History of Aromatase

33 Pages·2009·0.55 MB·English

R E V I E W
History of Aromatase: Saga of an Important
Biological Mediator and Therapeutic Target
R. J. Santen, H. Brodie, E. R. Simpson, P. K. Siiteri, and A. Brodie
DivisionofEndocrinologyandMetabolism(R.J.S.),UniversityofVirginia,Charlottesville,Virginia22908;Emeritus
Investigator(H.B.),Fulton,Maryland20759;PrinceHenry’sInstituteofMedicalResearch(E.R.S.),Melbourne3168,Australia;
DepartmentofObstetricsandGynecology(P.K.S.),UniversityofCaliforniaatSanFrancisco,SanFrancisco,California94143;
andDepartmentofPharmacology(A.B.),SchoolofMedicine,UniversityofMaryland,Baltimore,Maryland21201 D
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Aromataseistheenzymethatcatalyzestheconversionofandrogenstoestrogens.Initialstudies de
d
ofitsenzymaticactivityandfunctiontookplaceinanenvironmentfocusedonestrogenasacompo- fro
nentofthebirthcontrolpill.Atanearlystage,investigatorsrecognizedthatinhibitionofthisenzyme m
h
couldhavemajorpracticalapplicationsfortreatmentofhormone-dependentbreastcancer,alter- ttp
ationsofovarianandendometrialfunction,andtreatmentofbenigndisorderssuchasgynecomastia. s://a
Twogeneralapproachesultimatelyledtothedevelopmentofpotentandselectivearomataseinhib- c
a
d
itors.Onetargetedtheenzymeusinganalogsofnaturalsteroidalsubstratestoworkouttherelation- e
m
shipsbetweenstructureandfunction.Theotherapproachinitiallysoughttoblockadrenalfunctionas ic
.o
atreatmentforbreastcancerbutledtotheserendipitousfindingthatanonsteroidalP450steroido- u
p
genesisinhibitor,aminoglutethimide,servedasapotentbutnonselectivearomataseinhibitor.Proof .c
o
m
ofthetherapeuticconceptofaromataseinhibitioninvolvedavarietyofstudieswithaminoglutethim-
/e
ide and the selective steroidal inhibitor, formestane. The requirement for even more potent and drv
selectiveinhibitorsledtointensivemolecularstudiestoidentifythestructureofaromatase,todevel- /a
opmentofhigh-sensitivityestrogenassays,andto“mega”clinicaltrialsofthethird-generation rticle
aromatase inhibitors, letrozole, anastrozole, and exemestane, which are now in clinical use in -a
b
s
breastcancer.Duringthesestudies,unexpectedfindingsledinvestigatorstoappreciatetheim- tra
portantroleofestrogensinmalesaswellasinfemalesandinmultipleorgans,particularlythebone ct/3
andbrain.Thesestudiesidentifiedtheimportantregulatorypropertiesofaromataseactinginan 0/4
autocrine,paracrine,intracrine,neurocrine,andjuxtacrinefashionandtheorgan-specificenhanc- /3
4
3
ers and promoters controlling its transcription. The saga of these studies of aromatase and the /2
3
ultimateutilizationofinhibitorsashighlyeffectivetreatmentsofbreastcancerandforuseinrepro- 5
5
2
ductivedisordersservesasthebasisforthisfirstEndocrineReviewshistorymanuscript.(Endocrine 1
3
Reviews30:343–375,2009) b
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I. Introduction C. Comparison of antiestrogens vs. aromatase n
0
II. DiscoveryandCharacterizationofAromatase inhibitors 5
III. EAa.rlEyxPthraygsiloalnodguiclaarlSestutrdoigeesnofprAordoumctaitoanse DE.. KSteuydiinestetronaotpiotinmailzecoanrfoemreantcaeseinhibitortherapy April 2
B. Cell-specificsiteofaromataseinbreast VI. Preclinical Studies of Third-Generation Aromatase 019
Inhibitors
C. Insituproductionvs.uptake
A. Modelsystems
IV. SteroidalAromataseInhibitorDevelopment
B. Switchingstudiesinmodelsystems
A. Historicalaspects
VII. Clinical Studies of Third-Generation Inhibitor
B. 4-OH-androstenedione
Development
V. NonsteroidalAromataseInhibitorDevelopment
A. Historicalbackground VIII. “Mega”ClinicalTrials
B. Medicalvs.surgicaltherapy A. Comparisonofdifferenthormonaltherapies
B. Switchingtrials
ISSNPrint0021-972X ISSNOnline1945-7197
PrintedinU.S.A. Abbreviations:A,Androstene-3,17-dione;AG,aminoglutethimide;CC,clomiphene
Copyright©2009byTheEndocrineSociety citrate;Cox2,cyclooxygenase-2;DHEA,dehydroepiandrosterone;ER,estrogenreceptor;
doi:10.1210/er.2008-0016ReceivedApril29,2008.AcceptedMarch27,2009. HC,hydrocortisone;mTOR,mammaliantargetofrapamycin;4-OH-A,4-hydroxy-A;PGE2,
FirstPublishedOnlineApril23,2009 prostaglandinE2.
EndocrineReviews,June2009,30(4):343–375 edrv.endojournals.org 343
344 Santenetal. HistoryofAromatase EndocrineReviews,June2009,30(4):343–375
C. Management of toxicity due to aromatase males,suchasitsroleinenergybalance(6),maintenance
inhibitors of bone (7), regulation of pituitary gonadotropins (8),
D. Evaluationofemergingstrategieswitharomatase
modulation of spermatogenesis (9), and development of
inhibitors
gynecomastia(10).
