Document pmkwaEYJL2Xg44r2Q2BEebrg6

ly u I-Ay TERATOLOGY 50:19-26(1994) Prevention of Fluvastatin-InducedToxicity, Mortality,and Cardiac Myopathy in Pregnant Rats by Mevalonic Acl*dSupplementation ROMAN V. HRAB, HOWARD A. HARTMAN, AND RAYMOND H. COX, JR. RegulatoryToxicologyD,rug SafetyDepartmentS,andoz ResearchInstitutSea,ndozPharmaceuticalCsorporation. EastHanover,New Jersey07936 t)/ ABSTRACT Mevolonic acidisa productof the enzyme HMG-COA reductosewhich isessen- tialforcholesterolbiosynthesisF.luvastati(nSon- doz compound XU 62-320)isa potentinhibitorf thisenzyme and, hence, mevolonicacid produc- tion.Inthreeseparate studies,oraladministration of fluvastatiant 12 and 24 mg/kg/day to mated rats from dav I :)tqestatio-n-rtohugh weanino resuitedi nticiated maternalmortalitayt the time at arturitionand during lactationM.icro- scopicevn,int*npnertormeidn twostudierse- veoled significantcardiac myopothy in the d ing animals. Drua-related clini siqns- significonmf-n_ ternalbody weigwt lots,and an increaseimgillborn pups and neonatal mortalitywere also noted at one or b-o-m-cFo-seI!egy&13S.upplementation of fluvastatinadministrationwith ;)vwmq/Kq D.i.c.at me- volonic acid completely blocked and/or (@-m-erioro-t-e7Er-tRe--mao i , car ioc my (3-th-oh-vn--,&-oTF7eer oJv-e-rs-e-e-ffe-ichtess.e stud ies-indicote tha-t-tFe--oT- verse motemal 'effects observe with uvastatin be-ror-e-or7o7io-,,ing-parturirteisounlted rom exag- gerated phormacolo -cliacc ti*vi'tavt the dose levels administered,i.e.,inhibit-i-aTanth_ee_-_n.zy m e -HMG(Lo-A-re-@u-ct-@siet,limmediote product mevolon-ic acia, and 4-hol,a-rr-ol-biosynthesis. t 1994W-,IeT-L.-ss,-Inc. Fluvastatin (Sandoz compound XU 62-320) is a syn- theticpotent inhibitorof hydroxymethylglutaryl coen- zyme A (HMG-CoA) reductase,the rate-limitingenzyme in cholesterolbiosynthesis (Engstrom et al.,188; Kathawala et al.,'88:Kathawala,'91). Admini n of fluvastatinin rats do s.an mon - ind=u-aig- nificantreductii serum totalcholesterol,low-den- sity ipoproteincholesterol,an serumtriglycen e ev- e0TTs@-(J,uIv;n trom _eeta.tarll@u'-I@W.@T-Du-n@ng@e e@t@ya ssment o luvas:traomti@nn,onclinical studies were performed to evaluate the effectof this compound on fertilityr,epro- ductive performance, and teratogenicity in rats and rabbits.There were no adverse effectson fertilityor reproductive perform -c-e-up-t hest -sel@v-@Ts teste inm le(20mg/kg/day)andfemae(6mg/kg/day) rats,and no evidenceofteratogenicactivltvin ratsat opsesup to 36 mi,/-k-g/oZrary@-bviW-aTZsoe's up to 10 m,&/IkL/dZa(R.V. I Iunpu a . owever,in a perinatal/postn study (Segment 111),12 and 24 mg/kg/day of fluvastatinadministeredto pregnant rats from day 15 pc (postcoitus)through weaning resulted inmaternal mortalityat or near tem and duringthe ppo artu@@nr'oT-Tsi-s ri@qr@@ii.t.y--r i in a subsequent follow-upstudy, and scopi tissue -urrenc r io qp# y only in t animals. No cardiac i)atholo was , --.-gy-m-- etected in nonpregnant animal-s in ?Iuvas tin.As in previous rat studies(Stollet al.,'88)/'-, Forestomach epit e ia ype la is and h erkeratosis ;efr-eound. These have been shown to be spec ic r-oaents-andto be a resultof contact i itation y Nu-vastatin(Robison et aF 94). @Coadministration of mevalonic acid. the immediate product of the en-z-yffi-e-H?AG---Co@@d-ucta 'I@as-b@e-n shown to prevent or antagonize various organ @@Z3c-fii'is intiiicei-n ra d rabbitg with ther HMG-COA re- uctaseinhibitors(MacD)nald eta] et al.,'89).ln pregnant rats,ithas been shown that 500 -mj-/S-@-@val@onvalonic acid supp-re-ss-L47Fe--EFr-a-@g-e- nici@yof mevinolinic -a-c-iTa,n inhibitor of HIWG-RUA reductase-mathh=----aTmin-is-t(eFrpr-10F tao@n ap@pr-Txi- mately 5 hr afte nistraEQ-nv-io7fnorl--iv-'t,@nl-@.