Document 3eBvjeeL1wp2qB3mneoqm7BdJ

T V Uv t ) 0 '.c^ 'u l ^ f t ( * t c7 TERATOLOGY 50:19--26 (1994) A f W - 55^ Prevention of Fluvastatin-Induced Toxicity, Mortality, and Cardiac M yopathy in Pregnant Rats by M evalonic Acid Supplementation ROMAN V. HRAB, HOWARD A. HARTMAN, AND RAYMOND H. COX, JR. Regulatory Toxicology, Drug Safety Department, Sandoz Research Institute, Sandoz Pharm aceuticals Corporation, East Hanover, New jersey 07936 ABSTRACT Mevalonic acid is a product of the enzyme HM G -CoA reductase which is essen tial for cholesterol biosynthesis. Fluvastatin (San doz compound XU 62-320) is a potent inhibitor of this enzyme and, hence, mevalonic acid produc tion. In three separate studies, oral administration of fluvastatin at 12 and 24 mg/kg/day to mated rats from day 15 of gestation through weaning re sulted in unanticipated maternal mortality at the time of parturition and during lactation. M icro scopic evaluations performed in two studies re vealed significant cardiac myopathy in the dying animals. Drug-related clinical signs, significant ma ternal body weight loss, and an increase in stillborn pups and neonatal mortality were also noted a t one or born dose levels. Supplementation of fluvastann administration with 500 mg/ka b.i.d. ot mvclonic acid completely blocked and/or amelio rated the 'mortality, cardiac myopathy, and other adverse effects. I hese studies indicate that the adverse maternal effects observed with fluvastatin / before or following parturition resulted from exag gerated pharmacologic activity at the dose levels administered, i.e., inhibition of the enzyme H M G CoA reductase, its immediate product mevalonic acid, a nd cholesterol biosynthesis. 1994 Wiley-Liss, Inc) ~ ' Fluvastatin (Sandoz compound XU 62-320) is a syn thetic potent inhibitor of hydroxymethylglutaryl coen zyme A (HMG-CoA) reductase, the rate-limiting en zyme in cholesterol biosynthesis (Engstrom et al., '88; Kathawala et al., '88: Kathawala, '91). Administration of fluvastatin in rats, dogs, and monkevs induces sig nificant reductions in serum total cholesterol, low-den sity lipoprotein cholesterol, and serum triglyceride levels (Engstrom et al., '88). During the safety assessment of fluvastatin, nonclinical studies were performed to evaluate the effect of this compound on fertility, repro ductive performance, and teratogenicity in rats and rabbits. There were no adverse effects on fertility or reproductive performancejuP-to the highest dose levels tested in male (20 mg/kg/day) and female (6 mg/kg/day) rats, and no evidence of teratogenic activity in rats at doses up to 36 mg/kg/day or~rabbits~aTdoses up to 10 mg/kg/day (R.V. Hrab, unpublished data). However, in a perinatal/postnatal study (Segment III), 12 and 24 mg/kg/day of fluvastatin administered to pregnant rats from day 15 pc (post coitus) through weaning resulted in maternal mortality at or near term and during the postpartum period. This mortality was replicate3dn~a subsequent follow-up study, and microscopic tissue / evaluation rev'ealedThe'dccurrencFdf cardiomyopathy onTv~in the dying animals. No cardiac pathqlggy_was detected in nonpregnant animals in these studias_with - fluvastatin. As in previous rat studies (Stoll et al., '88)/ ' forestomach epithelial hyperplasia and hyperkeratosis were found. These have been shown to be speciti'c~to rodents a~nd to be a result of contact irritation by"flu- vastatin (Robison et al., '94K * Coadministration of mevalonic acid, the immediate product of the enzyme HMG-CoA reductase, has been shown to prevent or antagonize various organ toxicities induced in rats a'nd rabbits with other HMG-CoA re ductase inhibitors (MacDonald et al., '88: Kornbrua tet al., '85). In pregnant rats, it has been shown that 500 mg/kg'h i d. of mevalonic acid suppressed the teratoge nicity of mevinolinic acid, an inhibitor of HMu-CoA r eductase. ..when administered prior to and approxi mately 5 hr after administration of