IX. Resistance to Aromatase Inhibitors: Back from the
BedsidetotheBench Thestoryofthediscoveryofthearomataseenzymeand
X. FutureStrategiesforUsageofAromataseInhibitors its wide-ranging physiological actions provides insight
A. Aromataseinhibitorscombinedwithgrowthfac- intotheunexpectedcomplexitiesofhormonalregulation
torinhibitors andhighlightshowthepreparedmindcanprocessunex-
B. Blockadeofaromatasespecificallyinbreast
pected findings to generate new hypotheses, a scientific
C. Preventionofbreastcancer
process characterized in detail by Claude Bernard in the
XI. Purification,StructuralCharacterization,andRegula-
mid-19th century (11). This first “history” manuscript
tionofAromatase D
A. Purification publishedbyEndocrineReviewswillattempttocapture ow
n
B. Promoterregulation these aspects of the studies on aromatase and of the in- lo
a
C. Promoterswitching vestigatorswhocontributedtothisprocess. de
XII. BDi.oloTghirceael-FduimncetnisoinonoaflAstrroumctautraeseinSpecificOrgans d fro
m
A. Placenta h
B. Brain II. Discovery and Characterization ttp
C. Bones of Aromatase s://a
c
D. Testis a
Theisolationandbiochemicalcharacterizationofan- d
E. Prostate em
XIII. Clinical Use of Aromatase Inhibitors in Gynecology drogensandestrogensinthe1930sallowedrecognitionof ic
.o
andPediatrics the similarities between these two classes of compounds u
p
A. Anovulation and the speculation that C19 steroids might be directly .c
o
B. Endometriosis convertedtoC18estrogens(1–4,12).BernhardZondek m/e
C. Gynecomastia d
(12) speculated correctly in 1934 that “the female hor- rv
D. Shortstature /a
E. Aromataseexcessanddeficiencysyndromes monewhichisregularlypresentinthemaleorganismrep- rtic
XIV. LessonsLearnedfromStudiesofAromatase resentsanormalphysiologicalproductofthemetabolism le-a
A. Importanceofestrogensinthemale ofthesexhormonesespeciallysince—duetoourpresent bs
B. Estrogen action via autocrine, intracrine, para- chemicalknowledge—aconversionofthemalehormone trac
crine,juxtacrine,andneurocrinemechanisms intothefemaleoneappearstobequitepossible.”Steinach t/30
XV. SummaryandConclusions /4
andKun(13)in1937providedthefirstexperimentalev- /3
4
idenceofthisconversionbyadministeringunlabeledtes- 3
/2
3
tosteronepropionatetomenanddemonstratingenhanced 5
I. Introduction estrogenic activity in urine. Later, the focus on develop- 521
3
Theinitialdiscoveryofestrogensandtheirbioactivityin mentoforalcontraceptivesprovidedapracticalimpetus by
urinaryextractsinthe1920sprovidedtheimpetusto forintensivestudyofestrogensynthesisandmetabolism. gue
better understand the structure, biosynthesis, secretion, The Worcester Foundation for Experimental Biology st o
andfunctionofthevariousestrogens.Workingindepen- (WFEB), established in 1944 in Shrewsbury, Massachu- n 0
5
dently, Edward Doisy and Adolf Butenandt isolated es- setts, became an incubator for scientists interested in re- A
p
triol, estrone, and estradiol from pregnancy urine in the production, neurophysiology, and steroid biochemistry. ril 2
This ultimately led to the development of the oral contra- 0
early 1930s (1–5). These studies prompted attempts to 1
9
ceptivepillbyfounderGregoryPincus*andM.C.Chang.*
determinethespecificmechanismsofestrogensynthesis.
AmajorinterestoftheFoundation’sresearchwasthepro-
The initial discovery of the aromatase enzyme and the
gram focused on biosynthesis and metabolism of steroids
unravelingofitsvariedfunctionsrepresentanintriguing
producedbytheadrenalglands,testes,andovaries.
andillustrativeseriesofeventsandamajorsuccessstory
Recruitment of Ralph Dorfman* to the Foundation
inthefieldofendocrinologyregardingbreastcancerther-
(1951–1964) enhanced efforts to understand steroid
apy.Aromataseprovidedoneofthefirstmoleculartargets
metabolismandprovidednecessarybioassayexpertise.
forrationaldrugdevelopmentinthetreatmentofcancer.
Dorfman brought with him a team of scientists that in-
Comprehensive studies of aromatase demonstrated the
cludedtheenzymologist,MikaHayano,*whoselabora-
importance of extraglandular hormone synthesis and of
torybecameacenterforresearchonestrogenbiosynthesis.
intracrine, autocrine, paracrine, juxtacrine, and neuro-
crine mechanisms of estrogen action. Study of its physi-
*Aphotographofeachindividualidentifiedwithintextbyanasteriskappearsonlineonthe
ology revealed many surprising findings, particularly in AromataseHistoryWebSite(www.freewebs.com/aromatasehistory).
EndocrineReviews,June2009,30(4):343–375 edrv.endojournals.org 345
These investigations benefited from collaborations with sequencetobetestosterone319-hydroxyandrostenedi-
Andre Meyer, a Swiss scientist working with Pincus. one 3 19-oxoandrostenedione 3 estrogen. They also
Meyertookupthechallengeofidentifyingmetabolitesof notedthestrictrequirementofreducednicotinamidead-
4-androstene-3,17-dione(A)inincubationsofbovinead- enine dinucleotide phosphate (NADPH) and oxygen for
renalhomogenates.In1955,Meyermadethecrucialdis- theconversionofboth19-oxygenatedcompoundstoes-
coverythatandrostenedionewashydroxylatedbythebo- trogens.TheAringoftheestrogenmoleculethenreceived
vine adrenal to yield 19-hydroxy-A (19-OH-A) (14, 15) the experimental attention of Aubrey Thompson, Pentti
andnotedthat“theremovaloftheangularmethylgroup Siiteri,* and Paul MacDonald at University of Texas
atC19,asteprequiringintroductionofahydrogenmol- SouthwesternMedicalSchoolinDallas,JackFishmanand
eculetooxygen,facilitatedaromatizationofringAorBof his group at Rockefeller University, and Eliahu Caspi at
the steroid.” He recognized the enzymatic nature of 19- theWFEB(22–26).Theseinvestigatorsproposedseveral
D
hydroxylation and suggested that this was likely a first mechanisms,butdetailsofthereactionsequence,andin ow
n
step in the biosynthesis of estrogens from nonaromatic particularthethirdstep,haveeludedinvestigatorsuntil lo
a
d
steroids. He believed that aromatization involved more thisday(27,28),andnovelmechanismscontinuetobe e
d
thanoneenzymaticstepandtermedthisthe“aromatiza- suggested (29, 30). fro
m
tion process” (14, 15). Kenneth Ryan later agreed but AnotherpostdoctoralfellowfromtheNIHTrainingPro- h
speculatedaboutthepossibilityofasingleenzymaticstep gramforSteroidBiochemistrytojointheHayanolaboratory ttp
s
bystating:“Althoughthearomatizationofandrogensto wasHarryBrodie,*whoinitiallysoughttounderstandthe ://a
c
a
estrogensprobablyrepresentsamultienzymesystem,itis stereochemistryofhydrogeneliminationattheC-1position d
e
m
interestingtonotethatthetimecurveislinearforthefirst duringaromatization.InpreviousworkbyGual*etal.and
ic
hour”(16).Definitiveproofthatonlyasingleenzymewas Moratoetal.(20,21),anumberofsteroidstructureswere .ou
p
involvedawaitedpurificationofaromataseinthe1980s evaluatedaspossibleprecursorstoaromatizationofringA. .c
o
(seeSectionIX.A). Thesestudiessuggestedthatthe(cid:1)4-3-onestructureinringA m/e
Dorfman and Kenneth Savard later provided further was required but that the (cid:1)4-3-one structure was a poor drv
/a
insight into the “aromatization process.” They demon- initialprecursor,aswas19-norandrostenedione.Thepres- rtic
strated conversion of radiolabeled testosterone to estro- enceofaxial(cid:1)substituentsatC-11interferedwitharoma- le
-a
genbythehumanovaryinstudiescarriedoutattheWFEB tization,whereasthoseatthe11(cid:2)and9(cid:2)positionsdidnot. bs
incollaborationwithtwoHarvardscientists,LewisEngel The cumulative evidence suggested that 19-oxo-andro- trac
andBillyBaggett(17).Keytothesuccessofthesestudies stenedionerequiredhydroxylationandcofactorstobecon- t/30
/4
wastheuseofradiolabeledtracersteroidssynthesizedby vertedtoestrogenandthefinaloxidationstepinvolvedde- /3
4
the laboratory of Marcel Gut at the WFEB. Before the hydrogenationatC-1andC-2. 3/2
availability of radiolabeled steroids from commercial Because aromatization appeared to involve (cid:1)-face at- 35
5
sources, Gut’s radiolabeling expertise was critical to tacks,theprecisesite(i.e.,(cid:2)or(cid:1)hydrogen)wheretheste- 21
3
workingoutsteroidbiosynthesisandmetabolicpathways reochemical elimination of hydrogen at C-1 occurred be- b
y
g
(18). Meyer, Hayano, and co-workers further demon- cameofinterest.HarryBrodie,MoratoandGut,together u
e
s
strated formation of estrogen during incubation of 19- withothersintheHayanolab,preparedsamplesofandro- t o
hydroxyandrostendione with various tissues including stenedionelabeledwith3HatC-1ineitherthe(cid:2)or(cid:1)posi- n 0
5
ovaries and adrenals (14, 15). Nevertheless, the yields tion.Uponincubationwithplacentalmicrosomes,83%of A
p
were quite low, and it remained for Kenneth Ryan and tritiuminthe1(cid:1)positionwaslostduringconversiontoes- ril 2
LewisEngelatHarvardtoutilizehumanplacentalmicro- trone, whereas 85% of the 1(cid:2)tritium was retained, indi- 01
9
somalpreparationstoconvertandrogenstoestrogensin catingtheimportanceoflossofthe1(cid:1)-hydrogen(31,32).