-aiid t al.,'83).Since fluvastatin isan inh bfit@oorf the erniz e H -CoK-re-d-u-ctas-ean-d-ffi-e-r-eTCre--oT-me- va onic acid 2roduction a relationship was ctnsid@ered + --,-entnhe ee !eorrate im_T@-c@ro-p@c acttiinv- iiy ass @cia-ted-2@wi@7G A r3ductas@7i@ibiiion y . f ruv-a-st-a n tind the occurrence or Ea-5Fn-aT fn-oFElfiy and/or cardi-a-c-Te-si-o-n-s-.t-uTdEye reported hetrEegal-w-me&-to-de-Eer-mine wheth-er--c-o-a-d@@ini@st@a-iioofnme- ReceiveOdctober14.1993;acceptedFebruary25,1994. AddressreprintrequesttsoRoman V. Hrab,RegulatorTyoxicology. DrugSafetyDepartmentS,andozResearchInstitutSea,ndozPharmaceuticaClosrporationE,astHanover.NJ 07936. Thispapewrassubmittfeodrpublicatsihoonrtalfytetrheuntimely deathorDr.Cox inAugust1993. (@C(C)_1199994@4WII]LEY-LISS INC. 20 R.V. HRAB ET Al- Group I ii iri IV v vi TABLE 1.Dailydosingschedule Firstdoseadministration DeionizedH20 Mevalonicacid500 mg/kg Mevalonicacid500 mg/kg Mevalonicacid500 mg/kg Second doseadministration (approx.1/2hr.afterfirsdtose) 1% cmc 1% cmc Fluvastatin12 mg/kg/day Fluvastatin24 mg/kg/day Fluva3tatin12 mg/kg/day Fluvastati2n4 mg/kg/day Third dose administration (approx.5 hr.afterseconddosei DeionizedH2 MevalonicaCd0 500 ing/kg Mevalonic acid500 mg/kg Mevalonic acid500 mg/kg valonicacid with fluvastatinfrom_dqgyr_loLfog5estation tgMu--Zh day 21 _postidartumcould inhibitor,block the liver)F.ollowingdelivery(day 0 pp)through weaning adverse effectnzote(iwith fluvastatin. (day21 pp),the dose calculationswere based on the most recentlyrecordedbody weight. Controlrats re- ?4ATERIALS AND METHODS An,-als One hundred twenty femalerats(CharlesRiver CI)OSprague-Dawley-derived)obtainedfrom Charles River Breeding Laboratories,Inc.,Kingston,New York, were ceivedthreeseparatedoses:10 mi/kg b.i.do.fdeionized water and 10 ml/kg/day of 1% CMC, alsobased on the same body weight schedulesas in the dosegroups.Concentrationsof the dosing solutionswere assayed and verifiedfourtimes during the study. approximately 14 wk old at the startof the study.A group ofmales ofthe same strainand age,and from the same supplier,was used formating, during which each female was housed with one male. Observationofvaginalplugs the followingmorning was consideredevidence of mating and day 0 ofgestationor day 0 post coitus(pc).The day on which pups were born was designatedday 0 postpartum (pp),while day 21 pp was the day ofweaning. As the femaleswere mated, they were randomly assignedtosixgroups until20 ratsper group were mated. Aftermating, femaleswere housed individuallyinsuspended stainlessteelwire-bottomcages in a temperatureand humidity controlledroom with a 12-hrlightand 12-hrdark cycle.On day 20 ofgestation, the ratswere transferredtotransparentplasticages with bedding consistingof hardwood chips.Certified Purina rodentchow (pelletsa)nd water (providedvia automatic watering device)were suppliedad libitum. Exaniinations Individualclinicalobservationswere recordedat leastoncedaily.Body weights were recordedon days 0, 7, and dailyfrom days 15 through 20 of pregnancy. Aftereach animal delivered,maternal body weights were recordedon days 0, 7, 14, and 21 pp. Food consumption was recordedonly during gestationon days 0, 7, 15,and 20 PC. Followingdeliveryof each litterv,iabilityc,linical signs,and externalexaminations of pups were recordedon days 0 pp through 21 pp.The sex and individualweightsofallviablepups were recordedon days 0, 1,4, 7,14,and 21 pp. Pups