mevinolinic acid (Minsker et al.. '83). Since fluvastatin is an inhibitor of the enzvme HMG-CoA reductase and therelore of me valonic acid production, a relationship was considered to exist between the exaggerated pharmacologic activ ity associated with HMG-CoA reductase inhibition by fluvastatin and the~gecurrence oTlhaterhal mortality and/or cardiac lesions. The study reported hereinjwas- ' designed to determine whether coadministration of me- Received October 14, 1993; accepted February 25, 1994. Address reprint requests to Roman V. Hrab, Regulatory Toxicology, Drug Safety Department, Sandoz Research Institute, Sandoz Pharma ceuticals Corporation, East Hanover, NJ 07936. This paper was submitted for publication shortly after the untimely death of Dr. Cox in August 1993. 1994 WILEY-LISS, INC 000030 20 R.V. HRAB ET AL. Group I n m IV V VI TA BLE 1. D aily dosing sc h e d u le First dose administration Deionized H20 Mevalonic acid 500 mg/kg Mevalonic acid 500 mg/kg Mevalonic acid 500 mg/kg -- -- Second dose adm inistration (approx. 1/2 hr. after first dose) 1% CMC 1% CMC Fluvastatin 12 mg/kg/day Fluvastatin 24 mg/kg/dav Fluvastatin 12 mg/kg/day Fluvastatin 24 mg/kg/day Third dose adm inistration (approx. 5 hr. after second dose> Deionized H20 Mevalonic acid 500 mg/kg Mevalonic acid 500 mg/kg Mevalonic acid 500 mg/kg -- -- ) '''* [H L c\ r ;J- v y valonic acid with fluvastatin from day 15 of gestation Through day 21 postpartum could inhibit or block the adverse effects noted with fluvastatin. MATERIALS AND METHODS Animals One hundred twenty female rats (Charles River CDSprague-Dawley-derived) obtained from Charles River Breeding Laboratories, Inc., Kingston, New York, were approximately 14 wk old at the start of the study. A group of males of the same strain and age, and from the same supplier, was used for mating, during which each female was housed with one male. Observation of vag inal plugs the following morning was considered evi dence of mating and day 0 of gestation or day 0 post coitus (pc). The day on which pups were born was des ignated day 0 postpartum (pp), while day 21 pp was the day of weaning. As the females were mated, they were randomly assigned to six groups until 20 rats per group were mated. After mating, females were housed indi vidually in suspended stainless steel wire-bottom cages in a temperature and humidity controlled room with a 12-hr light and 12-hr dark cycle. On day 20 ofgestation, the rats were transferred to transparent plastic cages with bedding consisting of hardwood chips. Certified Purina rodent chow (pellets) and water (provided via automatic watering device) were supplied ad libitum. Compounds administered Lot 28 of fluvastatin (XU 62-320) was used in this experiment. Purity was determined by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to be 99 + %. DL-mevalonic acid q lactone was obtained from Fluka Chemical Corp. (Ronkonkoma, NY), and purity was found to be 96 + % as determined by HPLC. Dosage administration Dosing solutions of fluvastatin were prepared fresh daily in 1% carboxymethylcellulose (CMC). Mevalonic acid was prepared fresh twice daily in deionized water. Individual animal doses were calculated and adminis tered daily (to females only) via gavage from day 15 of gestation through day 21 postpartum at a volume of 10 ml/kg (Table 1). Dose calculations were based on the daily body weight from day 15 of gestation until delivery (or day 28 pc for those animals which did not de liver). Following delivery (day 0 pp) through weaning (day 21 pp), the dose calculations were based on the most recently recorded body weight. Control rats re ceived three separate doses: 10 ml/kg b.i.d. of deionized water and 10 ml/kg/day of 1% CMC, also based on the same body weight schedules as in the dose groups. Con centrations of the dosing solutions were assayed and verified four times during the study. E xam in ation s Individual clinical observations were recorded