high yields (16). This microsomal system became the Later,HarryBrodieandJohnTownsleyestablishedthat
mainstayforstudyingthearomatizationofandrogensto the2(cid:1)-hydrogenwasalsorequired(33).Theobservations
estrogensandthemechanismsinvolved(19). that 19-norandrostenedione and androsta-1,4-diene-3,17-
Investigation of the aromatization reaction benefited dione are poor substrates for conversion to estrogen sug-
fromthecriticalmassofrecruitedinvestigatorsandtrain- gested that hydrogens are removed after oxidation of the
ees attracted by the National Institutes of Health (NIH) C-19 methyl group to form the 19-hydroxy or 19-oxo
TrainingProgramforSteroidBiochemistryjointlyheldat compounds.
the WFEB and Clark University (20). Work from the Theseearlystudiesofaromataserequiredmultisteppu-
Hayano team led Tomas Morato and colleagues to pro- rificationsandmeticulousattentiontodetail.Apractically
pose 19-oxo-androstenedione as the intermediary be- importantadvancewasthedevelopmentofanaromatase
tween 19-OH-A and estrogen (21) and to postulate the assay requiring only one step. Knowledge of the 1(cid:1)-hy-
346 Santenetal. HistoryofAromatase EndocrineReviews,June2009,30(4):343–375
drogen loss stimulated Aubrey Thompson and Pentti Craig Jordan* at WFEB in 1972 added to an environ-
Siiteri*in1974(23,34)todevelopaone-stepradiometric ment focused on breast cancer treatment (43). Jordan
assayinwhichtritiumreleasefromthe1(cid:1)-tritium-labeled came to work with Michael Harper who had been in-
substratewasmeasuredastritiatedwater(3H O).Asval- volvedinoriginalworkontamoxifenwithArthurWal-
2
idation,theydemonstratedthattherateof3H Orelease poleintheUnitedKingdom(44,45).
2
andformationofisolatedestrogenproductweresimilar.
Thisassayfacilitatedstudyofpotentialaromataseinhibitors
inlaterexperiments(35–38).Bytheearly1970s,investiga- III. Early Physiological Studies of Aromatase
torsgenerallyagreeduponseveralfactsregardingthemech-
A.Extraglandularestrogenproduction
anismofaromataseaction.Conversionofandrostenedione
Inthe1970s,PaulMacDonald,PenttiSiiteri,andtheir
toestrogeninvolved19-OHand19-oxointermediates,but
Dallas collaborators made the sentinel discovery of ex- D
notthe19-oicacid,asprecursors.Thearomatizationofring ow
traglandulararomataseactivityinbothmenandwomen. n
Ainvolvedtheeliminationofthe1(cid:1)-and2(cid:1)-hydrogens(31, lo
Standarddogmabeforethattimedictatedthatsteroidhor- a
39,40),suggestingthataromataseactedonthe(cid:1)-faceofthe de
mones were only produced in endocrine glands such as d
substrate.Threemoleseachofoxygenandreducednicotin- ovary,testis,andadrenal.Theseinvestigatorsshowedthat from
asummideedapdeernminoeldeionfucalnedortoidgeenpshocospnhvaetrete(dNtAoDesPtHro)gwenerbeycothne- adiposetissuewasarichsourceofaromatase(10,22,23, http
humanplacentalaromatase(22,34).Thereactioninvolved 41, 46–53). These investigators demonstrated that pe- s://a
ripheral tissue provides themajorsourceofestrogensyn- c
a
a cytochrome P450-mediated enzyme, as evidenced by d
thesisinmenandinpostmenopausalwomenandthatthe e
m
blockadewithaminoglutethimide(AG),aknowninhibitor
reactioninvolvesaromatizationofadrenalandrostenedione. ic
ofadrenalP450-mediatedhydroxylation(41,42).Thearo- Studiesofpostmenopausalwomenshowedthattheconver- .oup
matizationreactionwasnotinitiallyconsideredtoinvolve .c
sion of androstenedione to estrogen was higher in obese o
m
P450becauseitcouldnotbeinhibitedbycarbonmonoxide, subjects, suggesting that adipose tissue might be the pri- /e
d
theclassictestatthattimeforP450-mediatedsteps. marysiteofaromatization(47).Toprovethishypothesis, rv/a
Asaresultofhisongoingmechanisticstudies,HarryBro- clinical studies demonstrated that total body aromatiza- rtic
die recognized the therapeutic potential of targeting aro- le
tion of plasma androstenedione increased from 0.5 to -a
mitoartsaisneathnedebaerglyan1d9e7v0esl.oIpnm1e9n7t3o,fhsieslegcrtoivuepapruobmliasthaesdeiinnhitiiba-l 1in0%wowmiethna(4r7is)e. Tinhbesoedydawtaeipghrotvfirdoemd1th0e0ftiors4t0in0dpicoautniodns bstrac
systematicstudiesonthedevelopmentofcompetitivesteroi- that adipose cells might express the aromatase gene and t/30
/4
dalaromataseinhibitors(17).Althoughanumberofclassical could readily explain how obesity increases the risk of /3
4
inhibitorsthatblockedmultiplecytochromeP450hydroxy- both endometrial and breast cancer in postmenopausal 3/2
3
lases had previously proved useful for mechanistic studies women.TogetherwithanObstetricsandGynecologyres- 55
2
(i.e.,cyanoketonep-hydroxymercuribenzoateandAG)(34, ident,A.Schindler,theSiiterigroupdirectlydemonstrated 13
35), none of these compounds was specific for aromatase thepresenceofaromatasewiththetritiumreleaseassayin by
g
(35,40).TheWFEBgroupreasonedthatsteroidalanalogsof surgicallyobtainedadiposetissuesfromwomen(54). ue
s
aromatasesubstrateswouldbemorespecificinhibitors,re- Breasttissuewasalsofoundtohavesubstantialaromatase t o
n
sultingfromtheirhigh-affinityinteractionswiththeactive activity.Basedontheclinicalinterpretationofdatafroma 0
5
siteoftheenzyme.Brodie’sgroupsystematicallyexamined youngboyreferredwithseveregynecomastiabutnofemi- A
p
more than 100 steroidal structures and substrate analogs nizingtumor,MacDonaldandSiiterifoundthat50%ofthis ril 2
(17)andinferredimportantstructuralcomponentsforinhi- patient’s plasma androstenedione was converted to estro- 01
9
bitionfromthesedata. gensthrougharomatization(10).BasedonstudiesofTom
Collaborationbetweeninvestigatorswithdifferentinter- Dao,Siiteripostulatedfromitsstructurethattestololactone
estsandexpertiseoftenleadstoabroaderexperimentalper- wasanaromataseinhibitorandsuggesteditsusetotreatthis
spective (17). As an example of this, a timely marriage of patient.Notably,estrogenlevelswerereducedby79%,and
concepts and individuals led to a biological focus on aro- subsequentstudiesbySiiteriand Aubrey Thompson, his
matase inhibitors for use in breast cancer patients. Angela graduatestudent,laterconfirmedthisactivityoftesto-
Brodie*hadarrivedattheWFEBin1962.Shesubsequently lolactoneusinganaromataseindex(e.g.,determination
marriedHarryBrodieandjoinedhislabasStaffScientistin of the ratio of androgens to estrogens) developed in
1971.AngelaBrodiehadpreviouslyworkedattheChristie collaboration with Howard Judd (46, 50).
