyictioodnonfoerfnoormrbautpioren-. FICig5u0rede5t.eIrnmgiennaetiroaln,sthfeorretwhaesCaYfaPir3Aag4repeamthewntayins tihseshraonwkinngisn. ic.ou
p
Metronidazole andalbendazole (datanotshown) werenotable In about one-half of the comparisons, the IC50 determinations .co
.cteixoo5cnn0etsr%po(tFliisonignwhusierb(eriFeti3igoi)nu.ndrOeiocn2fal)tyo.ivxOpeyonmoslyafoceTrocpDnohIanozfanoozleroelCfeionYarcPmnu3dbaAtma4ito,eicn2doDinwn6aiztHahonLldemMp2e.rBtPoh6odasudrieotciaenvcdee- apaitrnaievdtehibnwiunvaephyrrissbe,eintlwoyioripntphr.hoiopnxoeyrltcoioowndeaosltnte(o1ht5hi,ge2h8ree,s5pt0o,)rm,teaedstheKxamdpoefocnrteetdhineftoerrercmspoeemdcpitaievttee- m/jat/article
demonstrated TDI (Figure 2). We have not further explored -ab
s
mechanismsforthisTDI. Estimatesofinvivopotency trac
Oneofthesimplerequationstoextrapolateinvitrotoinvivo t/3
9
ScreenforrCYPinhibition inhibitionis: /5/3
7
CYP3A4isthemajorCYPpresentinHLM,andthemagnitudeof 4
inhibition found in HLM is a good indicator of inhibition of AUCi=AUCn ¼1þ½I(cid:6)=Ki ð1Þ /76
0
CYP3A4. All azoleswereselected forIC determinations with 6
CtuhsYainPtg3alACb4eYnpPda3atAhz4owlteaoyssausalsfcoucxroeidrdieninhangibdtitomortehytreaocfintniidvdiatiz5ny0ogglsereiwnateFeriregtufihrraesnt2s5c,0ree%xecnweepadst ciwnohnvecirveenoAtcrUaoCtniiocanenncdturAravUtieoCwnnioatrhfei(ntihh)eiabnaitrdoewraauitnnhddoeuKrtiti(hsnet)htiienmhienibhvieitborisrtu,io[sInp]licasostmnhsea- 92 by gue
tant.RatiosofAUC/AUC (cid:5)2areconsideredtobepotentially s
significant inhibitioins. Thnis equation is discussed in numerous t on
articles; Mao et al.(51) is a recentexample that studiedsome 06
azolesalongwithotherinhibitors.Whenoneassumescompeti- M
a
tiveinhibition,theKicanbeestimatedfromtheIC50usingthe y 2
Cheng-Prusoffequation(52): 01
9
K ¼IC =ð1þS=K Þ ð2Þ
i 50 m
whereSisthesubstrateconcentrationusedinvitroandK isthe
m
Michaelis–Mentonconstantforthereactionbeingstudied.
When equation (2) is applied to the calculated IC values
50
(Table II), the largest differences between K and K were
i m
foundforthepathwayswheretheK approximatesthe20-mM
m
substrate concentration, as is the case for CYP3A4 and 2C8
metabolismofbuprenorphineandCYP2B6metabolismofmeth-
adone.ThedifferencebetweenK andK islesssoastheK in-
Figure1. Plotofupperconcentrationofinhibitorusedtonotexceed1%DMSOin i m m
incubationversuslogoftheoctano-waterpartitioncoefficient (XLogP3).XLogP3 creases for CYP2D6 metabolism of oxycodone and CYP3A4
valuesarefromPubChem. metabolismofmethadone, and almostnonexistentforCYP3A4
AzoleInhibitionofOpioidMetabolism 377
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t/3
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6
9
2
b
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0
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Figure2. Inhibitionofmetabolismofbuprenorphinetonorbuprennorphine(Norbup),methadonetoR-EDDP(R-EDDP)andS-EDDP(S-EDDP),andoxycodonetonoroxycodone(Noroxy)
andoxymorphone(Oxymor).HLMswereincubatedwiththreeconcentrationsofantifungalazoleeitherwithout(openbars)orwitha15-minpreincubationofHLMwithaninhibitorand
anNADPH-GS.AcrudeapproximationofanIC wasmadeandisshownnexttotherespectivecondition.Albendazoleandmetronidazoledidnotexceed50%inhibitionandarenot
50
shown.
