Document Qkk56d2ByxdpYkywe0bDaZneE

TERATOLOGY 50:19-26(1994) Prevention of Fluvastatin-InducedToxicity, MortaHty, and Cardiac Myopathy in Pregnant Rats by Mevalonic Acid Supplementation ROMAN V. HRAE, HOWARD A. 4ARTMAN, ArM RAYMOND H. COX. JR. RegulatoryToxicologyD,rug SafetyDepartment,Sandat ResearchInstitutSea,ndoz PharmaceuticalsCorporation. East Hanover, New Jersey 07936 A.BSTRACT Mevolonic acidisa productof theenzyme HMG-COA reductosewhich isessen- d rats,and-npg-tv-iaenceof teratogenicacti'viity in ratsat --oseMsp to 36 M-g-lWg-loE-ary r@b-b-ia-af-aosesupto 10 tialfor cholesterolbiosynthesis.Fluvastatin(Son- m :&ida-y--(R-.-V-.-I-Ei-B,-u--@-p-ulatsht-d0w7-@d-vaelrI,T-iFnf doz compound XU 62-320) isa potent inhibitorof a perinatal/postnatalstudy (Segment M), 12 and 24 thisenzyme and, hence, mevolonic acid produc- mg/kg/day offluvastatinadministered to pregnant rats tion.In three separate studies,oral administration from day 15 pc (post coitus)through weaning resulted of fiuvastatinat 12 and 24 mg/kg/day to mated ratsfromdav 15 ofLae-st-at-io-n-ffwie'arnoiunggh re- sulted i ntici ted matemal mortalityat the in maternal mortality at or near term and during the ?KPa'dss-gt_-pLtir--r_t-u_n_m_p- '@is m oqrr@t@alliittyv.wyaAsg ireplliicc subsequent follow-up study, and microscopi u time of pa urition lactation.Micro- scope. performed in two studies re- evaiuaribn rev-eale e occurrence ca=r @rrrfybp-4thy 43-n-Fy-itn Lini@mals. No cardiac pathol.oxywas vealed significantcardiac myopathy in the dvin etectedin nonpregnant animals iD_Iheac-atudie&-wjth animals. Drug-relat@edclinicolsigns,significantma- rru-v4LstatiAns. in previous rat studies (Stollet al.,'88)/@', iernal gody--w-ei-gRTro-s-s,-o-n-ciTnoc-rnease in stillito--m Foreit-om-ach lialh I * ip!Ayperkeratosis l@,ups and neonatal mortalitywere also noted at were found. Thes-e-1;ave@been@ashlIlaa@-4n1mwtno be so&-iric-19 one or both close level5.Supplementation of fluv- r-ouents-andto be a result of contact irritat7ioony ?Fu- astafi-n-admi ation with5-00mo 9 1. .0 me- vastatin(Robisonet--9a4l).', va Fo-n-ic--a-ci-*acompletely blocked and/or omelio- @coa dministration of mevalonic acild the jMMedLate ro-t-e-d-tFe--mi-@o,-rFcaor@l c y Pothy, an-d-offi-er prauct of the -e-nz-y-m-e-'RNfU--CoA-re@uctas-hea-sI been a verse e ec s. ese stu iesindicatet e ad- shown toprevent or antagonize various orzan toxiiiii-es verse :@ot0er-onwale,ftfeects)-L@erved . with--ffu-v-o-s-fo-tininduced -iLn-ra-U-a-nr-abbits.ydth_othgr MQ.- goA re e ro@r-re"@ao'u owiinqTo npC,c's@rrttu.h,tion re ultedfrom exog- ductase inhibitors(MacD )nald et al.,'88-,Jigrmbrustet gerated phormocgLQ"l activ at t e ose levels al.,'89).In pregnant rats,it has been shown that 500 a-Um-inisteredi,.e.,inhibitionat the enzyme HMG- -Mj-L/El-u@- val@oni@cacid supp-re-ssL-4We-Te-r-a-@g-e- Co-A reductas@e,@, immediate product mevolonic fiicitoyf mevinolinic -a-ci-a&n, inhibitorof FRW-RUA acia, onci criotesterolbi snrnthe-qlq redu a minister prior to ana approxi- mately 5 hr afteradminist -v:,nigii,@a@id t al.,'83).Since fluvastatinisan inhibitorof Fluvastatin (Sandoz compound XU 62-320) isa syn- theticpotent inhibitorof hydroxymethylglutaryl coen- zyme A (HMG-CoA) reductase, the rate-limiti9n enzyme in cholesterolbiosynthesis (Engstrom et al.,'88; Kathawalaetal.,'88;Kathawala, f iflu-vastatin in rats logs.-an mon dmini n nducu-aig- aificantreductions in serum totalcholesterol,low-dien- the e e HMG-CoK-re-du-ctas-e an-Effi-er-ef6-re-or-me- va onic acid production, a relationship was considei7ed - fF-e-x-i'st-betwetehne exaggerate ._p kCE-O-!p-qI@t@i ity associated with HMG-COA r:?ductasW7i7iifiibiiio-n,-by fluv -s-utiz&iLad the occurrence of ma-fFfn-aT'ih-OFElfiy andio s. The study reported hetree R-to-ae-fer@minewhether coa i;@ini-sti-a-toifomne- sitylipoproteincholesterol,and serum triglyce-n-de-rev- eTs7En-ptrom e-t-aT.