CancerHospitalinManchester,UnitedKingdom,asiteof OriginalstudiesofWilliamMiller,*JohnAdams,Tom
activeinvestigationofhormonetherapiesforbreastcancer Dao,Y.J.Abul-Hajj,andDonaldKillingerdirectlydemon-
and later of tamoxifen development. The arrival of stratedlocalestrogensynthesisinbreastcancertissueusing
EndocrineReviews,June2009,30(4):343–375 edrv.endojournals.org 347
classicalproductisolationtechniques(55–57).Laterstudies plasmaandbreasttissue.Twogroups,thoseofWilliam
ofAlanLipton,*RichardSanten,*andAngelaBrodie(58– Miller*(77)andMichaelReed*(78),carriedoutthesecom-
60) used Siiteri’s (3H O) radiometric aromatase assay plexstudiesandconcurredthat50–70%oftheestrogenin
2
method(22,23)toquantitatethelevelsofaromataseina the breast resulted from local synthesis and the remainder
large number of breast cancers and correlate activity with fromuptake.AnitaDunbier,apostdoctoralresearchfellow
biologicalparameters.Theyfoundthatbothbenignandma- ofMitchDowsett,*recentlyconductedanelegantstudythat
lignantbreasttissuescontainaromatase(61–67). correlatedplasmaestradiollevelswithestrogen-responsive
geneexpressioninER(cid:2)breasttumorsandfoundfourgenes,
TFF-1,GREB1,RAGE,andAZGP1,withcorrelationcoef-
B.Cell-specificsiteofaromataseinbreast
For a number of years, controversy existed whether ficientsstatisticallysignificantatP(cid:3)0.001(79).Tumortis-
aromataseresidedpredominantlyinstromalorepithelial sueER(cid:2)levelsintumoralsocorrelatedwithgeneexpression. D
breast cancer cells (68, 69). The viewpoints of various Usingmultivariateanalysisoftwoindependenttumorsets, ow
n
investigators varied depending upon the methodology theyconcludedthatanaverageof19%ofthevariabilityof loa
d
used.Immunohistochemistrywithmonoclonalantibodies these genes was explained by plasma estradiol levels, and e
d
favoredanepithelialsource(69),whereaspolyclonalan- 13% from the levels of ER(cid:2)present. Interestingly, when fro
m
tibodiesorisolationofstromalandepithelialcellsintissue DunbierproposedthisstudytoDowsett,heinitiallythought h
culture and measurement of aromatase in these cells fa- thattheanalysiswasquiteunlikelytobeinformativeandwas ttps
voredfibroblasts(61,70).Convincingevidencethatboth notofhighenoughprioritytobedone. ://a
c
a
stromal and epithelial cancer cells contained aromatase William Miller’s group (80) and later Serdar Bulun* d
e
m
awaitedstudiesbyHironobuSasano*usinglasercapture andcolleagues(81)providedfurtherindirectevidencein ic
methodologywithquantitativePCRofaromatase.With favoroflocalestrogensynthesisinthebreast.Theydeter- .ou
p
this methodology, his group clearly demonstrated an minedtheactivityofaromataseanditsexpressioninquad- .c
o
m
equalroleforeachcompartmentinhumanbreastcancer rantswherebreasttumorsusuallyarose.Thequadrantof /e
d
tissue (71). Stromal and epithelial cells are not the only thebreastcontainingthetumorgenerallyhadthehighest rv
/a
sources of estrogen production in human breast cancer aromataseactivityandexpressionofmessage.Oneinter- rtic
tissue.GilMor*andhiscolleaguesdemonstratedthepres- pretation of these data is that a tumor is more likely to le
-a
enceofaromataseinmacrophages,acelltypecomprising develop in the region of the breast where aromatase ex- bs
upto25%ofthecellsfoundinbreasttumors(72,73). pressionishighest.Analternateexplanationisthatfactors trac
produced by the tumor stimulate aromatase expression t/30
/4
C.Insituproductionvs.uptake locally within the surrounding mesenchymal cells and /3
4
Animportantquestionaddressedwhetherbreasttumor withinthetumoritself.ProstaglandinE2(PGE2)isacan- 3/2
3
estrogenconcentrationsresultprimarilyfromlocalsynthesis didate factor because it is a powerful stimulator of aro- 55
2
inthetumorviaaromataseorfromplasmauptakeinpost- mataseexpressioninbreastadiposestromalcells(82,83). 13
menopausalwomen.Thepresenceofveryhighlevelsofes- PGE2isformedviacyclooxygenase-2(Cox2),whoseex- by
g
trogeninbreasttissueprovidedinferentialevidencethatlocal pressionishighinmanybreasttumorswhereitcorrelates ue
s
synthesis might predominate (74). Eleven studies reported withtumorsize,grade,Her-2positivity,andanunfavor- t o
n
meanestradiollevelsof46–480pg/ginbreastcancertissue abledisease-freeinterval.MoreoverCox2inhibitorshave 0
5
from postmenopausal women, levels substantially higher beenshowntoinhibitaromataseactivityofbreastcancer A
p
thanplasmaestrogenlevelsof2–10pg/mlaftermenopause cells(83).TherelationshipbetweenCox2andbreastes- ril 2
(75, 76). However, the maintenance of higher tissue than trogenlevelsisofinterestbecauseofepidemiologicalstud- 01
9
plasmalevelsafterthemenopausecouldalsoreflectuptake iesinpatientsreceivingaspirinornonsteroidalantiinflam-
againstagradientmediatedbyhigh-affinityestrogenrecep- matory drugs and development of breast cancer. As
tors (ERs). The first direct assessment of this question in- reviewedbyRobertBrueggemeier(83),regularuseofsuch
volvedaxenograftcastratemousemodelsystemdeveloped drugs correlates with up to a 50% reduction in breast
byWeiYue,*aformertraineeofAngelaBrodie(62).Cas- cancer risk over a 5-yr period. Use of acetaminophen,
trationwasusedinthisandsubsequentstudiestomodelthe whichdoesnotblockCox2,wasusedasacontrolandwas
postmenopausal state in women. Her data suggested that associatedwithnoreductioninbreastcancerrisk(84).