378 Moodyetal.
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Figure3. Effectofpreincubationoninhibitionbythepositivecontrols5mMtroleandromycin(Trol),5mMgemfibrozilglucuronide(Gem),0.5mMparoxetine(Parox)and10mM m
ThioTEPA. /ja
t/a
rtic
le
-a
metabolismofoxycodone(TableII).Whenequation(1)isthen negligible,theratiodecreasesdramatically.When fu,pl(cid:3) fu,rCYP, bs
used,wefindtheestimatedratiosofAUCi/AUCnexceed2forke- the ratio is essentially the same as when binding was not tra
c
toconazoleandvoriconazoleonCYP3A4pathwaysandforvori- considered. t/3
conazole on the CYP2B6 pathway. If the level of concern is 9
/5
loweredtoafactorof1.5,theimpactofitraconazole,fluconazole /3
7
and posaconazole is also seen with a number of the CYP3A4 4
pathways, as well as ketoconazole and CYP2B6 metabolism Discussion /76
0
(Table II). This modeling system would not predict even Theoverridingaimofourstudiesistocharacterizetheinhibitory 69
2
1.5-foldfactorchangesinCYP2D6orbyplasmaconcentrations potentialofgroupsofdrugsbasedontheirinvitroinhibitionof b
y
publishedforthemucosalandtopicalformulations.Thetopical metabolic pathways of oxycodone, methadone and buprenor- g
u
formulation did not exceed this factoreven when the plasma phine. While manyof the potential inhibitors weare studying e
s
concentrationswereincreased10-foldtomimicaseriousover- havebeen characterized to acertain extent, this has generally t o
n
dosesituation(TableII). been done using model substrates for various CYP gene prod- 0
6
Severalmodificationscanbemadetoequation(1)asincreas- ucts. Here, we are using mass spectrometric quantification to M
a
inglycomplexmodelsarederivedforinvitrotoinvivoextrap- study specific pathways in the metabolism of opioids, which y
2
olations. This is also discussed in numerous articles; again see are among those contributing to a decade long epidemic of 0
1
Maoetal(51)forarecentexample.Oneofthemainmodifica- opioid-relatedfatalities(1–4). 9
tions is use of free (unbound) fraction of inhibitor in plasma At thistime, we have nowexamined the in vitroeffect of a
(f ) and in enzyme source (f or f ). These were groupofanti-protozoanazolesonthemetabolismofbuprenor-
u,pl u,HLM u,rCYP
not calculated for this study. Plasma protein-binding data are phine,methadoneandoxycodone.Thepathwaysstudiedinclude
available in the literature (51), but (f or f ) for the a CYP3A4-mediated N-dealkylation foreach drug, a CYP2C8-,
u,HLM u,rCYP
azoles are not available. With the lowprotein content used in 2B6-and2C18-mediateddealkylationofbuprenorphine,metha-
our incubations, non-specific binding is relatively reduced. done and oxycodone, respectively, and a CYP2D6-mediated
Also, non-specific binding to membranes is often less than to O-demethylationof oxycodone(15,16, 24,27–29).Thisstudy
plasma protein [e.g., we found that f for buprenorphine included11azoledrugsand1predominantmetabolite.Thefocus
u,HLM
was0.42,while f is0.01(55)].Wethereforehaveusedahy- was on the seven azoles administered byoral and/or iv routes,
u,pl
potheticalrangeof f of1.0–0.5.Theseunboundfractions whichareassociatedwith1–10mMplasmaconcentrations;three
u,rCYP
havenowbeenusedtoshowhowproteinbindingeffectsthera- representativeazolesusedtotreatmucosaloraland/orvaginalcav-
tiosofAUC/AUC (TableIII).When f issmalland f is ities, whichareassociatedwith (cid:3)20nMplasmaconcentrations,
i n u,pl u,rCYP
AzoleInhibitionofOpioidMetabolism 379
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Figure4. InhibitionoftheCYP450-mediatedmetabolismusingthehighestachievableconcentrationfornon-CYP3A4pathways,andhighestconcentrationusedinIC determinations
50
forCYP3A4pathways.