-,%Bi)).unng the safety ssment of fluvastafin,-nonclinicalstudies were performed to evaluate the effectof thiscompound on fertilityr,eproductive performance, and teratogenicityin rats and rabbits.There were no adverse effectson fertilityor re!S@@proidnucmratlieiv(e2rifog2.a-i--up-to-h"@ig est@dose@v @-S t@e!st@iendzaale (20@lmzgfkglday)and female (6 mg/kg/day) ReceiveOdctober14,1993;accepteFdebruary25,1994. AddressreprinrtequesttsoRoman V. Hrab,RegulatorTyoxicology, DrugSafetDyepartmentS,andozResearchInstitutSea,ndozPhceuticaClosrporatioEna.stHanover.NJ 07936. Thispaperwas submittedforpublicatiosnhortlyaftertheuntimely deathofDr. Cox inAugust 1993. (:::(C:)1_9914994WE ILEY-LISS, INC. 20 Group I 11 m IV v vi R.V. HRAB ET AJ- TABLE 1.Daily dosing schedule Firstdose administration Deionized H20 Mevalonic acid500 mg/kg Mevalonic acid500 mg/kg Mevalonic acid500 mg/kg Second dose administration tapprox.1/2 hr.afterfirstdose) 1% cmc 1% cmc Fluvastatin12 mg/kg/day Fluvantatin24 mgfkgtday Fluvastatin12 mg/kg/day Fluvastatin24 mglkg/day Third dose administration (approx.5 hr.aftersecond closei Deionized12 Mevalonic i0d500 mg/kg Mevalonic acid500 mg/kg Mevalonic acid 500 mg/kg valonicacid with fluvastatinfrom day 15 of gestation th could inhibitor block the a&v-erse effectsnoted wi'th fluvastatin. MATERIALS AND METHODS Animals One hundred twenty female rats(CharlesRiver CI)OSpmgue-Dawley-derived) obtained from Charles River Breeding Laboratories,Inc.,Kingston, New York, were approximately 14 wk old at the startof the study.A group of males ofthe same strainand age, and from the same supplier,was used formating, during which each female was housed with one male. Observation ofvaginal plugs the followingmorning was consideredevidence of mating and day 0 of gestationor day 0 post coitus(pc).The day on which pups were born was designated day 0 postpartum (pp),while day 21 pp was the day of weaning. As the females were mated, they were randomly assigned to sixgroups until20 ratsper group were mated. After mating, females were housed individuallyin suspended stainlesssteelwire-bottom cages in a temperature and humidity controlledroom with a 12-hr lightand 12-hr dark cycle.On day 20 ofgestation, the ratswere transferredto transparent plasticcages with bedding consistingof hardwood chips.Certified Purina rodent chow (pelletsa)nd water (providedvia automatic watering device)were suppliedad libitum. Compounds administered Lot 28 of fluvastatin(XU 62-320)was used in this experiment. Purity was determined by thin-layerchromatography (TLC) and high-performanceliquidchromatography (HPLC) to be 99+%. DL-mevalonic acid ? lactone was obtained from Fluka Chemical Corp. (Ronkonkoma, NY), and puritywas found to be 96 + % as determined by HPLC. Dosage admixiistration Dosing solutionsof nuvastatin were prepared fresh daily in 1% carboxymethylcellulose(CMC). Mevalonic acid was prepared freshtwice dailyin deionizedwater. Individualanimal doses were calculatedand administered daily(tofemales only)via gavage from day 15 of gestationthrough day 21 postpartum at a volume of 10 ml/kg (Table 1).Dose calculationswere based on the dailybody weight from day 15 of gestationuntildelivery (orday 28 pc for those animals which did not de- liver)F.ollowingdelivery(day 0 pp) through weaning (day 21 pp), the dose calculationswere based on the most recentlyrecorded -body weight. Control rats receivedthreeseparatedoses:10 ml/kg b.i.do.fdeionized water and 10 ml/kg/day of 1% CMC, alsobased on the same body weight schedules as in the dose groups. Concentrationsof the dosing solutions were assayed and verifiedfour times