componentsofbothuptakeandlocalsynthesisarephysio- Anadditionalcomplexityregardinglocalestrogensyn-
logically important. Attempts to determine this directly in thesisinbreastistherelativeroleofaromatasevs.forma-
postmenopausal women with breast cancer involved infu- tionfromcirculatingestronesulfateviathesulfataseen-
sion of 3H-androstenedione and 14C-estrone for recovery zyme (85–87). The isotopic kinetic methods using
corrections and determinations of radioisotope ratios in tritiatedandrostenedioneassubstratedescribedinSection
348 Santenetal. HistoryofAromatase EndocrineReviews,June2009,30(4):343–375
III.A.donotestimatelocalformationviasulfatase,and Site-directedmutagenesisdataidentifiedthreeimportantad-
consequentlyradiolabeledestronesulfatemustbeusedto ditional regions in the active site of aromatase, S478 and
assess local synthesis directly (88). With this technique, H480(inthe(cid:1)-4sheetatthecarboxy-terminus)andD309
approximately 19–50% of the estrogen found in rodent (in the I helix). These sites are thought to participate in a
breastcancertissuesfromcastrateanimals(i.e.,postmeno- chargerelaysystemthatleadstothearomatizationoftheA
pausal)originatedfromlocalenzymaticcleavageofestrone ring of the androgen substrate. It has been proposed that
sulfate to free estrone via sulfatase as shown by Shigeru exemestane is converted to reactive intermediates by the
Masamuraandcolleagues(88).Nohumandatausingdirect heme through hydroxylation of the C-19 group, aided by
isotopictechniquesareasyetavailable. D309andT310.Theintermediatesthenbindirreversiblyto
theenzyme,causingsuicideinhibitioninwhichD309may
alsobeinvolved(28).
D
IV.SteroidalAromataseInhibitorDevelopment ow
n
B.4-OH-androstenedione lo
A.Historicalaspects Focusingon4-OH-Aasaleadcompoundafterextensive ad
e
Systematic structure/function studies by the Brodies d
(Harry and Angela) examined nearly 100 steroidal aro- cdoumctpioanriosofnesstarmogoennglienvheilbsiitnorrso,dthenetBsr.oAdsiepsrdoeomfoonfsbtiroaltoegdicrae-l from
mataseinhibitorsandledtotheidentificationof1,4,6-an- efficacy,4-OH-Acausedregressionof7,12-dimethylbenz[a] http
drostatrienedione(19)and4-hydroxy-androstenedione(4- anthracene rat mammary tumors (a model developed by s://a
OH-A) (89) as the most promising candidate inhibitors. c
CharlesHuggins)moreeffectivelythantamoxifen(89).In a
d
Theirfindingsencouragedacadreofinvestigatorswork- e
contrasttotamoxifen,4-OH-Awasnotestrogeniconother m
ingindependentlyatotherinstitutionstoidentifyawide ic
tissuessuchastheratuterus.Studiesinnonhumanprimates .o
mvaoraientdyroofgesntesro(i9d0a)l,in7h-i(cid:3)b-iatomrisn.oTphheesneyilnthcilouadneddr1o6st(cid:2)e-nberdoi-- demonstratedthat4-OH-Ainhibitedperipheral(nonovar- up.co
ian)estrogensynthesisusingradiolabeledtracerstudiescar- m
one (91), and 1-methyl-1,4-androstadiene-3,17-dione /e
riedoutincollaborationwithChristopherLongcopeatthe d
(92).Testololactone,usedforovertwodecadesasatreat- WFEB(104).AngelaBrodiethenestablishedcollaborations rv/a
ment for breast cancer, was found to inhibit aromatase withtwomedicaloncologistsinLondon,CharlesCoombes* rticle
(50). C-19-Substituted aromatase inhibitors include thi- andPaulGoss,*andaclinicalchemist,MitchDowsett.This -ab
iranesandoxiranes(93,94),epoxysteroids(94),andthiol collaborative group enabled the first clinical trials with stra
andaminoanalogs(95,96).PotentAring-bridgedsteroid c
4-OH-Afortreatmentofbreastcancerinwomen.Pilotstud- t/3
inhibitors(97,98)thatconsistofanalogscontainingcar- 0
iesdemonstratedsignificantresponsesto4-OH-A,although /4
bon, oxygen, nitrogen, or sulfur atoms have also been /3
manywomenhadrelapsedfromprevioustamoxifentreat- 4
3
described.ReviewsbyBrueggemeierprovideextensivede- ment. Coombes and Brodie then proposed to Ciba-Geigy /23
tail regarding these and other inhibitors and the investi- thatfurtherclinicaldevelopmentof4-OH-Abeundertaken. 552
gatorsinvolved(37,99). ThelateStuartHughesatCIBA-Geigyquicklyappreciated 13 b
Someoftheinhibitorsdescribedabovewerelaterfoundto y
that selective aromatase inhibitors could have distinct ad- g
cause enzyme inactivation (100) by functioning as mecha- vantagesovertamoxifenandAG.Withpharmaceuticalcom- ues
nism-basedinhibitors.Becausetheseinhibitorsbindtothe pany support, further clinical trials proceeded in the mid- t on
activeenzymaticsite,theyarequitespecificandshouldalso 0
1980sand4-OH-A,renamedformestane,becamethefirst 5
havelastingeffectsinvivoasaresultofinactivatingtheen- A
“selectivearomataseinhibitor”tobecomeavailableforthe p
zyme.Thefirstcompounddesignedasamechanism-based treatmentofbreastcancer. ril 2
0
inhibitorofaromatasewas10-propargy1-4-estrene-3,17-di- 1
9
one(MDL18,962),designedandsynthesizedindependently
by the three research groups of Cecil Robinson, O’Neal
V. Nonsteroidal Aromatase Inhibitor
Johnston,andDougCovey*(38,101,102).Othermecha-
Development
nism-basedinhibitorswereidentifiedamongthemostpotent
steroidalinhibitorsoriginallythoughttobecompetitivein- A.Historicalbackground
hibitors including 4-OH-A, l-methylandrosta-1,4-diene- SirGeorgeBeatsonintroducedbilateraloophorectomy
3,17-dione (92), and 6-methyleneandrost-1,4-diene-3,17- fortreatmentofpremenopausalwomenwithbreastcan-
dione(exemestane). cerin1896(105),andthisapproachgainedcommonusage,
RecentstudiesbyShuianChen*andcolleagues,utilizing asdidsurgicaladrenalectomyandhypophysectomy(106).In
thethree-dimensionalstructuralaromatasemodelgenerated the 1950s and 1960s, several endocrinologists including
byFaviaetal.(103),proposedanewclampingmechanism Mortimer Lipsett, Olaf Pearson, Albert Segaloff, B. J.
ofsteroidalsubstrate/inhibitorbindingtotheactivesite(28). Kennedy,AnneCarter,andothersfocusedondevelopment
EndocrineReviews,June2009,30(4):343–375 edrv.endojournals.org 349
of medical alternatives to these surgical modalities for
treatment of breast cancer. Termed “hormone additive
therapy,” these included administration of glucocorti-
coids, androgens, and even estrogens (107–111). Ralph
Cashsuggestedanotherapproach:directinhibitionofad-
renalsteroidsynthesis.HeknewthatAGcouldblockcho-
lesterol side chain cleavage (112) and reasoned that AG
mightreplacesurgicaladrenalectomyforthetreatmentof
breastcancerinpostmenopausalwomen.Objectivetumor
regression in a single patient with breast cancer treated in
1967providedproofofhisconcept(112).