380 Moodyetal.
andonerepresentativetopicalapplicationazole,whichisassociated Wenowreportnovelfindingsthatanumberoftheazolesinhibit
with0.3–3nMplasmaconcentrations(40–42). the orphan CYP2C18 with relative potencyof ketoconazole(cid:3)
Intermsofexploringinvitroinhibitionbybothanumberof econazole . miconazole (cid:3) posaconazole . terconazole.vor-
azolesandanumberofCYPpathways,thisstudyexceedsprevious iconazole . fluconazole. For CYP2D6, we found econazole.
studies.Zhangetal.studiedeightCYPpathways,butonlywith miconazole.ketoconazole.fluconazole. The potent inhibi-
clotrimazole and miconazole (56), Niwaet al. studied six CYP tionofCYP2D6bymiconazolehasbeennotedinotherstudies
pathways,butwithonlyfourazoles(57,58),Jeongetal.studied (56, 58, 60), as has the relative ineffectiveness of fluconazole
eightCYPpathways,butonlywithvoriconazole(59),andZientek (58). This isthe firststudy to report an IC for CYP2D6 with
50
etal.(60)studiedfiveCYPpathways,butonlywithmiconazole. econazoleandketoconazole.
Severalstudieshavefocusedontheeffectofselectedazoleson Clinical studies havebeen performed studying drug interac-
single CYPs. A numberof studies examined the effects of 3–4 tionofanti-fungalazoleswith oxycodoneand methadone.The
azolesonCYP3A4;cumulatively,dataontheeffectofeightazoles clinicalstudiesperformedwithmethadonewereforinteractions
onCYP3A4havebeenreported(56,58,61–66).Additionalstudies withfluconazoleandvoriconazole.Withfluconazole,thechange
have provided information on specific CYPs: four azoles with inracemicmethadoneAUCwas1.35(73);thiscloselymatches D
o
CYP2B6 (67), five azoles with CYP2C8 (68), six azoles with the extrapolated value forfluconazole and CYP3A4 (Table III). w
n
CYP2C9(69)andfiveazoleswithCYP19(aromatase)(70). Inourstudy,fluconazolewasabouta15-foldmorepotentinhib- lo
a
Priortostartingexperimentsoninhibition,itwasnecessaryto itorofCYP3A4thanCYP2B6.ItshouldbenotedthatKharasch d
e
opprotidmuicztefoinrcmuabtaiotinonlinceoanrdoitvieorntshteotkimeeepcsouubrssteraoteftlhoessin,c1u0b%atiaonnd. eistianhl.ib(7it4e,d7i5n)vhivaovebudtemmeotnhsatdraotneedNe-xdaemmpeltehsylwathioenreisCnYoPt3afAfe4c/t5- d from
Whilewefoundwecouldreadilymeettheseconditionswitha ed.TheseandotherfindingsleadthemtosuggestthatCYP2B6is h
relatively low substrate concentration (2mM at 0.5mg/mL moreimportantincontrollingmethadoneclearance.Whilethis ttps
HLM) for oxycodone and methadone (38), the turnover rate isnotclearlyevidentfromthefluconazolestudy,theresultsfrom ://a
c
forbuprenorphineinHLMsorrCYPswastoohighatthisconcen- theinteraction with voriconazole supportasignificantrole for a
d
tration.A10-foldincreaseinsubstrateconcentrationalongwitha CYP2B6.Invitroinhibitionbyvoriconazolehasaboutanequal em
10-folddecreaseinproteinwasrequired tomeetthesubstrate effect on CYP2B6 and CYP3A4. However, when voriconzole ic
.o
depletioncriteria.Toconductoxycodoneandmethadoneincu- was co-administered with methadone, the change in AUC of u
p
bationsatacomparablesubstrateconcentration,HLMandrCYP R-methadone was only 1.37, while that of S-methadone was .c