during the study. Exslrninations Individualclinicalobservationswere recorded at leastonce daily.Body weights were recordedon days 0, 7,and dailyfrom days 15 through 20 of pregnancy. After each animal delivered,maternal body weights were recorded on days 0, 7, 14, and 21 pp, Food consumption was recordedonly during gestationon days 0, 7,15, and 20 pc. Following deliveryof each litterv,iabilityc,linical signs,and external examinations of pups were recorded on days 0 pp through 21 pp. The sex and individualweights of allviablepups were recorded on days 0, 1,4, 7, 14, and 21 pp. Pups stillbornor found dead during the postpartum periodand not autolyzed or cannibalizedwere examined, identifieda,nd preserved in neutralbuffered 10% formalin.Dam dying spontaneouslyor sacrificedmoribund were necropsied.Animals which did not deliverwere necropsied on day 28 pc. On day 21 pp, all remaining dams and pups were euthanized,necropsied,and examined. Pups with lesionsas well as severalcontrolgroup pups were identifiedand saved in neutralbuffered10% formalin.All other pups were discarded. Post mortem examinations Following inductionof deep surgicalanesthesia using excess C02, animals were euthanized by severing the axillaryvesselsforexsanguination.Terminal body weight was recorded foreach dam and a thorough dissectionwas performed on allsurviving dam at weaning (day 21 pp).All grosslesionsand approximately 40 representativetissuespecimens were collectedand preserved in neutral buffered10% formalin. Spontaneously dying animals were dissectedand a complete set of tissuespecimens, including the gravid reproductivetracts,were collectedand preserved. Nonpregnant animals were sacrificedon day 28 pc. Since the treatment-relatedcardiacfindingsoccurredonly in MEVALONATE AND PREGNAW RAT FLUVASTATIN TOXICITY 21 TABLE 2.Cause of death ofspontaneouslydyinganimals Dose group I Control IIMevalonicacid500 mgag IIIFluvastati1n2 mg/kg. mevalonicacid500 mg/kg IV Fluvastati2n4 mgfkg, mevalonicacid500 mglkg V Fluvastati1n2 mg/kg VI Fluvastati2n4 mgikg Totalnumber offemales thatdied on study 0 2 1 2 3 8 Pregnant females thatdied beforedelivery on day 22 pc Pregnant females thatdied postpartum 0 0 0 1 (sacm.oribundday I pp)" 0 0 Nonpregnant females dyingduring experiment 0 1 (day26 pc)-l 1 (day18pc)l 0 0 1 (day 19 pC)3 I (electivseac.day 15 pe)-, il 1 (day4 pp)l 0 1 (day13 pp)G 12 1 (day2 pp)2 0 1 (day5 pp)2 1 (day6 pp)2 2 (sacm.oribundday 10 pp)2 1 (day12 pp)2 1 (day14 pp)2 'Causecouldnotbe determined. 2Heartlesion(vacualadregenerationa,nd/ormyocarditipsrobablyrelatedtodeath). 'Intubatioanccidentl@esion3seenby grossormicroscopiecxamination. 'Intubatioanccidenbtased on clinicaolbservationasnd grossormicroscopiecxamination. 'Possiblientubatioanccidenta,lthoughmicroscopifcindingwsere not conclusive. "Intubatioanccidentbasedon clinicaolbservationasl.thoughmicroscopifcindingwsere notconclusive. "Broken,malalignedincisorsa;nimalinpoorhealth. pregnantanimals,data from thenonpregnantanimals inutero.Othersdiedwithinthefirs2t wk postpartum. were consideredseparately. The causeofdeathofthe 8 animals administerednu- Based on resultsfrom previousstudiesm,icroscopic vastatinat24 mg/kgtdaywas relatedtothe presenceof evaluationswere limitedtothe heartsand stomachs cardiomyopathycharacterizebdy hyaline,granularor from allanimals.For spontaneouslydyingor animals vacuolardegenerationa,nd/ormyocarditis(Fig.2-5). sacrificemdoribund, the tissueevaluationincluded The causeofdeathof the 2 ratsadministeredfluvas- grosslesionsl,ungs,trachea,thymus with mediasti- tatinat 12 mgfkgtdaycouldnotbe determined. num, and liver. Necropsyexaminationofthe 2 animalsthatdiedon Statisticalnalysis day 22 ofgestationrevealeddead,morphologicallynormal full-terfmetusesin