D
RichardSanten,*duringhisendocrinologyfellowship ow
n
in 1971, evaluated a breast cancer patient who was se- lo
a
d
verelyCushingoidfromhighdosesofprednisoneusedto e
d
suppress the adrenal. Not aware of Cash’s case report, FIG1. Left,Lyticmetastases(arrows)inpelvisandcervicalvertebra fro
m
Santenenvisionedtheuseofdirectadrenalinhibitionwith beforearomataseinhibitortherapy.Right,Healingoflyticlesions h
AGtoavoidthesideeffectsofhigh-doseprednisone.Upon duringadministrationofthearomataseinhibitor,AG,incombination ttp
s
recruitmenttothenewmedicalschoolatPennStateUni- withHC.[ReproducedwithpermissionfromR.J.Santen,E.Ramojlik, ://a
T.J.Worgul1982Aminoglutethimide.ScientificProfile.In:R.J.Santen, c
a
versity,heandamedicaloncologist,AlanLipton,*initi- I.C.Henderson,eds.Pharmanual:Acomprehensiveguidetothe d
e
atedclinicalstudieswithAGin1972.Independentgroups therapeuticuseofaminoglutethimide.2ndRevisedEdition,Basel:S. m
ic
headedbyThomasHallandH.H.(Dicky)Newsomealso Karger;101–161.] .ou
p
begantoexamineAGasbreastcancertreatmentatabout .c
o
aromatase in postmenopausal women (48). Santen’s m
thesametime(113,114). groupthenusedthismethodologyanddemonstrated95– /ed
The adrenal inhibitory properties of AG necessitated rv
98% inhibition in postmenopausal patients with breast /a
use of replacement glucocorticoid, and a long-acting cancer (Fig. 2 (117). This observation focused emphasis rtic
agent, dexamethasone, was chosen (115). However, AG le
uponthearomataseinhibitorypropertiesofAGandledto -a
acceleratedthemetabolismofdexamethasoneandlimited bs
itsglucocorticoidpotency(115).Toovercomethisprob- trac
lem,hydrocortisone(HC)wassubstitutedbecauseitsme- t/30
/4
tabolism was not significantly altered by AG. With the /3
4
availability of a practical AG/HC regimen, pilot studies 3/2
3
withthisregimenwereundertaken.Largelyonthebasisof 5
5
2
anecdotalobservationsoftumorregressions(Fig.1),inves- 1
3
tigatorsbecameconvincedthatAG/HCwaseffective,and b
y
g
multiple clinical trials conducted by Trevor Powles,* Ian u
e
s
Smith,* Adrian Harris,* Robin Murray, and others later t o
n
providedcompellingevidence(106,116). 0
5
A chance meeting between Pentti Siiteri and Richard A
p
Santen led to the realization that AG effectively blocked ril 2
total body aromatase in postmenopausal women. Siiteri 01
9
hadpreviouslydemonstratedin1969thatAGcouldblock
aromatase in vitro (41) and was aware of the reports of
selective and potent aromatase inhibitors by the Brodies
(19). He had also envisioned the potential use of aro-
mataseinhibitorsfortreatmentofbreastcancer(22,23,
34,41,50)andsuggestedthatmanyofthethen-current
treatments for breast cancer worked through aromatase FIG2. TheeffectofAGonaromatizationofandrostenedione((cid:1)-4A)
toestrone(E1)asmeasuredinblood.Thedatapointsrepresent
inhibition(22,23,50).InvitedtolectureatthePennState
individualpatientsstudiedbeforeandduringtreatmentwith1000mg
Medical School in 1974, Siiteri suggested that AG most ofAGand40mgofHCdaily.Thehorizontallinesrepresentthemean
likely was working as an aromatase inhibitor for breast valuesforallfivepatients.Thedownslopinglinesconnectindividual
patientsbeforeandduringtreatment.[Reproducedwithpermission
cancerandadviseduseoftheisotopickinetic,totalbody
fromR.J.Santenetal.:JClinEndocrinolMetab47:1257–1265,1978
aromatizationmethodtodirectlyprovethatAGinhibited (117)©1978TheEndocrineSociety.]
350 Santenetal. HistoryofAromatase EndocrineReviews,June2009,30(4):343–375
its later designation as a “nonselective first-generation” D.Keyinternationalconference
aromataseinhibitor(118–126). MotivatedbythesuccessfulclinicaltrialswithAG/HCin
William McGuire* chaired the Breast Cancer Task the1970sandrecognitionofitsaromataseblockingprop-
ForceoftheNIHatthistimeandstronglyencouragedthe erties,AlanLipton,HaroldHarvey,andRichardSantencon-
PennStategroup,nowcomprisingHaroldHarvey,*Law- ceived and organized an international symposium at Key
Biscayne,Florida,inDecemberof1981todiscussabroad
renceDemers,*SteveSantner,andEugeniuzSamojlik*(in
range of the basic and clinical aspects of aromatase. One
addition to Santen and Lipton), to pursue further work
majorconclusionwasthatthepredominatemechanismof
with AG (127). McGuire facilitated a key collaborative
AGinbreastcancerpatientswasaromataseinhibition,not
arrangementbetweenthePennStategroupandtheBreast
suppression of adrenal steroidogenesis (137, 138). This
Cancer program at Duke University, headed by Samuel
meetingservedasacatalystforinvestmentofmajorresources
Wells.*Thiscollaborationfacilitatedrapidexpansionof D
intotheclinicaldevelopmentofhighlyselectiveandpotent o
thescopeofstudieswithAG. w
aromataseinhibitors(137,138).Severalpresentationsalso nlo
Precisequantitationofthelowestrogenlevelscharacter- a
highlightedtheimportanceofaromataseforarangeofbio- d
e
isticofmenopausalwomen(i.e.,2–10pg/ml)(76)provided d
amajorchallengeforthestudyofaromataseinhibitors.Eu- logicalprocessesinthebrain,ovary,testis,andplacenta.A fro
series of eight follow-up International Aromatase Confer- m
geniuszSamojlik*(128–130)developedRIAsthatmeasured encesbroughttogetherinvestigatorsinthisfieldoverthe http
estrogensinconcentratedurineandinplasmaafterpuri- next25yr,whichhasledtomultiplecollaborativestudies. s://a
ficationbycelitecolumnchromatography,techniquesde- c
a
d
velopedbySiiteri10yrearlier.Themostpracticalmethod E.Studiestooptimizearomataseinhibitortherapy e
m
took advantage of the 100-fold higher levels of estrone AdrianHarris,*MitchDowsett,andtheSantengroup ic.o
sulfateinplasmathanfreeestrogenandinvolvedaspecific attemptedtoreducethedoseofAGoruseitsD-stereoiso- up
.c
methodformeasuringestronesulfate(128,130). mertoreducesideeffectsandeliminatetheneedforHC. om
However,theseeffortswerenotsuccessful(139–143)and /e
d
emphasized the need for selective aromatase inhibitors rv/a
B.Medicalvs.surgicaltherapy suchas4-OH-A.Severalclinicaltrialsdemonstratedthe rtic
ThenextphaseofclinicaltrialscomparedAG/HCwith le
efficacy of an im formulation of 4-OH-A in producing -a
thheeadstaconmdapradrhisoornmsobneatwlmeeondAalGiti/eHsCthaenndavsuairlgaibclael.aHderaendatlo- othbijsecbtaivseis,b4re-aOsHt c-aAn,cnerowregrreensasmioends (f1o2rm6,es1t4a4ne–,14w8a)s. aOpn- bstrac
ectomy were facilitated by the collaboration of a breast provedbytheNationalHealthServiceintheUnitedKing- t/30
/4