o
protein concentrations were also lowered for these drugs. 2.16 (76). The greater inhibition of S-methadone clearance is m
/ja
Reducingenzymeproteinhadthebenefitofminimizingtheim- consistentwithagreaterimpactonCYP2B6. t/a
pact of non-specific substrate binding to the microsomes (71, Instudieswithoxycodone,therespectivechangesinplasma rtic
72).Whilewewereabletokeeptheoxycodoneandmethadone AUC after treatment with ketoconazole were 1.84, 0.86 and le
-a
concentrations considerably lower than their reported Km(28, 3.46 foroxycodone, noroxycodoneandoxymorphone, respec- bs
50), assuring minimal substrate depletion for buprenorphine tively(33).Anotherstudyreporteda2.46-foldincreaseinoxyco- tra
c
resultedinatrade-offtouseofasubstrateconcentrationalmost doneplasma AUC(77).TherespectivechangesinplasmaAUC t/3
twicethereportedK valuesforCYP3A4and2C8(15).Asseen, after treatment with voriconazolewere 3.57, 0.52 and6.97 for 9
m /5
thiswasreflectedingenerallyhigherIC50valuesforinhibitionof oxycodone, noroxycodone and oxymorphone, respectively /37
buprenorphinecomparedwithoxycodoneandmethadone. (78). These changes are consistent with a strong inhibition of 4
/7
Forthetwomostpotent inhibitors,ketoconazole and clotri- CYP3A4with little or no effectonCYP2D6.The magnitude of 6
0
6
mazole, therewaslittle difference among the IC50 values with thechangesisrelativelyclosetothoseseeninourextrapolations 92
CYP3A4 foroxycodone,methadoneandbuprenorphine. Forall afteraccountingforunboundconcentrations(TableIII).There- b
y
otherazoles,thevalueswerehigherforbuprenorphine.Asimilar spectivechangesinplasma AUCaftertreatmentwith itracona- g
u
rank of potency order, however, was seen with all three sub- zole were 2.25, 0.52 and 3.20 for oxycodone, noroxycodone e
s
strates,witheconazole(cid:3)itraconazole(cid:3)miconazole(cid:3)tercona- and oxymorphone, respectively (79). This is also consistent t o
n
zole (cid:3)voriconazole.posaconazole..fluconazole and no withstrong inhibitionof CYP3A4, but was notconsistentwith 0
6
noted effect from albendazole sulfoxide or metronidazole. ourextrapolations.Thismaybeexplained,inpart,asthemetab- M
a
These findings are consistent with other in vitro results with olitesofitraconazolehavebeenfoundtocontributetoitsinhibi- y
2
CYP3A4substrates(56,58,61–66).WithCYP2B6,wefoundpo- tionofCYP3A4pathways(80).Therespectivechangesinplasma 0
1
tenciesofvoriconazole(cid:3)miconazole.econazole.ketocona- AUCaftertreatmentwithmiconazoleoralgelwere1.64,1.27and 9
zole ..fluconazole. Walsky et al. (67) found a similar rank 0.25foroxycodone,noroxycodoneandoxymorphone(81).This
orderforvoriconazoleandketoconazole,butfounditraconazole isconsistentwithamodestinhibitionofbothCYP3A4and2D6.
and clotrimazole to be more potent than we observed. Zhang Whilewefoundmiconazoletobearelativelygoodinhibitorof
et al. (56) concurred that miconazole was more potent than bothoftheseCYPs,theplasmaconcentrationsreportedfollow-