stillbornor found dead duringthepostpartum periodand not autolyzedor cannibalizedwere examined, identifieda,nd preserved in neutralbuffered10% formalin.Dams dying spontaneouslyorsacrificemdoribund were necropsied.Animals which didnot deliverwere necropsiedon day 28 pc.On Compounds administered Lot 28 of fluvastati(nXU 62-320)was used in this experiment.Puritywas determined by thin-layerchromatography (TLC) and high-performanceliquidchro. matography (HPLC) to be 99 + %. DL-mevalonic acid day 21 pp,allremaining dams and pups were euthanized,necropsied,and examined. Pups with lesionsas well as severalcontrolgroup pups were identifieadnd saved inneutralbuffered10% forinalinA.ll otherpups were discarded. lactonewas obtained from Fluka Chemical Corp. Post mortem examinations (Ronkonkoma, NY), and Puritywas found tobe 96 + % as dete.mined by HPLC. Followinginductionof deep surgicalanesthesiaus ing excessC02, animals were euthanizedby severing Dosage administration Dosing solutionsof fluvastatinwere prepared fresh dailyin 1% carboxymethylcellulos(eCMC). Mevalonic acidwas prepared freshtwicedailyindeionizedwater. Individualanimal doses were calculatedand administereddaily(tofemales only)viagavage from day 15 of gestationthrough day 21 postpartum ata volume of10 mllkg (Table 1).Dose calculationwsere based on the dailybody weight from day 15 ofgestationuntildelivery (orday 28 pc forthoseanimals which did not de- theaxillaryvesselsforexsanguination.Terminal body weight was recordedforeach dam and a thorough dissectionwas performed on allsurvivingdarn at weaning(day21 pp).Allgrosslesionsand approximately40 representativteissuespecimens were collectedand preservedin neutralbufrered10% fonnalin. Spontaneouslydying animals were dissectedand a completesetoftissuespecimens,includingthe gravid reproductivetractsw,ere collectedand preserved.Nonpregnant animals were sacrificedon day 28 pc.Since thetreatment-relatecdardiacrindingsoccurredonly in MEVALONATE AND PREGNANT RAT FLUVASTATIN TOXICITY 21 TABLE 2.Cause of deathof spontaneouslydyinganimals Dose group I Control IIMevalonicacid500 mg/kg IIIFluvastati1n2 mg/kg, mevalonicacid500 mg/kg IV Fluvastati2n4 mg/kg, mevalonicacid500 mg/kg V Fluvastati1n2 mglkg VI Fluvastati2n4 mg/lcg Totalnumber offemales thatdied on study 0 2 1 2 3 8 Pregnantfemales thatdied beforedelivery an day 22 pc Pregnant females thatdied postpartum 0 0 0 1 (sacm.oribundday 1 pp)" 0 0 Nonpregnant females dyingduring experiment 1 (day26 pc)s 1 (day18pc)" 0 0 1 (day19 pc)3 1 (electivseac.day 15 pc)-- 1(day4 pp)l 0 1(day13 pp)' 12 1 (day2 pp)2 1 (day5 pp)2 0 I (day6 pp)2 2( lay10 pp)2 1 (day12pp)2 1 (day14pp)2 'Causecouldnotbe determined. 2Heartlesion(vacuoladregenerationa,nd/ormyocarditipsrobablyrelatedtodeath). 3Intubationaccidentl@esionseen by grow ormicroscopicexamination. "Intubatioanccidentbased on clinicaolbservationasnd grossor microscopiecxamination. 'Possiblientubatioan=ident,althoughmicroscopifcindingwsere notconclusive. 'Intubatioanccidentbased on clinicaolbservationasl,thoughmicroscopifcindingswere not conclusive. 