at least once daily. Body weights were recorded on days 0, 7, and daily from days 15 through 20 of pregnancy. After each animal delivered, maternal body weights were recorded on days 0, 7, 14, and 21 pp. Food con sumption was recorded only during gestation on days 0, 7, 15, and 20 pc. Following delivery of each litter, viability, clinical signs, and external examinations of pups were re corded on days 0 pp through 21 pp. The sex and indi vidual weights of all viable pups were recorded on days 0, 1, 4, 7, 14, and 21 pp. Pups stillborn or found dead during the postpartum period and not autolyzed or can nibalized were examined, identified, and preserved in neutral buffered 10% formalin. Dams dying spontane ously or sacrificed moribund were necropsied. Animals which did not deliver were necropsied on day 28 pc. On day 21 pp, all remaining dams and pups were eutha nized, necropsied, and examined. Pups with lesions as well as several control group pups were identified and saved in neutral buffered 10% formalin. All other pups were discarded. Post mortem examinations Following induction of deep surgical anesthesia us ing excess C02, animals were euthanized by severing the axillary vessels for exsanguination. Terminal body weight was recorded for each dam and a thorough dis section was performed on all surviving dams at wean ing (day 21 pp). All gross lesions and approximately 40 representative tissue specimens were collected and pre served in neutral buffered 10% formalin. Spontaneously dying animals were dissected and a complete set of tissue specimens, including the gravid reproductive tracts, were collected and preserved. Non pregnant animals were sacrificed on day 28 pc. Since the treatment-related cardiac findings occurred only in 000031 MEVALONATE AND PREGNANT RAT FLUVASTATIN TOXICITY 21 TA B LE 2. C ause o f d e a th o f sp o n ta n e o u sly dying an im a ls Dose group Total number Pregnant females of females th a t died th a t died before delivery on study on day 22 pc Pregnant females that died postpartum Nonpregnant females dying during experiment I Control II Mevalonic acid 500 mg/kg 0 2- 0 0 00 1 (sac. moribund day 1 pp)4 1 (day 26 pc)5 III F luvastatin 12 mg/kg, mevalonic acid 500 mg/kg 1 0 0 1 (day 18 pc)4 IV F lu v astatin 24 mg/kg, mevalonic acid 500 mg/kg 2 0 0 1 (day 19 pc)3 1 (elective sac. day 15 pc)T V F luvastatin 12 mg/kg 3 l1 1 (day 4 pp)1 1 (day 13 pp)6 0 VI F lu v astatin 24 mg/kg 8 l2 1 (day 2 pp)2 1 (day 5 pp)2 1 (day 6 pp)2 2 (sac. moribund day 10 pp)2 1 (day 12 pp)2 1 (day 14 pp)2 0 `Cause could not be determined. ^ e a r t lesion (vacuolar degeneration, and/or myocarditis probably related to death). 3lntu b atio n accident, lesions seen by gross or microscopic examination. `Intubation accident based on clinical observations and gross or microscopic examination. sPossible intubation accident, although microscopic findings were not conclusive. 6Intubation accident based on clinical observations, although microscopic findings were not conclusive. 'Broken, malaligned incisors; anim al in poor health. pregnant animals, data from the nonpregnant animals were considered separately. Based on results from previous studies, microscopic evaluations were limited to the hearts and stomachs from all animals. For spontaneously dying or animals sacrificed moribund, the tissue evaluation included gross lesions, lungs, trachea, thymus with mediasti num, and liver. Statistical analysis All statistical evaluations of data compared treat ment groups with controls atP^O.05 andP<0.01 levels of significance, with a two-tailed analysis. Maternal body weight, duration of gestation, number of pups de livered, live pups per litter, pup weight, and implanta tion sites were evaluated by analysis of variance (ANOVA) followed by Dunnett's Test. Fisher's Exact Test was used to evaluate maternal fertility and repro ductive performance, numbers of dead pups, and sex ratio, as well as neonatal necropsy