clotrimazole,andJeongetal.(59)alsofoundthatvoriconazole ingoralgelusearetoolowtoindicatetheseamountsofinhibi-
wasa potent inhibitor. With CYP2C8, we found miconazole(cid:3) tionwithourextrapolations.Analmost1000-foldincreasewould
econazole.ketoconazole.voriconazole..fluconazole. berequired toextrapolatethe magnitude ofinhibition seenin
Ongetal.(68)studiedinhibitionofCYP2C8atasingleconcen- the Gronlund et al. study. Miconazole plasma concentrations
tration,100mM.Theyfoundthatclotrimazole,whichwecould werenotreportedinthatstudy(81).Whilewenotethisdiscrep-
studyonly up to 30mM, was most potent, but otherwise con- ancy between anticipated miconazole plasma concentrations
curred that econazole and miconazole had similar potencies fromuseasanoralgelandthepotentialforinteractionwithoxy-
while fluconazole was not inhibitory at this concentration. codone, the fact that an interaction mayoccur should not be
AzoleInhibitionofOpioidMetabolism 381
TableI
InVitroReversibleInhibitionofCYP-MediatedMetabolismofMethadone,BuprenorphineandOxycodonebyAzoleAntifungalAgents
AntifungalAzole CYPpathwaysandproductsa
3A4 2B6 2C8 2C18 2D6
R-EDDP S-EDDP Norbup Norox R-EDDP S-EDDP Norbup Norox Oxymor
IC50(mM)
Albendazolesulfoxide 224b 224b .300 .300 .300 .300 .300 .300 .300
Clotrimazole 0.348 0.350 0.354 0.303 .30 .30 .30 .30 .30
Econazole 2.38 2.18 4.94 2.58 9.46 6.75 6.04 1.04 1.22
Fluconazole 16.1 16.3 65.8 7.69 313 361 1240 17.2 1000
Itraconazole 2.48b 2.45b .5 3.16b .5 .5 .5 .5 .5
Ketoconazole 0.0793 0.0853 0.0935 0.0320 11.7 31.2 77.6b 0.976 182b
Metronidazole .500 .500 .500 .500 .500 .500 .500 .500 .500
Miconazole 2.34 2.28 7.59b 3.73 2.76 2.78 5.34 3.10 5.89
Posaconazole 3.44 3.41 19.8 3.82 .30 .30 .30 3.82 .30 D
Terconazole 1.70 1.53 .10 0.481 .10 .10 .10 8.13b .10 o
Voriconazole 2.28 2.89 14.6 0.397 2.40 2.53 170 12.7 .300 wn
lo
aPathwaysstudied:CYP3A4and2B6—methadoneconversiontoR-EDDPandS-EDDP;CYP3A4and2C8—buprenorphineconversiontonorbuprenorphine(Norbup);CYP3A4and2C18—oxycodoneconversion ad
e
tonoroxycodone(Norox)andCYP2D6—oxycodoneconversiontooxymorphone(Oxymor). d
bDeterminedbyextrapolation. fro
m
h
TableII ttp
s
ExtrapolationofIC50DeterminationstoKiandInhibitoryPotentialatAverageand10(cid:4)AveragePlasmaConcentrationofAzoleInhibitors ://a
c
Inhibitor [I]pl(mM)a IC50(mM) Ki(mM)b AUCi/AUCnc AUCi10/AUCn IC50(mM) Ki(mM) AUCi/AUCn AUCi10/AUCn ad
e
OxycodonebyCYP3A4 OxycodonebyCYP2D6 m
FItlruaccoonnaazzoollee 41..2948 73..6196d 73..4046 11..5675 67..7407 1—000 6—66 1—.01 1—.06 ic.ou
Ketoconazole 7.53 0.0320 0.0310 244 2433 182d 121 1.06 1.62 p.c
Posaconazole 1.86 3.82 3.70 1.50 6.03 — — — — o
m
Voriconazole 10.9 0.397 0.384 29.4 285 — — — —
Clotrimazole 0.029 0.303 0.293 1.10 1.99 — — — — /ja
Miconazole 0.024 3.73 3.61 1.01 1.07 5.89 3.92 1.01 1.06 t/a
Terconazole 0.019 0.481 0.465 1.04 1.41 — — — — rtic
Econazole 0.0029 2.58 2.50 1.00 1.01 1.22 0.812 1.00 1.04 le
R-methadonebyCYP3A4 S-methadonebyCYP3A4 -a
Albendazolesulfoxide 4.90 224d 190 1.03 1.26 224d 195 1.03 1.25 bs
Fluconazole 16.1 13.7 1.31 4.10 16.3 14.2 1.30 3.98 tra