'Broken,malalignedincisorsa;nimal in poorhealth. pregnantanimals,data from thenonpregnantanimals inutero.Othersdiedwithinthefirs2t wk postpartum. were consideredseparately. The causeofdeath ofthe 8 animalsadministeredflu- Based on resultsfrom previousstudiesm,icroscopic vastatinat 24 mg/kg/daywas ivlatedtothepresenceof evaluationswere limitedtothe heartsand stomachs cardiomyopathycharacterizebdy hyaline,granularor from allanimals.For spontaneouslydyingor animals vacuolardegenerationa,nd/ormyocarditis(Fig.2-5). sacrificemdoribund, the tissueevaluationincluded The causeof deathofthe 2 ratsadministeredfluvas- grosslesionsl,ungs,trachea,thymus with mecliasti-tatinat 12 mg/kgldaycouldnotbe determined. num, and liver. Necropsy examinationofthe 2 animals thatdiedon Statisticalnalysis Allstatisticeavlaluationsof data compared treatment groupswith controlsatP:SO.05and P:50.01levels of significancwei,th a two-taileadnalysisM.aternal body weight,durationofgestationn,umber ofpups deliveredl,ivepups per litterp,up weight,and implantation siteswere evaluatedby analysisof variance (ANOVA) followedby Dunnett'sTest.FisheesExact Testwas used toevaluatematernalfertiliatnyd reproductiveperformance,numbers of dead pups,and sex ratioa,s wellas neonatalnecropsyexaminationdata. Maternal terminalbody weightswere evaluatedby a one-way ANOVA followedby Duncan'sMultipleRange Test. day 22 ofgestationrevealeddead,morphologicallynormal full-terfmetusesin the uterus,indicatintghat theywere probablyaliveup tothe time ofthe dam's death.Furthermore,drug-relatecdlinicaolbservations were seen in the 24 mgtkg/dayfluvastatiannimal which includeddecreasedlocomotoractivity(ondays 20 and 21 pc),splayed(extended)hindlimbs,tremors, ataxia,laboredbreathings,alivationl,acrimal-ona,nd disorientation day 21 pc.Similarsignswere seen in the 12 mg/kg/dayanimalon day 22 pc priortodeath. Other animals from the 12 and/or24 mg/kg/dayflu- vastatingroups alsoexhibitedvariousdrug-related clinicaslignsincludingataxia,impairment/lososf rightingreflexd,ecreasedlocomotoractivityh,unched or flattenedbody positions,played(extended)hind RESULTS limbs,ptosist,remors,disorientatiolna,boredbreath- Maternal mortalityand clinicalobservations ing,skinpallora,nd dehydration. Drug-relatemdaternalmortalityoccurredinanimals Maternal body weight and food consumption receiving12 and 24 mglkg/day offluvastatiwnhere 2 No treatment-relateddifferenceisnmaternal body outof17 and 8 outof17 pregnantanimals,respectively,weightgain were notedduring gestationdays 15-20, diedorwere sacrificemdoribund (Table2).One animal the initiapleriodof administratioonf fluvastatin from each ofthesegroups diedon day 22 ofgestation and/ormevalonicacid(Table3).During thefirs7t days withoutcompletingdeliverybut had full-terfmetuses afterdeliverya,transients,tatisticalsliygnificanmta- p 22 R.v'.HRAB ET AL. el Ara 4r c F,,V 1*4 OW Of Figs. 1-4. MEVALONATE W, 0-1 AND PREGNANT RAT FLUVASTATIN TOXICITY 23 mglkgtday dose leveloffluvastatinsupplemented with mevalonicacid.During the next 7 days (days-s-14pp). a body weight gain was evidentinallgroups.However, consideringthe firs2t wk postpartum (days0-14),an overallbody weight losswas neverthelessapparent in the 2 fluvastatignroups(Table3). Subsequently,during days 14-21 pp,a slightmater. I nal body weight losswas observed in allgroups includ- ing controls.Maternal weight 16-',sis not uncommon duringthistime periodand isprobablydue toa greater nutritionadlemand on the dams resultingfrom in- creasedlactationc,ombined with more ingestionoffeed by the pups and thereforelessby the dams. Although not statisticalsliygnificanta,slightdecreasein food consumption ofapproximately 11% was evidentduring the treatmentperiodfrom days 15-20 of gestationin both groups administered24 mg/kg/day offluvastatin, with and withoutmevalonic acid(Table3). Fertilityand reproductive performance No remarkablevariationin pregnancyrateoccurred among the groups.Consideringthe mean lengthofthe gestationperiodofthose animals completingdelivery, there was no treatment-relatedvariationamong groups(Table4).A slightlylower