surgeonatDukeUniversity,SamuelWells,andthePenn domandrepresentedthefirst“selectivearomataseinhib- /3
4
Stategroup.AG/HCdemonstratedequalefficacyincom- itor”andfirstagentapprovedspecificallyforthispurpose. 3/2
3
parisonwithbothsurgicaladrenalectomyandhypophy- However, with further studies, it became apparent that 55
2
sectomy, both in clinical responses and in estrogen sup- formestane did not block aromatase sufficiently to im- 13
b
pression (131, 132) and led to abandonment of the proveefficacyoverAG,andmorepotentinhibitorswere y
g
surgicalablativemethods.AromataseinhibitionwithAG, deemednecessary(144,149,150). ue
s
however,couldnotreplacesurgicaloophorectomyinpre- Recognizingthepotentialofaromataseinhibitors,sev- t o
n
menopausalwomenbecauseAG/HCdidnotblockovar- eralpharmaceuticalcompaniescontributedsubstantially 0
5
ianfunctionduetointerruptionofestradiolnegativefeed- totheidentificationandclinicaldevelopmentofmorepo- Ap
backwithreflexrisesinLHandFSH(133). tentsteroidalandnonsteroidalselectivearomataseinhib- ril 2
0
itors.Ciba-Geigy(nowNovartis),Merrill-Dow,Imperial 1
9
Chemical Industries (now Astra-Zeneca), Lilly, Pharmi-
C.Comparisonofantiestrogensvs.aromataseinhibitors
talia, Jannsen, and Yamaguchi pharmaceuticals all initi-
Tamoxifenwasbeingdevelopedatapproximatelythe
atedaromataseinhibitordevelopmentprograms.Thefirst
sametimeasAGinthe1970s,anddirectcomparisonsof
agenttoemergewasthesecond-generationinhibitorCGS-
the two approaches were deemed necessary. Four ran-
16949A, later renamed fadrozole, a compound initially
domizedclinicaltrialscomparedAGwithtamoxifen(106,
studiedbyRonaldSteeleatCiba-Geigy(151).Thegroups
134–136)andfoundequalclinicalefficacybutfewerside ofLawrenceDemers*atPennStateandPatrickTrunetat
effectsandtoxicitywiththeantiestrogen.Asanadditional Novartisconcomitantlydemonstrateditsabilitytoinhibit
disadvantage,useofAGrequiredcoadministrationofre- estrogensinwomenbutunexpectedlyuncoveredaldoste-
placementHC.Basedontheseconsiderations,tamoxifen rone-blocking properties (152, 153). This limited the
became established as first-line hormonal therapy for dosesoffadrozolethatcouldbegiventoblockaromatase
breastcancer,apositionheldforthenexttwodecades. moreeffectivelythanwithAGorformestane(152,154).
EndocrineReviews,June2009,30(4):343–375 edrv.endojournals.org 351
Although approved for use in Japan, fadrozole never gonadotropins.Thus,inhibitorstargetingaromataseand
achievedsubstantialusage(155).Morepotentandselective alsoantiestrogenscouldbestudiedintumorsformedfrom
aromataseinhibitorswereclearlyneeded.Usinganarrayof these cells. The third-generation aromatase inhibitors,
resources, the pharmaceutical companies then used struc- letrozoleandanastrozole,reducedtumorvolumesignifi-
ture/function analysis, animal models, and sophisticated cantlymoreeffectivelyandforalongerdurationthanta-
hormoneassaystoidentifyleadcompounds.Thisresultedin moxifen. When combined with tamoxifen, these agents
thedevelopmentandlicensingoftwononsteroidalreversible causedsuppressionsimilartotamoxifenalone(165).Be-
inhibitors, anastrozole and letrozole, and one mechanism- causetheantiestrogenfulvestrantcausesERdegradation,
basedsteroidalinhibitor,exemestane.Toemphasizetherole the combination of this antiestrogen with letrozole was
of classical endocrinology in this process, Ajay Bhatnager, postulatedtobemoreeffectivetreatmentthanwitheither
areproductiveendocrinologistworkingatCiba-Geigy,used compound.Alltreatmentssuppressedtumorgrowth(P(cid:3)
D
agamutofbio-andRIAtechniquestoidentifyletrozoleasa 0.001),butletrozoleappearedbetterthanthepureanties- o
w
n
highlypromisingcompound. trogenfulvestrant(1mg/d).However,whenthetwodrugs lo
a
were combined, tumor suppression was significantly de
d
greaterthantreatmentwitheitherletrozoleorfulvestrant fro
VI. Preclinical Studies of Third-Generation alone(P(cid:3)0.0001)(166).Theadditiveeffectontumors m h
Aromatase Inhibitors treated with the combination of these two compounds ttp
s
suggested that some transcription via the ER may occur ://a
A.Modelsystems c
with fulvestrant treatment alone that is not completely a
d
Historically, rodent model systems played an impor- e
blockedbytheantiestrogen. m
tantroleintheinitialdevelopmentofaromataseinhibitors ic
.o
as well as antiestrogens (156), but these represented the B.Switchingstudiesinmodelsystems up.c
premenopausalhormonalstate.Seventypercentofhuman o
Theinitialstudieswithfirst-generationaromataseinhib- m
breasttumorsoccurinpostmenopausalwomeninwhom /e
itorsinvolvedbreastcancerpatientswhohadrelapsedafter d
localestrogensynthesisinthetumorisakeycomponent priorendocrinetherapiessuchasoophorectomy,high-dose rv/a
(asnotedpreviously,aromataseinhibitorsdonotinhibit estrogens,andtamoxifen.Accordingly,inthexenografttu- rtic
le
estrogens in premenopausal women). Therefore, human mormodel,micereceivedtamoxifenfirst,withalaterswitch -a
b
models more comparable to the postmenopausal breast toletrozole.Althoughefficacious,thisstrategyprovedinfe- stra
cancerpatientwereneededtoexaminetheantitumoref- c
riortotreatmentwithletrozoleasfirst-linetreatment(167). t/3
fects of the third-generation aromatase inhibitors being 0
Bothtamoxifenandfulvestrantwereineffectiveassecond- /4
developed.Useofathymicmousexenografts,atechnique /3
linetherapyafterletrozoletreatment.Thesestudiesprovided 4
3
developedinthe1980s(157),allowedinvivogrowthof insightintocurrentswitchingtrialsthatareexaminingthe /23
tumorsfromhumancarcinomacelllines(156,158–160). correctsequencesofhormonaltherapy(166). 552
AlthoughtheathymicmousewithMCF-7tumorsproved 13
b
tobeanexcellentmodelforstudyingantiestrogensunder y
g
premenopausalconditions,itisnotusefulforinvestigat- VII. Clinical Studies of Third-Generation ues
ing aromatase inhibitors in postmenopausal patients. Inhibitor Development t on
Accordingly, xenografts with tumors containing aro- 0
5
matase grown in castrate animals were needed. The three Food and Drug Administration (FDA)-ap- A
p
Shuian Chen (161), from the City of Hope (Duarte, proved third-generation inhibitors (118), anastrozole, ril 2
0
CA),createdtheneededcells(MCF-7Ca)bystablytrans- letrozole, and exemestane, possess 100- to 10,000-fold 1
9
fectingaromataseintowild-typeMCF-7cells,whichthen higherpotencyandgreaterefficacythanAG,formestane,
served as a local source of estrogen by aromatizing an- and fadrozole, and less toxicity than AG and fadrozole.