examination data. Maternal terminal body weights were evaluated by a one-way ANOVA followed by Duncan's Multiple Range Test. RESULTS Maternal mortality and clinical observations Drug-related maternal mortality occurred in animals receiving 12 and 24 mg/kg/day of fluvastatin where 2 out of 17 and 8 out of 17 pregnant animals, respectively, died or were sacrificed moribund (Table 2). One animal from each of these groups died on day 22 of gestation without completing delivery but had full-term fetuses in utero. Others died within the first 2 wk postpartum. The cause of death of the 8 animals administered flu vastatin at 24 mg/kg/day was related to the presence of cardiomyopathy characterized by hyaline, granular or vacuolar degeneration, and/or myocarditis (Fig. 2-5). The cause of death of the 2 rats administered fluvas tatin at 12 mg/kg/day could not be determined. Necropsy examination of the 2 animals that died on day 22 of gestation revealed dead, morphologically nor mal full-term fetuses in the uterus, indicating that they were probably alive up to the time of the dam's death. Furthermore, drug-related clinical observations were seen in the 24 mg/kg/day fluvastatin animal which included decreased locomotor activity (on days 20 and 21 pc), splayed (extended) hindlimbs, tremors, ataxia, labored breathing, salivation, lacrimation, and disorientation on day 21 pc. Similar signs were seen in the 12 mg/kg/day animal on day 22 pc prior to death. Other animals from the 12 and/or 24 mg/kg/day flu vastatin groups also exhibited various drug-related clinical signs including ataxia, impairment/loss of righting reflex, decreased locomotor activity, hunched or flattened body position, splayed (extended) hind limbs, ptosis, tremors, disorientation, labored breath ing, skin pallor, and dehydration. Maternal body weight and food consumption No treatment-related differences in maternal body weight gain were noted during gestation days 15-20, the initial period of administration of fluvastatin and/or mevalonic acid (Table 3). During the first 7 days after delivery, a transient, statistically significant ma- 000032 22 R.V. HRA B ET AL. Figs. 1-4. BEST COPY AVAILABLE 0C003 MEVALONATE AND PREGNANT RAT FLUVASTATIN TOXICITY 23 mg/kg/day dose level of fiuvastatin supplemented with mevalonic acid. During the next 7 days (days 7-14 ppi, a body weight gain was evident in all groups. However, considering the first 2 wk postpartum (days 0-14), an overall body weight loss was nevertheless apparent in the 2 fiuvastatin groups (Table 3). Subsequently, during days 14-21 pp, a slight mater nal body weight loss was observed in all groups includ ing controls. Maternal weight loss is not uncommon during this time period and is probably due to a greater nutritional demand on the dams resulting from in creased lactation, combined with more ingestion of feed by the pups and therefore less by the dams. Although not statistically significant, a slight decrease in food consumption of approximately 11Sr was evident during the treatment period from days 15-20 of gestation in both groups administered 24 mg/kg/day of fiuvastatin, with and without mevalonic acid (Table 3). Fig. 5. 24 mg/kg: death on day 14 pp. In some high-dose animals dying in the later postpartum period, myocardial lesions encountered were often more complex, frequently characterized by areas of myofiber disintegration accompanied by inflammatory infiltrates and in terstitial edema. The affected areas ranged from limited to widespread. Not seen in earlier myocardial lesions was the occurrence of conspic uous cytoplasmic vacuolization illustrated in this figure ( x 400). ternal body weight loss of 5% and 9% was apparent in the 12 and 24 mg/kg day fiuvastatin dose groups, re spectively, compared to a 4% gain in controls. A 2% body weight loss occurred in animals receiving the 24 Fig. 1. Control animai: typical appearance of noncreated control myocardial tissue i x 200 . Fig. 2. 