gestationindex in Fig.S. 24 mgikg-death on day 14 pp. Insome high-doseanini&6 Group VI (fluvastaiant 24 mg/kg/day)reflecttsheone dyinginthe laterpostpartumperiod.myocardiallesionesncountered animalwhich had no viablepups,5 stillborpnups,and were oftenmore complex.frequentlycharacterizebdy areasofmyo- 7 dead pups ofunknown viabilitsytatusat birth. fiberdisintegratioanccompaniedby irdlammatoryinfiltrataensd in- terstitieadlema.The afrecteadreasrangedfromlimitetdowidespread. Not seenin earliermyocardiallesionswas theoccurrencoefconspic- uouscytoplasmicvacuolizatioinllustrateidnthisfigure(x 400). Postnatal litterdata The mean number oflivebornpups was lowerinboth ternalbody weight lossof5% and 9% was apparentin the 12 and 24 mg/kg.;dayfluvastatindose groups,respectivelyc,ompared to a 4% gain in controlsA. 2% body weight lossoccurredin animals receivingthe 24 groupsadministeredfluvastatinalone(Groups V and VI)when compared to-oncurrentcontrols(Table5).Am increasedincidenceofstillbornpups occurredinthe 12 and 24 mg/kg/day fluvastatinGroup V (7%) and Group VI (22%),compared to a range of2-3% in controlsand Groups II,III,and IV.There was no evidencetosuggest any remarkable increasein in uteropostimplantation Fig. 1. Controlanimal: @-picaalppearance ofnontreatedcontrol embryo/fetalmortality(i.e.e,arly or lateresorptions) myocardialtissuetx 200:. inany ofthe groups,eitherbeforeor afterinitiatioonf Fig. 2. 24 mgikg: death on day 21 pc beforedeliveryA.n example fluvastatinand/ormevalonic acidadministration(Ta- illustratintghe significantbut focalnature of myocardialhyaline degenerationi.nterstitieadlema. and evidenceofactualmyoriberlose encounteredindying high-doseanimals.Lesionsofthistype(lefthalf ofillustratiowne)re detectedonlyinanimalsdyingduringdeliveryor ble 5).Neonatal mortality of 15% and 56% was observedthrough weaning in both 12 and 24 mg/kg/day nuvastatin Groups V and VI, respectively(Table5), immediately postpartum. The -ryocardiumin the righthalfofthis with a statisticalsliygnificanitncreasenoted at 8-14 illustratioinsunaffected ( 200 days pos'Partum in Group V and as earlyas 1-4 days Fig.3. 24 mg/kg: death on day 22 pc beforedeliveryD.iscretearea postpartum in Group VI. Although the overallneonaofsignificanmtyocardialnecrosiswith mononuclearphagocytic infil- ta mortalityin Group IV (24 mg/kg/day fluvastatin tration. Affected myofibers have undergone granular degeneration. supplemented with mevalonic acid)was alsostatisti- Adjacent myofibers are normal. Niultiple foci of this type were seen elsewhere in the myocardium of this animal i x 200). callysignificanti,twas minimal. Drug-relatedlower birth weights as wellas lower Fig. 4. 24 mg,kg- sacr@ilced noribund on day lopp.conspicuousneonatalbody weightsduring the lactatiopneriodwere myoriber cytoplasmic vacuolization was frequently noted in animals evidentin both 24 mg/kg/day nuvastatindosegroups, dying later in the postpa:-turn period. These cvtoplasmic alterations were unaccompanied by ir.,"Ilamination or other evidence of myoriber degeneration. The distribution of affected fibers ranged from limited areas to widespread regions or rnyocardium. The vacuoles themselves withand withoutmevalonicacidsupplementation. The most prominent neonatal clinicaolbservations occurredin Group VI (24 mg/kg/day of fluvastatin) were found to be devoid a,- a- o, glycogen 4001. where severallittersw,hose dams were adverselyaf- 24 R.V. HRAB ET Al@ TABLE 3. Percent body weight change and relative food cOnsummPtiOn cosupared to controls Group 1 Control 17 Mevalonic Acid ni rv Fluvastatin+ Mevalonic Acid 12 mg/kg/day 24 mg/kg/day v vi Fluvastatin % body weight change: days 15-20 pc 0-7 pp 0-14 pp 7-14 pp 14-21 pp Relativefood consumption: davs 15-20 pc *p :s0.01. **P :s0.05. 