the uterus,indicatintghat Allstatisticeavlaluationsofdata compared treat- theywere probablyaliveup to the time of the dames ment groupswith controlsatP:sO.05and P:sO.01levels death.Furthermore,drug-relatecdlinicaolbservations of significancwei,th a two-taileadnalysisM.aternal were seen in the 24 mg(kg/dayfluvastatiannimal body weight,durationofgestationn,umber ofpups de- which includeddecreasedlocomotoractivity(ondays liveredl,ivepups per littepru,p weight,and implanta- 20 and 21 pc),splayed(extended)hindlimbst,remors, tion siteswere evaluatedby analysisof variance ataxial,aboredbreathing,salivationl,acrimationa,nd (ANOVA) followedby Dunnett'sTest.FisheesExact disorientation day 21 pc.Similarsignswere seenin Testwas usedtoevaluatematernal fertiliatnyd repro- the12 mg/kg/dayanimal on day 22 pc priorto death. ductiveperformance,numbers of dead pups, and sex Other animalsfrom the 12 and/or24 mg/kg/dayflu- ratioa,s well as neonatalnecropsyexaminationdata. vastatingroups alsoexhibitedvariousdrug-related Maternal terminalbody weightswere evaluatedby a clinicaslignsincludingataxia,impairment/lososf one-way ANOVA followedby Duncan'sMultipleRange rightingreflexd,ecreasedlocomotoractivityh,unched Test. or flattenedbody positions,played(extended)hind RESULTS Maternal mortalityand chnicalobservations limbs,ptosist,remors,disorientatiolna,boredbreathing,skinpallor,and dehydration. Drug-relatemdaternalmortalitoyccurredinanimals Maternal body weight and food consumption receiving12 and 24 mglkg/day offluvastatiwnhere 2 No treatment-relateddifferenceisn maternalbody outof17 and 8 outof17 pregnantanimals,respectively,weightgain were notedduringgestationdays 15-20, diedor were sacrificemdoribund (Table2).One animal the initiapleriodof administratioonf fluvastatin from each of thesegroups diedon day 22 of gestation and/ormevalanicacid(Table3).During thefirs7t days withoutcompletingdeliverybut had full-terfmetuses afterdeliverya,transients,tatisticalsliygnificanMtR- 22 R.@,'H.RAB ET AL. rt .4x -*4dmlb- .7 14 Figs. 1-4. MEVALONATE AND PREGNANT RAT FLUVASTATIN TOXICITY 23 wrl cv7p, mglkg/day dose leveloffluvastatinsupplemented with mevalonicacid.During thenext 7 days(days7-14 pp), a body weightgainwas evidentinallgroups.However, considerintghe firs2t wk postpartum(days0-14),an overallbody weightlosswas neverthelessapparentin the2 fluvastatignroups(Table3). Subsequentlyd,uringdays 14-21 pp,a slightmater- nalbody weight losswas observedinallgroupsinclud. ing controlsM.aternal weight lossisnot uncommon duringthistimeperiodand isprobablydue toa greater nutritionadlemand on the dams resultinfgrom in- creasedlactationc,ombined with more ingestionoffeed by thepups and thereforelessby the dams. Although 10 not statisticalsliygnificanat,slightdecreasein food consuxnptioonfapproximately11% was evidentduring thetreatmentperiodfrom days 15-20 ofgestationin bothgroupsadministered24 mg/kg/dayoffluvastatin, withand withoutmevalonicacid(Table3). Fertilitaynd reproductive performance No remarkablevariatioinnpregnancyrateoccurred among the groups.Consideringthe mean lengthofthe gestationperiodof thoseanimals completingdelivery, there was no treatment-relatevdariationamong groups(Table4).A slightllyowergestationindexin Fig. 5. 24 mgikg: death on day 14 pp. In some high-dosaenimals Group VI (fluvastaiant 24 mg/kg/day)reflecttsheone dyingin thelaterpostpartum period.myocardiallesionesncountered animalwhich had no viablepups,5 stillborpnups,and were oftenmore complex.frequentlcyharacterizbeyd areasOfmyO- 7 deadpups ofunknown viabilyitstatusatbirth. fiberdisintegratiaocncompaniedby irinammatoryinfiltrataensd in- terstitieadlema. The affecteadreasranged fromlimitedtowidespread. Not seen in earliermyocardial lesionswas the occurrenceof conspic- PostnatalUtterdata uouscytoplasmviaccuolizatiolnlustraitnetdhisfigur(ex 400). The mean number oflivebornpups was lowerinboth groupsadministeredfluvastatianlone(GroupsV and ternalbody weightlossof5% and 9% was apparentin VI)when comparedtoconcurrentcontrol(sTable5).An the 12 and 24 mglkg!day fluvastatidnosegroups,re- increasedincidenceofstillborpnups occurredinthe 12 spectivelyc,ompared to a 4% gain in controlsA. 