24 mg/kg: death on day 21 pc before delivery. An example illustrating the significant but focal nature of myocardial hyaline degeneration, interstitial edema, and evidence of actual myofiber loss encountered in dying high-dose animals. Lesions of this type (left half of illustration i were detected only in animals dying during delivery or immediately postpartum. The myocardium in the right half of this illustration is unaffected * 200 Fig. 3. 24 mg/kg: death on day 22 pc before delivery. Discrete area of significant myocardial necrosis with mononuclear phagocytic infil tration. Affected myofibers have undergone granular degeneration. Adjacent myofibers are normal. Multiple foci of this type were seen elsewhere in the myocardium of this animal i x 200). Fig. 4. 24 mg kg: sacrificed moribund on day 10 pp. Conspicuous myofiber cytoplasmic vacuolization was frequently noted in animals dying later in the postpartum period. These cytoplasmic alterations were unaccompanied by inflammation or other evidence of myofiber degeneration. The distribution of affected fibers ranged from limited areas to widespread regions of myocardium. The vacuoles themselves were found to be devoid of fat or glycogen ! * 400). Fertility and reproductive performance No remarkable variation in pregnancy rate occurred among the groups. Considering the mean length of the gestation period of those animals completing delivery, there was no treatment-related variation among groups (Table 4). A slightly lower gestation index in Group VI (fluvastain at 24 mg/kg/day) reflects the one animal which had no viable pups, 5 stillborn pups, and 7 dead pups of unknown viability status at birth. Postnatal litter data The mean number of liveborn pups was lower in both groups administered fiuvastatin alone (Groups V and VI) when compared to concurrent controls (Table 5). An increased incidence of stillborn pups occurred in the 12 and 24 mg/kg/day fiuvastatin Group V (7%) and Group VI (22%), compared to a range of 2-3% in controls and Groups II, III, and IV. There was no evidence to suggest any remarkable increase in in utero postimplantation embryo/fetal mortality (i.e., early or late resorptions) in any of the groups, either before or after initiation of fiuvastatin and/or mevalonic acid administration (Ta ble 5). Neonatal mortality of 15% and 56% was ob served through weaning in both 12 and 24 mg/kg/day fiuvastatin Groups V and VI, respectively (Table 5), with a statistically significant increase noted at 8-14 days postpartum in Group V and as early as 1-4 days postpartum in Group VI. Although the overall neona tal mortality in Group IV (24 mg/kg/day fiuvastatin supplemented with mevalonic acid) was also statisti cally significant, it was minimal. Drug-related lower birth weights as well as lower neonatal body weights during the lactation period were evident in both 24 mg/kg/day fiuvastatin dose groups, with and without mevalonic acid supplementation. The most prominent neonatal clinical observations occurred in Group VI (24 mg/kg/day of fiuvastatin) where several litters, whose dams were adversely af- BEST COPY AVAILABLE 000034 24 R.V. HRAB ET AL. TABLE 3. P e rc e n t body w eight change an d relative food consum ption co m p ared to controls Group % body weight change: days 15-20 pc 0 -7 pp 0 -14 pp 7-14 pp 14-21 pp Relative food consumption: days 15-20 pc I Control 0 mg/kg/day 18 4 8 4 -5 100* II Mevalonic Acid 500 mg/kg b.i.d. 18 3 4 1 -3 100* ni rv Fluvastatin + Mevalonic Acid 12 mg/kg/day + 500 mg/kg b.i.d. 24 mg/kg/day + 500 mg/kg b.i.d. 18 16 2 -2 42 24 -4 -3 96* 89* V VI Fluvastatin 12 mg/kg/day 16 -5 -1** 4 -3 93* 24 mg/kg/day 16 -9* -3* 1 -1 89* *P 0.01. **P 0.05. TABLE 4. F ertility a n d reproductive p erfo rm an ce Group No. of anim als m ated/group # Pregnant Pregnancy rate1 Gesitation index3 No. of anim als completing delivery Mean length gestation of animals completing delivery (days) Range (days) I Control 0 mg/kg/day 20 18 