0 mg/kg/day 18 4 8 4 -5 100% 500 mg/kg b.i.d. is 3 4 1 -3 100% 500 mg/kg b.i.d. 18 2 4 2 -4 96% 500 mg/kg b.i.d. 16. -2 2 4 -3 89% 12 mg/kg/day 16 -5* -1** 4 -3 93% 24 mg/kg/dav 16 -go -3* 1 -1 89% TA13LE 4.FertiHty and reproductive perfornlance Group I Control n Mevalonic Acid m rv Fluvastatin+ Mevalonic Acid Y2-mg/kgtday 24 mg/kg/day v vi Fluvastatin No. ofanimals mated/group # Pregnant Pregnancy rate' Gestation indeX2 No. ofanimals 0 mg(kg/day 20 18 90% 100% 500 mg/kg b.i.d. 20 17 85% 100% 500 mi/kg b.i.d. 20 15 75% 100% 500 mg/kg b.i.d. 20 18 90% 100% 12 rng/kg/day 20 17 85% 100% 24 mg/kg/day 20 17 85% 94% Mecaonmplleentgitnhggdeesltiavteiroynof 18 163 15 18 16 16 animals completing delivery(days) Range(days) 21.9 21-22 21.8 21-22 22.0 21.7 22.1 22.2 22 21-22 22-23 21-23 'Percent mat inp resultingin pregnancy;includesanimals which died. 32Percentpregnanciesresultinginlitterwsith one or more viableofrspringd-o.es not includeanimals which died. Does not includeone anirn-qwlhich delivered4 stillborn4,dead,and 6 viablepups,but was sacrificedin a moribund condition on day I pp followinga probableintubationaccidenton day 22 pc. TABLE Group Mean no. ofpups delivered Mean no. oflivebornpups Mean no. ofimplantationsites In uteropostimplantationloss Overallneonatalmortalitythrough weaning *P:s 0.01. **p :s0.05. 5.Neonatal littesrizeand mortality Control 15.4 15.1 16.4 6.1% 3% u 16.1 15.9 17.3 6.9% 5% III 17.1 16.7 18.2 6.0% 6% IV 15.6 15.2 16.7 6.6c,', 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, andtor 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 anirnsal No macroscopic evidence of treatment-relatedeffects was noted other than the anticipatedforeowmach thickeningpresent in 2 of 3 and 7 of8 animals from the 12 and 24 mg/kglday fluvastatingroups, respectively. which died or were sacrificedmoribund. MEVALONATE AND PREGNANT RAT FLUVASTATIN TOXICITY 25 TABLE 6.Time and incidenceoftreatinent-relamtaetdernalmortalitIy Study Dose Img/kg) 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 @00 12 + 500 24 - 500 12 24 1 1 1 1 6 'To facilitatseimultaneouspresentationofdata,the controlgroups(allnegative)were omitted. Microscopicfindings consistedof a variety of myocardiallesionsin alldying Group VI (fluvastatiant 24mg/kg/day) animals. The cardiomyopathy was characterizedby focalmyocarditis and lesionsofmyofiber hyaline,granular and vacuolar degeneration (Table 2). These findingswere consideredtreatment-relatedand imilarto those seen in a previousstudy in pregnant or lactatinganimals dying during or afterparturition. Two types of myocardial lesionswere detectedmicroscopicallyI.n animals dying at and within a few days afterparturitiont,he lesionswere characterizedby hyaline degeneration of isolatedgroups of myofi'bers accompanied by slightinterstitiaeldema and hemorrhage (Fig.2 and 3).The lesionstypicalof animals dying 3 -4 days afterparturitionor laterwere characterizedby widespread cytoplasmicvacuolar changes in the myofibers, i.e.t,he occurrence of large and small clearlydefinedround empty vacuoles (Fig.4 and 5). Occasionally these were accompanied by a sarcolemmal response and inflammatory cellinfiltratesw,hich suggested a myofiber proliferativoer reparativeresponse to the inductionof the myocardial injury. Cardiomyopathy was not present in the spontaneously dying animals which had been given a lower dose (12 mgtkglday) offluvastatinalone.All animals