2% and 24 mg/kg/dayfluvastatiGnroup V (7%)and Group body weightlossoccurredin animals receivintghe 24 VI (22%),compared toa range of2-3% in controlsand Groups II,HI,and IV.There was no evidencetosuggest any remarkableincreasein in uteropostimplantation myFoicga.r1d.iaClotnitsrsouleiaxni2m0a0l:.typicalappearanceofnontreatedcontrolembryo/fetamlortality(i.e.e,arlyor lateresorptions) in any ofthegroups,eitherbeforeor afterinitiatioonf Fig.2. 24 mgtkg:deathon day 21 pc beforedeliveryA.n example fluvastatiannd/ormevalonicacidadministratio(nTa- illustratitnhge signif'icabnutt focalnatureof myocardialhyaline degenerationi.nterstitieadlema. and evidenceofactualmyoriberloss encounteredin dving high-doseanimals.Lesionsofthistype(lefhtalf ofillustratiowne)re detectedonlyinanimalsdyingduringdeliveryor immediatelypostpar-um.The -ryocardiumin the righthalfof this ble 5).Neonatal mortalityof 15% and 56% was observedthroughweaning in both 12 and 24 mg/kg/day fluvastatiGnroups V and VI, respectivel(yTable5), witha statisticaslilgynificanitncreasenotedat 8-14 illustratiiosnunaffectedv,200 @ days poepartum in Group V and as earlyas 1-4 days Fig.3. 24 mgkg: deathon da.-,2-2 pc beforedeliveryD.iscretearm postpartumin Group VI. Although theoverallneonaofsignificanmtvocardialnecrossiwithmononuclearphagoeyticizir talmortalityin Group IV (24 mg/kg/dayfluvastatin tration.Affectedmyorik>ershave undergonegranulardegeneration. supplementedwith mevalonic acid)was alsostatisti- Adjacentmyofit>erasre normal. Aultiplefociofthistypewere men callysignificanitt,was minimal. elsewhereinthemyocardium of-hisanimal(x 200). Drug-relatedlower birthweights as well as lower Fig.4. 24 mglcg-sacrificemdoribundon day lopp.conspicuousneonatalbody weightsduring the lactatiopneriodwere myoribercytoplasmicvacuoliza-.iwoans frequentlnyotedin animals evidentinboth 24 mg/kg/day fluvastatidnose groups, dwyeirnegulnaatcecroimnptahneipeodsbtypiarr.t1u,r1-D1.e'-aimordn.Toahrteoistoehnceyrteovpildaesnmcieoacflmtyeroartiibon*srwith and withoutmevalonicacidsupplementation. degenerationT.he distributioonfafr@cterdibersrangedfrom limited The most prominent neonatalclinicaolbservations areastowidespreadregionso(:nvocardiumT.he vacuolesthemselves Occurred in Group VI (24 mg/kg/dayof fluvastatin) were found to be devoid o-.-',ao-,-glvcogenIx 400). where severallittersw,hose dams were adverselyaf- 24 R.V.HRAB ET AL TABLE 3.Percent body weight change and relativefood consumption compared to controls Group I Control a Mevalonic Acid m IV FluvLsUtin + MevalonicAcid 12 mg/kg/day 24 mg/kg/day v vi Fluvastatin % body weight change: days 15-20 pe 0-7 pp 0-14 pp 7-14 pp 14-21 pp Relativefood consumption: days 15-20 pc *P:s 0.01. **p s 0.05. 0 -mg/kg/Oay 18 4 8 4 -5 100% 500 mglkg b.i.d. is 3 4 1 -3 100% 500 mgfkg b.i.d. 18 2 4 2 -4 96% M mg/kg b.i.d. 16 -2 2 4 -3 a9 12 24 ing(kg/day mg/kg/da 16 -5* -1** 4 -3 93% 16 -9* -3* 1 -1 89ey TABLE 4.FertiHtyand reproductiveperformance Group 1 Control 12 Mevalonic Acid m IV Fluvastatin+ Mevalonic Acid 12 mg/kg/day 24 mglkg/day v vi Fluvastatin No. ofanimals matedigroup # Pregnant Pregnancy rate' Gestation indeX2 No. ofanimals completingdelivery Mean length gestationof animals completing delivery(days) Range (days) 0 mg/kg/claX 20 is 90% 100% is 21.9 21-22 500 mg/kg b.i.d. 20 17 85% 100% 16 3 21.8 21-22 500 