IKtreatcoocnoanzaozloele 20..40879d3 02..016073 111.934 11102.40 02..04855d3 02..017444 110.923 11001.34 ct/3
9
Posaconazole 3.44 2.92 1.64 7.37 3.41 2.97 1.63 7.26 /5
Voriconazole 2.28 1.93 6.63 57.3 2.89 2.52 5.33 44.3 /3
Clotrimazole 0.348 0.295 1.10 1.98 0.350 0.305 1.10 1.95 74
Miconazole 2.34 1.99 1.01 1.12 2.28 1.99 1.01 1.12 /7
6
Terconazole 1.70 1.44 1.01 1.13 1.53 1.33 1.01 1.14 0
Econazole 2.38 2.02 1.00 1.01 2.18 1.90 1.00 1.02 69
R-methadonebyCYP2B6 R-methadonebyCYP2B6 2
Fluconazole 313 210 1.02 1.20 361 185 1.02 1.23 by
Ketoconazole 11.7 7.86 1.96 10.6 31.2 16.0 1.47 5.71 g
u
Voriconazole 2.40 1.61 7.76 68.6 2.53 1.30 9.41 85.1 e
MEciocnoanzaozloele 29..7466 16..8366 11..0010 11..1030 26..7785 13..4426 11..0020 11..1071 st on
BuprenorphinebyCYP3A4 BuprenorphinebyCYP2C8 0
FKleutcoocnoanzaozleole 605.0.8935 02.60.3678 120.106 219.5991 7172.460d 42795.7 11..0215 13..0594 6 Ma
Posaconazole 19.8 8.01 1.23 3.32 — — — — y
Voriconazole 14.6 5.91 2.84 19.4 170 65.1 1.17 2.68 20
Clotrimazole 0.354 0.143 1.20 3.02 — — — — 19
Miconazole 7.59d 3.07 1.01 1.08 5.34 2.04 1.01 1.12
Econazole 4.94 2.00 1.00 1.01 6.04 2.31 1.00 1.01
aPlasmaconcentrations([I]pl)fortheoralazolesweretakenfromliteratureC valuesasfollows:albendazolesulfoxide(53),fluconazole,itraconazoleandvoriconazole(41),ketoconazole(40)and
max
posaconazole(54);themucosalformulationsclotrimazole,miconazoleandterconazolewerecalculatedfromanestimatedCmaxof10ng/mLandthetopicaleconazolefromanestimatedCmaxof
1.0ng/mL.
bKiwasestimatedfromtheIC50usingtheCheng-Prusoff,equation,whereKi¼IC50/(1þS/Km).Kmvalues(mM)werefromtheliteratureasfollows:oxycodonebyCYP3A4(600)and2D6(39.8),(28)
R-methadonebyCYP3A4(112)and2B6(13.6)andS-methadonebyCYP3A4(136)and2B6(12.4),(50)andbuprenorphinebyCYP3A4(13.6)andby2C8(12.4)(15).
cInhibitorypotentialAUCi/AUCn¼1þ[I]/Ki,whereAUC¼areaundertheconcentrationcurveinthepresenceofinhibitor(i),10-timesinhibitor(i10)ornoinhibitor(n).
dInsufficientpointswereavailableforcalculationofIC bynonlinearregression;valuewasdeterminedbyextrapolationfromthecurve.
50
ignored.Mikietal.(82)discussanumberofcasestudiesforin- administration of the two drugs should be avoided. Indeed,
teractionsbetweenoralgelmiconazoleandwarfarin,andmodel withwarfarin,casereportshavealsobeenpresentedforinterac-
thepotentialforapharmacokinetic interactionconcluding co- tionswithtopicallyappliedmiconazole(83)andeconazole(84).
382 Moodyetal.
system (51), good agreement was found with several clinical
studies.Theexceptionbeingitraconazole,whichhascontribut-
ing inhibitory metabolites, and oral gelmiconazole, where the
anticipated plasma concentrations from gel delivery did not
seemsufficienttocauseaninvivointeraction.Non-CYPfactors
havebeen proposedtocontributeto theinvivoeffectsofthe
azoles on drug disposition. These include inhibition of drug
transports(85),antagonismofPXRreceptors(86)andinhibition
ofUDP-glucuronosyltransferases(87).Thecontributionofthese
andotherfactorscannotberuledout,butagoodcorrelationto
theeffectofazolesonCYPactivityisaconsistentfinding.