90* 100* 18 21.9 21-22 n Mevalonic Acid 500 mg/kg b.i.d. 20 17 85* 100* 163 21.8 21-22 in IV Fluvastatin + Mevalonic Acid 12 mg/kg/day + 500 mg/kg b.i.d. 24 mg/kg/day + 500 mg/kg b.i.d. 20 15 75* 100* 20 18 90* 100* 15 18 22.0 21.7 22 21-22 V VI Fluvastatin 12 mg/kg/day 20 17 85* 100* 16 24 mg/kg/day 20 17 85* 94* 16 22.1 22-23 22.2 21-23 P e rc e n t m atings resu ltin g in pregnancy; includes anim als which died. P e rc e n t pregnancies resu ltin g in litters with one or more viable offspring; does not include anim als which died. 3Does not include one anim al which delivered 4 stillborn, 4 dead, and 6 viable pups, but was sacrificed in a moribund condition on day 1 pp following a probable intubation accident on day 22 pc. TABLE 5. N eo n atal litte r size a n d m o rta lity Group Mean no. of pups delivered Mean no. of livebom pups Mean no. of im plantation sites In utero post im plantation loss Overall neonatal m ortality through weaning Control 15.4 15.1 16.4 6.1% 3* n 16.1 15.9 17.3 6.9* 5% m 17.1 16.7 18.2 6.0* 6* *P 0.01. **P 0.05. IV 15.6 15.2 16.7 6.6* 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 lactation period, or were culled prior to weaning, showed that both groups administered fluvastatin alone (Group V and VI) had a higher incidence of pups without milk visible in the stomach. Macroscopic and microscopic evaluation of spontaneously dying animals No macroscopic evidence of treatment-related effects was noted other than the anticipated forestomach thickening present in 2 of 3 and 7 of 8 animals from the 12 and 24 mg/kg/day fluvastatin groups, respectively, which died or were sacrificed moribund. qQG035 MEVALONATE AND PREGNANT RAT FLUVASTATIN TOXICITY 25 TABLE 6. Tim e an d incidence of tre a tm e n t-rela te d m atern al m o rta lity 1 Study_____________ Segm ent III_______ Segment III follow-up____________ Segment III m evalonate supplementation Fluvastatin Fluvastatin MEV Fluvastatin + MEV Fluvastatin * MEV Fluvastatin Dose (mg/kg) 2 12 24 2 6 12 24 500 12 * 500 24 -* 500 12 24 Day 15-21 pc 0 pp = 22 pc 23 pc 1 -4 pp 5 -15 pp 2 24 11 1 16 2 4 2 13 11 11 6 `To facilitate sim ultaneous presentation of data, the control groups (all negative) were omitted. Microscopic findings consisted of a variety of myo cardial lesions in all dying Group VI (fluvastatin at 24mg/kg/day) animals. The cardiomyopathy was charac terized by focal myocarditis and lesions of myofiber hy aline, granular and vacuolar degeneration (Table 2). These findings were considered treatment-related and similar to those seen in a previous study in pregnant or lactating animals dying during or after parturition. Two types of myocardial lesions were detected micro scopically. In animals dying at and within a few days after parturition, the lesions were characterized by hy aline degeneration of isolated groups of myofibers ac companied by slight interstitial edema and hemor rhage (Fig. 2 and 3). The lesions typical of animals dying 3 -4 days after parturition or later were charac terized by widespread cytoplasmic vacuolar changes in the myofibers, i.e., the occurrence of large and small clearly defined round empty vacuoles (Fig. 4 and 5). Occasionally these were accompanied by a sarcolemmal response and inflammatory cell infiltrates, which suggested a myofiber proliferative or reparative re sponse to the induction of the myocardial injury. Cardiomyopathy was not present in the spontane ously dying animals which had been given a lower dose (12 mg/kg/day) of fluvastatin alone. AH animals dying in the 24 mg/kg/day fluvastatin group had cardiac le sions. These lesions were considered related to the cause of death in these animals. Terminal sacrifice animals The only treatment-related effect noted occurred in all the groups which received fluvastatin. Most of the animals in Group III (14 out of 19), Group IV (17 out of 18), Group V (14 out of 17), and Group VI (11 out of 12) had varying degrees of forestomach thickening. No