mg/kg b.i.d. 20 15 75% 100% 15 22.0 22 500 mg/kg b.i.d. 20 18 90% 100% 18 21.7 21-22 12 mg/kg/day 20 17 85% 100% 16 24 mg/kgtda 20 17 85rk 94% 16 22.1 22-23 22.2 21-23 'Percentmatinp resultinginpregnancy;includesanimals which died. 2Percentpregnanciesresultingin litterwsith one or more viableoffspringd-o.esnot includeanimals which died. 3Does not includeone animal which delivered4 stillborn4,dead,and 6 viablepups,but was sacrificedin a moribund condition on day 1 pp followinga probableintubationaccidenton day 22 pe. TABLE 5.Neonatal Uttersizeand mortauty Group Mean no. ofpups delivered Mean no.of livebom pups Mean no. ofimplantationsites In uteropostimplantationloss Overallneonatalmortalitythrough weaning Control 15.4 15.1 16.4 6.1% 3% ii 16.1 15.9 17.3 6.9% 5% ni 17.1 16.7 18.2 6.0% 6% *p s 0.01. **P :r.0.05. IV 15.6 15.2 16.7 6.6c/'t 7%** v 13.6 12.6 15.2 10.5% 15%* vi 14.9 11.1* 16.3 8.6% 56%* fected,had pups which appeared pale, thin, weak, and/or dehydrated. Necropsy examination of pups which were found dead during the lactationperiod,or were culledpriorto weaning, showed that both groups administered fluvastatinalone (Group V and VI) had a higher incidenceof pups without milk visiblein the stomach. Macroscopic and microscopic evaluation of spontaneously dying arkimal No macroscopic evidence of treatment-related effects was noted other than the anticipatedforestornach thickeningpresent in 2 of 3 and 7 of8 animals from the 12 and 24 mg/kg/day fluvastatingroups, respectively. which died or were sacrticed moribund. New., MEVALONATE AND PREGNANT RAT FLUVASTATLN TOXICITY 25 TABLE 6.Time and incidenceof treatinent-relatmeadternal mortality' Study Dose (mglkg) Day 15-21 pc 0 pp - 22 pc 23 pc 1-4 pp 5-15 pp Segment III Fluvastatin 2 12 24 2 2 4 1 1 1 1 6 Segment IIIfollow-up Fluvastatin 2 6 12 24 2 4 2 1 3 Segment M mevalonate supplementation Fluvastatin Fluvastatin MEV + MEV + MEV Fluvastatin Soo 12 + 500 24 - 500 12 24 1 1 1 1 6 'To facilitatesimultaneous presentation of data, the control groups (allnegative) were omitted. Microscopicfindingsconsistedof a varietyof myo- plasticlesions.The hyperplasia ranged from mild to cardiallesionsin alldying Group VI (fluvastatiant 24. mg/kg/day) animals. The cardiomyopathy was charac. terizedby focalmyocarditisand lesionsofmyofiber hyaline,granular and vacuolar degeneration(Table 2). These findingswere consideredtreatment-relateadnd moderate in degree.In the absence of specificquantitativemeasurements, there appeared to be correlation, with greater degreesof hyperplasia seen at the higher dosesof fluvastatinin Groups IV and VI (24 mg/kgl day)compared totheresponseseen inGroups IIIand V similarto those seen in a previous study in pregnant or lactatinganimals dying during or afterparturition. Two types of myocardial lesionswere detectedmicro- (12 mg/kg/day). There did appear to be a lesserdegree of hyperplasia/hyperkeratosisin the animals administeredmevalonate and 12 mg/kg/day fluvastatin(Group scopicallyI.n animals d3ringat and within a few days M) compared to the low dose of fluvastatinalone afterparturition,the lesionswere characterizedby hy- (Group V). aline degeneration of isolatedgroups of myofibers accompanied by slightinterstitiaeldema and hemorrhage (Fig.2 and 3).The lesionstypicalof animals dying 3-4 days afterparturitionor laterwere characterizedby widespread cytoplasmic vacuolar changes in the myofibers,i.e.t,he occurrence of large and small Histologicalevidence offluvastatin-inducedmyocardialfiberinjuryan&or inflammation was not present in the mevalonate-supplemented Groups III and IV. Likewise,in the survivingGroup V and VI (fluvastatin at 12 and 24 mg/kgtday) animals, myocardial lesions were not detected. clearlydefined round empty vacuoles(Fig.4 