drostenedione.ThePennStategroupinitiallyshowedthat Testingoftheseagentsusedisotopickinetic,radioimmu-
androstenedione could stimulate proliferation of these nological and ultrasensitive biological assays for assess-
(MCF-7Ca) cells in vitro and that aromatase inhibitors mentofaromataseblockadeandphaseI,II,andIIIclinical
couldblockthisgrowth(162).WeiYue*inAngelaBro- trialsfordeterminationofantitumorefficacy.
die’s group then developed an in vivo xenograft model Tofacilitateclinicalstudiesofestrogensuppression,sub-
withthesearomatasetransfectedMCF-7cells(160,163) stantialfurtherattentionwasdirectedtowardthedevelop-
asarobustmeansofconductingpreclinicalstudies(164, mentofultrasensitiveassaysformeasurementofplasmaes-
165).Becausetheyweredevoidofovaries,animalsbear- trogens,particularlybythegroupsofKarenKlein,Mitch
ingMCF-7Cacellsmimickedthepostmenopausalsitua- Dowsett,LawrenceDemers,JurgenGeisler,*andPerLon-
tionwherearomataseisnotunderfeedbackregulationby ning*(168–171).Theplasmaestronesulfateassays,the
352 Santenetal. HistoryofAromatase EndocrineReviews,June2009,30(4):343–375
ultrasensitivebioassays,andtheisotopickineticmethods the largest number of subjects into specific protocols.
appeared superior to RIAs of estradiol to determine the These trials enrolled up to 10,000 participants each and
degreeofsuppressionofaromatase.Withthemostprecise werepoweredtodetectdifferencesinseveraldiseasepa-
method,isotopickineticmeasurement,aromataseinhibi- rameters. One individual involved in these trials, Paul
tionexceeded98%witheachoftheseinhibitors(Fig.3), Plourde,*isofparticularinterest.Hetrainedasaclinical
with letrozole seemingly slightly more potent than the endocrinology fellow at Penn State during the develop-
otheragents(171–173).BioassayandstandardRIAdata mentofAGandlaterspearheadedtherapiddevelopment
alsodemonstratedprofoundsuppressionofestradiollev- ofanastrozole.HiseffortsledtoitsapprovalbytheFDA
elswiththeseagents(168–170,174–176).PhaseIandII in what proved to be record time (i.e., 43 d) after sub-
clinical trials were then conducted with very promising mission of the request for approval. Anastrozole then
results(176–185). became the initial third-generation inhibitor approved
D
worldwide. o
w
n
Results among studies with each aromatase inhibitor lo
a
VIII. “Mega” Clinical Trials werenearlyuniformanddemonstratedsuperiorefficacy de
d
of the third-generation aromatase inhibitors when com- fro
A.Comparisonofdifferenthormonaltherapies pared with AG/HC and the progestin, megestrol acetate m
Largetrialshavenowbeenundertakentodemonstrate (186–188). Five large, multicenter, multinational, ran- http
the superiority of third-generation aromatase inhibitors domized trials then directly compared the aromatase in- s://a
overtamoxifenandprogestinswithrespecttoefficacyand c
hibitors with tamoxifen in advanced disease (189). All a
d
toxicity. These studies established the superiority of one e
demonstratedthesuperiorityofthearomataseinhibitors m
endocrinetherapyoveranotherforthefirsttime.Asop- ic
inclinicalefficacy,withimprovedresponsesrangingfrom .o
posed to earlier studies generally conducted by single 2 to 13% (Fig. 4). The differences were statistically sig- up.c
groups,thecollaborativeeffortsofseveralinvestigatorsat o
nificantinallbutonetrialinwhichthereceptorstatuswas m
multinationalsiteswererequiredtoconductthesestudies, /e
unknownin55%ofpatients(190). d
and scientific advisory committees generally assumed Toxicitiesrelatedtotamoxifendifferedfromthosewith rv/a
leadershiproles.Firstauthorsoftheresultingpublications thearomataseinhibitors.Pooleddataindicatethattamox- rticle
generallyrepresentedtheinvestigatorswhohadenrolled ifenusewasassociatedwithdeepvenousthrombosesand -ab
s
pulmonaryemboli(7.6vs.4.5%)significantlymorefre- tra
c
quently than the aromatase inhibitor, anastrozole (190, t/3
0
191), whereas nausea, hot flashes, and gastrointestinal /4
/3
distress were comparable. Aromatase inhibitor use was 4
3
/2
associated with an increase in osteoporosis, osteopenia, 3
5
arthralgias,andmyalgia.However,intheonetrialcom- 52
1
paringaromataseinhibitorswithplacebo,itissurprising 3 b
y
howcommonlymostoftheseproblemswerenotedinthe g
u
placebo group (192). Accordingly, the risks causally at- es
tributabletothearomataseinhibitorsarelessthanappre- t on
0
ciated from the comparative trials. For example, in one 5
A
p
ril 2
0
1
9
FIG3. Aromataseactivityremainingduringtheadministrationoffirst-,
second-,andthird-generationaromataseinhibitors(milligramsperday).
Dataareexpressedonalogscaletoemphasizetheexpectedlogdose
responsecharacteristicsofpharmacologicalinhibitors.Withthemost
potentinhibitor,only1%ofaromataseactivitypersistsduringtherapy.
Thedegreeofsuppressionwasdeterminedbyanisotopickineticmethod
using3H-androstenedioneand14C-estronetoassesstheRhovaluebefore
andduringtherapy.TheRhovaluerepresentsthepercentageconversion
ofandrogenstoestrogensinthetotalbodyunderequilibriumconditions. FIG4. Comparisonoftamoxifenvs.aromataseinhibitorsinfive
ANA,Anastrozole;EXE,exemestane;FAD,fadrozole;FOR,formestane; randomized,controlledstudiesinadvanceddisease.[Reproducedwith
LET,letrozole.[ReproducedwithpermissionfromH.Kronenberg,S. permissionfromH.Kronenberg,S.Melmed,K.Polonsky,P.R.Larsen
Melmed,K.Polonsky,P.R.Larsen2008WilliamsTextbookof 2008WilliamsTextbookofEndocrinology,11thEdition,Philadelphia:
Endocrinology,11thEdition,Philadelphia:Saunders(239).] Saunders(239).]