dying in the 24 mg/kg/day fluvastatingroup had cardiaclesions.These lesionswere consideredrelated to the cause of death in these animals. Terminal sacrificeanimals The only treatment-relatedeffectnoted occurred in allthe groups which receivedfluvastatin.Most of the animals in Group 111(14 out of 19),Group IV (17 out of 18),Group V (14 oiitof 17),and Group VI (11 out of 12) had varying degrees of forestomach thickening.No evidence of this effectwas detectedin Group H (mevalonic acid alone)or the controls.In Groups IIIand rV, supplementation with mevalonate did not influence the incidenceoffluvastatin-inducedforestomach thickening. Microscopically,the forestomach thickening was characterizedby epithelialhyperplasia/hyperkeratosis in all groups which receivedfluvastatin,including those receivingthe mevalonic acid supplementation. No evidence of ulcerationaccompanied these hyper- plasticlesions.The hyperplasia ranged from mild to moderate in degree.In the absence of specificquantitativemeasurements, there appeared to be correlation, with greater degrees of hyperplasia seen at the higher dosesof fluvastatinin Groups IV and VI (24 mg/kg/ day)compared tothe responseseen inGroups M and V (12 mg/kg/day). There did appear to be a lesserdegree ofhyperplasia/hyperkeratosisin the animals administeredmevalonate and 12 mg/kg/day fluvastatin(Group M) compared to the low dose of fluvastatinalone (Group V). Histologicaelvidence offluvastatin-inducedmyocardialfiberinjuryand/orinflammation was not present in the mevalonate-supplemented Groups M and IV. Likewise,in the survivingGroup V and VI (fluvastatin at 12 and 24 mg/kg/day) animals, myocardial lesions were not detected. DISCUSSION The maternal toxiceffectsinduced by fluvastatinreported herein had been encountered in two previous studiesat similar dose levels,and consistedof maternal mortalityat deliveryand during the postpartum period (Table 6). Microscopic evidence of associated myocardial lesionswas detected in the follow-upstudy. An increasein stillbornpups was observed with no evidenceof earlierin utero fetalmortality,suggesting a toxiceffecton the dam-q inunediatelypriorto,or during,parturition.The absorptionand dispositionof fluvastatinhave been studied in nonpregnant rats(Tse et al.,'90)and in pregnant and lactatingrats,and in sucklingpups (A. Schweitzer,unpublished data).In all casesfluvastatinwas rapidlyeliminatedwith littloer no accumulation in the tissues.Limited placental transferresultedin very low levelsin embryos or fetuses.Fluvastatinwas detected in maternal milk and 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 maternal lactationand lack oflittercare as influenced by maternal toxicity. The resultsindicatethat fluvastatin-inducedmaternal and neonatal mortalitycould be completelyblocked 26 R.V. HRAB ET AL. and/oramelioratekdv coadministratioofnmevalonic acid.Adverse effectson maternal body weight were nonexistentwith mevalonic acidsupplemented fluvastatinat 12 mg/kg/day and markedly improved at 24 mg/kgtday, although food consumption remained slightlydecreasedat 24 mg/kg/day despitesupplementationwith mevalonic acid.The decreasedneonatal body weight gain auring lactationat24 mg/kg/day may be relatedtothe reduced maternal foodintakenoted at thisdose level. While mevalonate supplementationprevented mortalityand the development ofheartlesionsin Groups IIIand IV (fluvastatiant 12 and 24 mg/kg/clay)i,tdid notreduce the directeffectoffluvastationn the gastric forestomachsquamous epithelium,although the de-' gree did appear somewhat lower in Group M. This is