and 5). Occasionally these were accompanied by a sarcolemmal response and inflammatory cellinfiltratewsh,ich DISCUSSION suggested a myofiber proliferativoer reparativere- The maternal toxiceffectsinduced by fluvastatinre- sponse to the inductionof the myocardial injury. ported herein had been encountered in two previous Cardiomyopathy was not present in the spontane- studiesat similardose levels,and consistedof mater- ously dying animals which had been given a lower dose nal mortalityat deliveryand during the postpartum (12 mg/kgtday) of fluvastatinalone.All animals dying period (Table 6). Microscopic evidence of associated in the 24 mg/kg/day fluvastatingroup had cardiacle- myocardial lesionswas detectedin the follow-upstudy. sions.These lesionswere consideredrelatedto the An increasein stillbornpups was observed with no cause of death in these animals. evidence of earlierin utero fetalmortality,suggesting Terminal sacrificeaniinals a toxiceffecton the dams immediately priorto,or during,parturitionT.he absorptionand dispositioonf flu- The only treatment-relatedeffectnoted occurred in vastatinhave been studiedin nonpregnant rats(Tseet allthe groups which receivedfluvastatin.Most ofthe animals in Group 111(14 out of 19),Group IV (17 out of 18),Group V (14 oiltof 17),and Group VI (11out of 12) al.,'90)and in pregnant and lactatingrats,and in sucklingpups (A.Schweitzer,unpublished data).In all casesfluvastatinwas rapidlyeliminatedwith littloer had varying degrees of forestomach thickening.No ev- no accumulation in the tissues.Limited placental idence of this effectwas detectedin Group II (meval- transferresultedin very low levelsin embryos or fe- onic acid alone)or the controls.In Groups M and IV, tuses.Fluvastatinwas detectedin maternal milk and supplementation with mevalonate did not influence the incidenceof fluvastatin-inducedforestomach thickening. Microscopically,the forestomach thickening was subsequently at measurable levelsin suckling pups. However, the neonatal mortality observed soon after parturitiona,s well as during the lactationperiod,May be a reflectionof adverse effectsrelativeto deficient characterizedby epithelialhyperplasia/hyperkeratosis maternal lactationand lack oflittercare as influenced in all groups which receivedfluvastatini,ncluding by maternal toxicity. those receiving the mevalonic acid supplementation. The resultsindicatethat fluvastatin-inducemdaterNo evidence of ulcerationaccompanied these hyper- nal and neonatal mortalitycouldbe completelyblocked 26 R.V. HRAB ET Al- and/or amelioratedby coadministrationof mevalonic Itwould notbe unexpected.thereforet,hatinhibitioonf acid.Adverse effectosn maternal body weight were thiscriticaelnzyme would influenceor disturbthedv- nonexistentwith mevalonicacidsupplementedfluvas- namic eventsoccurringin lategestationand at part'u- tatinat 12 mg/kg/day and markedly improvedat 24 ritionT.he datafrom Groups IIIand IV clearlyindicate mg/kg/day,although food consumption remained thatmevalonatesupplementationpreventsmortality slightldyecreasedat 24 mg/kglday despitesupplemen- and the developmentof heart lesionsI.tisnot clear tationwith mevalonic acid.The decreasedneonatal from thesestudiesexactlywhat the effectisin the bodyweightgainduringlactatioant24 mg(kg/daymay myocardialtissueswhich allows developmentofthe be relatedtothe reducedmaternal foodintakenotedat heartlesionsa,nd why the restoredor maintainedlev- thisdose level. elsofmevalonatesuppressor blockthe developmentof While mevalonate supplementationpreventedmor- theselesions. talitaynd the developmentofheartlesionsin Groups LITERATURE CITED IIIand IV (fluvastatiant12 and 24 mg/kg/day)i,td@d notreducethe directeffectoffluvastationn the gastric Alberts.A.W. (1988)Discovery.biochemistrvand