Conclusions D
o
w
Manyof the azole agents inhibited the in vitro metabolism of n
Figure5. RankingofIC50determinationsforCYP3A4-mediatedpathways. oxycodone, methadone and buprenorphine. Ketoconazole and loa
d
clotrimazole had sub-micromolar IC values for the CYP3A4- e
50 d
mediatedpathways,asdidterconazoleandvoriconazoleforoxy- fro
codone metabolism by CYP3A4. Severalazoles had IC values m
TableIII 50 h
ExamplesoftheImpactofPlasmaand(Hypothetical)MicrosomeDrugBindingonCalculationof between1 and10mMfor the CYP3A4 pathways. Twoto three ttp
IAnzhoibleitoryPotentialfua,pl fu,rCYP(hypothetical) [I]u,pl(mM) Kiu,rCYP(mM) AUCi/AUCn 2oazCfo1tlh8eesanaaldzsoo2lDehsa6dwpIaeCtrh5ew0uvaysaeslu.dWe,sthhienenrtehuinwsbaroasunnfgadeirpfoalagrsrmteheaemcCoeYnncPte2bnBet6rta,wt2ieoCen8ns, s://acad
e
OxycodonebyCYP3A4 extrapolationpredictedincreasesinAUCandthoseseeninthe m
Fluconazole 0.89 1.00 3.77 7.44 1.51 ic
0.50 3.72 2.01 literatureforclinicalstudies.Notedexceptionswerewithitraco- .o
u
Itraconazole 0.01 1.00 0.0198 3.06 1.01 nazole,whichhascontributinginhibitorymetabolites,andwith p
0.50 1.53 1.01 .c
oral gel use of miconazole. The impact of orallyadministered o
Ketoconazole 0.01 1.00 0.0753 0.0310 3.43 m
0.50 0.0155 5.86 azoles is likely to be far more significant as our correlations /ja
Voriconazole 0.42 1.00 4.58 0.384 12.9 show.Potentialimpactfrommucosalandtopicalazoleswasmin- t/a
Miconazole 0.10 10..0500 0.0024 30..61192 241..800 imalinourcalculations,butsomecautionmustalsobeapplied rticle
0.50 1.80 1.00 basedoncasereportsoftoxicityintheliterature.Whileopioid -a
1.00 2.40b 3.61 1.66 fatalities most often arise from the toxic effect of the opioid bs
OMxicycoondaozonleebyCYP02.1D06 1.00 0.0024 3.92 1.00 itself, our studies suggest that further considerations may be trac
0.50 1.96 1.00 required if evidence is found for co-use of drugs such as the t/3
1.00 2.40b 3.92 1.61 azolesthatcancausepharmacokineticinteractions. 9/5
R-methadonebyCYP3A4(valueswouldbesimilarforS-methadone) /3
Fluconazole 0.89 1.00 3.77 13.7 1.28 7
4
Itraconazole 0.01 10..0500 0.0198 26..1805 11..0515 Supplementarydata /76
0
0.50 1.05 1.02 6
Supplementary data are available at Journal of Analytical 9
Ketoconazole 0.01 1.00 0.0753 0.0673 2.12 2
0.50 0.0336 3.24 Toxicologyonline. b
y
Voriconazole 0.42 1.00 4.58 1.93 3.37 g
0.50 0.965 5.74 u
e
Miconazole 0.10 1.00 0.0024 2.02 1.00 Funding s
0.50 1.01 1.00 t o
1.00 2.40b 2.02 2.19 ThisworkwassupportedbyanawardfromtheNationalInstitute n 0
FRl-umcoenthaazdoolenebyC0Y.8P92B6(v1a.l0u0eswouldbesimilarfo3r.7S7-methadone)210 1.02 ofJustice,OfficeofJusticePrograms,U.S.DepartmentofJustice 6 M
0.50 105 1.04 (awardno.2011-DN-BX-K532).Theopinions,findingsandcon- ay
Ketoconazole 0.01 1.00 0.0753 7.86 1.01 clusionsorrecommendationsexpressedinthisarticlearethose 2
0
Voriconazole 0.42 10..0500 4.58 13..6913 31..8042 of the author and do not necessarily reflect those of the 19
0.50 0.805 6.69 DepartmentofJustice.
aPlasmaproteinbindingfromMaoetal.(51).
bHypotheticalmiconazoleunboundplasmaconcentrationneededtoachievesignificantpredicted References
inhibition. 1. Maxwell,J.C.,McCance-Katz,E.F.(2010)Indicatorsofbuprenorphine
andmethadoneuseandabuse:whatdoweknow?AmericanJournal
Smallchangesinplasmaconcentrationsoftheopioidswestud- onAddictions,19,73–88.
iedmaynotbeasclinicallyimportantasthoseinvolvingwarfarin, 2. CDC(2012)Vitalsigns:riskforoverdosefrommethadoneusedfor
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cautionarynote.
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While our extrapolations have not used the more detailed
theopioidreplacementtherapyandanalgesicdrugs:methadone
equationsthattakeintoaccountfractionmetabolizedbydiffer- and buprenorphine. Expert Opinion on Drug Metabolism and
entCYPs,andcontributionofmetabolisminthegastrointestinal Toxicology,9,679–697.
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