ev idence of this effect was detected in Group II (meval onic acid alone) or the controls. In Groups III and IV, supplementation with mevalonate did not influence the incidence of fluvastatin-induced forestomach thick ening. Microscopically, the forestomach thickening was characterized by epithelial hyperplasia/hyperkeratosis in all groups which received fluvastatin, including those receiving the mevalonic acid supplementation. No evidence of ulceration accompanied these hyper plastic lesions. The hyperplasia ranged from mild to moderate in degree. In the absence of specific quanti tative measurements, there appeared to be correlation, with greater degrees of hyperplasia seen at the higher doses of fluvastatin in Groups IV and VI (24 mg/kg/ day) compared to the response seen in Groups III and V (12 mg/kg/day). There did appear to be a lesser degree of hyperplasia/hyperkeratosis in the animals adminis tered mevalonate and 12 mg/kg/day fluvastatin (Group III) compared to the low dose of fluvastatin alone (Group V). Histological evidence of fluvastatin-induced myocar dial fiber injury and/or inflammation was not present in the mevalonate-supplemented Groups III and IV. Likewise, in the surviving Group V and VI (fluvastatin at 12 and 24 mg/kg/day) animals, myocardial lesions were not detected. DISCUSSION The maternal toxic effects induced by fluvastatin re ported herein had been encountered in two previous studies at similar dose levels, and consisted of mater nal mortality at delivery and during the postpartum period (Table 6). Microscopic evidence of associated myocardial lesions was detected in the follow-up study. An increase in stillborn pups was observed with no evidence of earlier in utero fetal mortality, suggesting a toxic effect on the dams immediately prior to, or dur ing, parturition. The absorption and disposition of flu vastatin have been studied in nonpregnant rats (Tse et al., '90) and in pregnant and lactating rats, and in suckling pups (A. Schweitzer, unpublished data). In all cases fluvastatin was rapidly eliminated with little or no accumulation in the tissues. Limited placental transfer resulted in very low levels in embryos or fe tuses. Fluvastatin was detected in maternal milk and subsequently at measurable levels in suckling pups. However, the neonatal mortality observed soon after parturition, as well as during the lactation period, may be a reflection of adverse effects relative to deficient maternal lactation and lack of litter care as influenced by maternal toxicity. The results indicate that fluvastatin-induced mater nal and neonatal mortality could be completely blocked 000036 26 R.V. HRAB ET AL. and/or ameliorated by coadministration of mevalonic It would not be unexpected, therefore, that inhibition of acid. Adverse effects on maternal body weight were this critical enzyme would influence or disturb the dy nonexistent with mevalonic acid supplemented fluvas- namic events occurring in late gestation and at partu tatin at 12 mg/kg/day and markedly improved at 24 rition. The data from Groups III and IV clearly indicate mg/kg/day, although food consumption remained that mevalonate supplementation prevents mortality slightly decreased at 24 mg/kg/day despite supplemen and the development of heart lesions. It is not clear tation with mevalonic acid. The decreased neonatal from these studies exactly what the effect is in the body weight gain during lactation at 24 mg/kg/day may myocardial tissues which allows development of the be related to the reduced maternal food intake noted at heart lesions, and why the restored or maintained lev this dose level. els of mevalonate suppress or block the development of While mevalonate supplementation prevented mor these lesions. tality and the development of heart lesions in Groups III and IV (fluvastatin at 12 and 24 mg/kg/day), it did not reduce the direct effect of fluvastatin on the gastric forestomach squamous epithelium, although