consideredfurtherevidence that hyperplasia/hyper- Itwould notbe unexpected,thereforet.hatinhibitioonf thiscriticaelnzyme would influenceor disturbthe dv. namic eventsoccurringin lategestationand at part'uritionT.he datafrom Groups IIIand IV clearlyindicate thatmevalonate supplementationpreventsmortality and the development of heart lesions.It is not clear from thesestudiesexactlywhat the effectis in the myocardialtissueswhich allows dl$'velopineonftthe heart lesionsa,nd why the restoredor maintained levelsofmevalonate suppress or blockthe development of these lesions. LITERATURE CITED Alb.erts.A.W. (19881Discovery.biochemistryand biolog.vof lovasta- Am. J.Cardial.6.2:10-15. Brto'nwM'.nS,..and J.L.Goldstein(1980)Multivalentfeedbackregula- tionofHMG CoA reducum. a controlmechanism coordinatingisopretwidsynthesisand cellgrowth. J. Lipid.Res..21:505-517. keratosisisthe resultofdirector contactirritatioii-ObrEynVmmm, &G.. D.B. Winswin. F.G. Kathawala. T. Scallen.J.B. fluvastatinon the sqtilartkofuosrestomachepithelium. The fluvastatin-induceedarlycardiaclesionschw- Esknon, bLL Rucker,& Miserindino.and J.Babiak (1998)HypolipoprotainemiacctivitoyfXU 62-320,a potentcompetitiveinhibiwr ofHMG-COA reductase.Eighth InternationaSlymposium on acterizedby hyalinedegeneration(beforeor at partu- AtherosclerosiRso,me. October9-13, 1988:230 (abstract). ritionr)esembled thoseinducedby isoprotereno(lpreg- Kathawala, F.G..T. Scallen.R-G. Engstrom, D.B. Weinstein.H. nant rats are uniquely sensitive)The postnatal lesions(vacuolarsarcoplasmicchanges and myocarditis)appeared tobe the resultofsarcoplasmicfluidac- Schuster,R. Stabler,J. Kratunis,J.R. Wareing. W.F. Jewell.L. Widler.and S.Wattanasin (1988)XU 62-320,an HMG-COA reductaseinhibitorm.ore potentthan compactin and lovastatinE.ighth InternationaSlymposium on Atherawlerosis,Rome. October9-13. cumulation possiblyrelatedto alteratioonf cellularV 1988;445 (abstract). membrane structures.T'he relationshioprprogression A-Hathawala.F.G.(1991)HMG-COA reductaseinhibitorsA-n exciting between pre- and postpartum lesions,ifany, is not developmentinthe treatmentof hyperlipoproteinemiaM.ed. P.M Rev.,ZI:121-146. clear.Likewise,it isnot known at what pointin theirfrxornbrust, D.J.,J.S. MacDonald, C.P. Peter,D.M. Duchai. R.J. development eitherwould be reversible. Stubbs,J.LGermershausen. and A.W. Alberts(igsg)Toxicityofihe Severalfactorsexistedwhich probablycontributedto HMG-Coenzyme A reductaseinhibitorl.ovastatint,o rabbits.J. Pharmacol.Exp. Ther..248:498-505. the development ofmyocardial damage and/ordeath of MacDonald. J.S..R.J.Gerson.D.J.Kornbrust,M.W. Kloss.S.Pmhalthenuvastatin-treatedanimals.In additiontothe con- ada.P.H.Berry.A.W. Alberts.and D.L. Bokelman (1988)Preclincurrentstressesof pregnancy,parturitiona,nd lacta- icalevaluationoflovastatinA.m. J.Cardiol.6,2:16-27. tion,the exaggeratedpharmacologicactivitoyfflultrafs--,Minsker,D.H.,J.S.MacDonald, PT. Robertson.and D.L. Bokelman tatin,i.e.,inhibitionof mevalonate and cholesterol (19831Mevalonate supplementationin pregnant rats suppresses biosynthesisw,as a significanftactor.HMG-COA re- theteratogenicitoyfmevinolinicacid,an inhibitoorf3-hydroxy-3- methylglutaryi-coenzymAe reductase.Teratology3,8:449-456. ductaseand itsimmediate productmevalonic acidplay Robison.P-L, W. Suter.and PH. Cox (1994)Carcinogenicitaynd a key rolein cholesterolbiosynthesisand are essential mutagenicitystudieswith fiuvastatina, new entirelysynthetic forthe synthesisofsteroidhormones, cellmembranes, HMG-COA reductaseinhibitorF.und. Appi. 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