biologvof lovasta- un. Am. J.Cardiol.6.2:10-15. forestomachsquamous epithelium,althoughthe de-' BrownM,.S.. and J.L GoicL-jwin(1980) Multivalent feedback regula- greedidappear somewhat lowerin Group M. This is tioonfHMG CoAreducuisaec.ontrmoelchaniscmoordinatingiso- consideredfurtherevidencethat hyperplasia/hyper- prenoidsynthesisand callgrowth. J.Lipid.Res.,21.505-517. keratosisis the resultof director contact irritationby IVEngstrom,R.G..D.B. Weinmin. F.G. Kathawala. T. Scallen,J.B. fluvastationn thesqlljamoufsomswmach epithelium. Esknon. M.L Rucimr.R. Miserendino.and J.Babiak (19881Hypo- lipaproteiancatmivciotfXyU 62-TA, a pot*ntcompetitiveinhib- The fluvastatin-induceeadrlycardiaclesionschar- itoorfHMG-COA reducum. Eighth InternationalSymposium on acterizebdy hyalinedegeneration(beforeor at partu- dl Atherosclwwis, Rome. October 9-13. 1988. 230 tabstmet). ritionr)esembledthoseinducedby isoproteren(oplre9 - "Kathawala,F.G.,T. Scallen.FLG. Engstrom. D.B. Weinstein.H. nant rats are uniquelysensitive)T'h.e postnatal Schuster.R. Stabler,J. Kratunis,J.P, Wareing. W.F. Jewell.L. Widler.and S.Wattanasin (1988)XU 62-320,an HMG-COA reduc- lesions(vacuolarsarcoplasmicchanges and myocardi- tan iwbitor. more potentthan compactinand lovastatinE.ighth tis)appearedto be the resultofsarcoplasmifcluidac- InternationalSymposium on AtherosclerosisR,ome. October 9-13, cumulationpossiblyrelatedto alteratioonf cellular4LI 1988;445 tabvxact). membrane structures.T'he relationshioprprogression4Rathawala. F.G. (1991)HMG-COA reductaseinhibitorsA:n exciting betweenpre-and postpartumlesionsi,fany,is not dReevvel1o1p:m1e2n1t-i1n4t6h.e treaunent ofhyperlipoproteinemiaM.e& PA& clearL.ikewise,itisnotknown atwhat pointintheirIfKo;;@;,;@,D.J.,J.S. MacDonald. C.P. Peter. D.M. Duchai. R.J. developmenteitherwould be reversible. Stubbs,J.LGermenhauseii, and A.W. Alberts(1989)Toxicityofthe Severalfactorsexistedwhich probablycontributedto the developmentofmyocardialdamage and/ordeath of thefluvastatin-treataendimals.In additiotnothecon- HMG-Coenzyme A reductaseinhibitorl.ovastatint.o rabbits.J. Pharmacol.Exp. Ther..248:498-505. MacDonald, J.S.,R.J.Gerson.D.J. Kornbrust,M.W. Kloss,S.Prahalada.P.H.Berry,A.W. Alberts.and D.L. Bakelman (1988)Preclin- currentstressesof pregnancy,parturitiona,nd lacta- calevaluationoflovastatinA.m. J.Cardiol.6,2:16-27. tion,the exaggeratedpharmacologicactivitoyffluvas- nsker,D.H.,J.S.MacDonald, R.T.Robertson,and D.L. Rokelman tatin,i.e.i,nhibitioonf mevalonateand cholesterol (1993)Mevalonate supplementationin pregnant ratssuppmsm biosynthesisw,as a significanftactorH.MG-COA re- theteratogeniciotfymevinolinicacid.an inhibitoorf3-hydroxy-3methylglutaryi-coenzymAe reductase.Temtoiogy,38:449-456. ductaseand itsirnmetiiatperoductmevalonicacidplay Robison.&L, W. Suter,and ILH. Cox (1994)Carcinogenicitaynd a key rolein cholesterobliosynthesiasnd areessential mutagenicitystudieswith fluvastatina, new entirelysynthetic forthe synthesisofsteroidhormones,cellmembranes, HMG-COA reductaseinhibitorF.und. Appi.Taxicol.(manuscript cellgrowth,and normal cellulamretabolism(Brov4rn acceptedforpublication). and Goldstein',80;Alberts,'88;MacDonald etal.,'88). StoU.R.E.,DJ. Mt. bL Evans, 0. Hockwin, S.Roberts,& Robison,L Rubin,and C.Smith (1988)Subchronictargetorpn toxicitoyfXU 62-320in the rat.mouse. dog and monkey. Eighth International Symposium on AtherosclerosisR,ome. October9-13, 1998,902(ab- stract). Tse. F.L.S.,H.T.Smith.F.H. Ballard.and J. Nicollett(i1990)Dispo 'Wagner. B.M.. Depu%v Director,Nathan S. Kline lnstitt:tNeY,U. sitioonffluvastatina.n inhibitorofHMG-COA reductasei.nmouse. Consultationat Sandoz. East Hanover, N.J. December 29,1989. rat,dog,and monkey. Biopharm. Drug DiSP03., 11:519-531.