the de LITERATURE CITED Alberts. A.W. (1988) Discovery, biochemistry and biology of lovastatin. Am. J. Cardiol., 52:10-15. Brown, M.S.. and J.L. Goldstein (1980) Multivalent feedback regula gree did appear somewhat lower in Group III. This is tion of HMG CoA reductase, a control mechanism coordinating iso- considered further evidence that hyperplasia/hyper- prenoid synthesis and cell growth. J. Lipid. Res., 22:505-517. keratosis is the result of direct or contact irritation by fluvastatin on the squamous forestomach epithelium. ^/Engstrom, R.G., D.B. Weinstein, F.G. Kathawala. T. Scallen. J.B. Eskesen, M.L. Rucker, R. Miserendino. and J. Babiak (1988) Hypolipoproteinemic activity of XU 62-320. a potent competitive inhib The fluvastatin-induced early cardiac lesions char itor of HMG-CoA reductase. Eighth International Symposium on acterized by hyaline degeneration (before or at partu Atherosclerosis, Rome. October 9-13, 1988: 230 (abstract). rition) resembled those induced by isoproterenol (preg *Kathawala, F.G., T. Scallen, R.G. Engstrom, D.B. Weinstein, H. nant rats are uniquely sensitive)1. The postnatal Schuster, R. Stabler, J. Kratunis, J.R. Wareing. W.F. Jewell, L. Widler. and S. Wattanasin (1988) XU 62-320, an HMG-CoA reduc lesions (vacuolar sarcoplasmic changes and myocardi tase inhibitor, more potent than compactin and lovastatin. Eighth tis) appeared to be the result of sarcoplasmic fluid ac International Symposium on Atherosclerosis, Rome. October 9-13, cumulation possibly related to alteration of cellular membrane structures.1The relationship or progression between pre- and postpartum lesions, if any, is not clear. Likewise, it is not known at what point in their 1988; 445 (abstract). athawala. F.G. (1991) HMG-CoA reductase inhibitors: An exciting development in the treatm ent of hyperlipoproteinemia. Med. Res. ,, Rev., 22:121-146. Kornbrust, D.J., J.S. MacDonald. C.P. Peter, D.M. Duchai, R J. development either would be reversible. Stubbs, J J. Germershausen, and A.W. Alberts (1989) Toxicity ofthe Several factors existed which probably contributed to the development of myocardial damage and/or death of the fluvastatin-treated animals. In addition to the con HMG-Coenzyme A reductase inhibitor, lovastatin, to 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. Bokelman (1988) Preclin- current stresses of pregnancy, parturition, and lacta , ical evaluation of lovastatin. Am. J. Cardiol., 62:16-27. tion, the exaggerated pharmacologic activity of fluvas tatin, i.e., inhibition of mevalonate and cholesterol biosynthesis, was a significant factor. HMG-CoA re ductase and its immediate product mevalonic acid play Minsker, D.H., J.S. MacDonald. R.T. Robertson, and D.L. Bokelman (1983) Mevalonate supplementation in pregnant rats suppresses the teratogenicity of mevinolinic acid, an inhibitor of 3-hydroxy-3methylglutaryl-coenzyme A reductase. Teratology. 38:449-456. Robison. R.L., W. Suter, and R.H. Cox (1994) Carcinogenicity and a key role in cholesterol biosynthesis and are essential mutagenicity studies with fluvastatin. a new entirely synthetic for the synthesis of steroid hormones, cell membranes, cell growth, and normal cellular metabolism (Brown and Goldstein, '80; Alberts, '88; MacDonald et al., '88). HMG-CoA reductase inhibitor. Fund. Appl. Toxicol, (manuscript accepted for publication). Stoll, R.E., DJ. Ball, M. Evans, O. Hockwin. S. Roberts, R. Robison, L. Rubin, and C. Smith (1988) Subchronic target organ toxicity of XU 62-320 in the rat. mouse, dog and monkey. Eighth International Symposium on Atherosclerosis, Rome. October 9-13, 1988:902 (ab stract). Tse, F.L.S., H.T. Smith, F.H. Ballard, and J. Nicolletti (1990) Dispo 'Wagner. B.M.. Deputy Director, Nathan S. Kline Institute, NYU. sition of fluvastatin. an inhibitor of HMG-CoA reductase, in mouse, Consultation at Sandoz, East Hanover, N.J. December 29, 1989. rat, dog, and monkey. Biopharm. Drug Dispos., 22:519-531. 000037