Animal safety and toxicology of simvastatin and related hydroxy-methylglutaryl-coenzyme a reductase inhibitors

Animal Safety and Toxicology of Simvastatin and Related Hydroxy-methylglutarylCoenzyme A Reductase Inhibitors RONALD J. GERSON, Ph.D.,JAMES S. MACDONALD, Ph.D.,ALBERT W. ALERTS, M.S., DOUGLAS J. KORNBRUST, Ph.D., JAMES A. MAJKA, D.V.M.,R. JOHN STUBBS, Ph.D.,DELWIN L. BOKELMAN, D.V.M.,Ph.D. westpoint,Pennsy/vania

Simvastatin, a hydroxy-methylglutaryl-coenzyme A reductase’ inhibitor intended for use as a hypocholesterolemic agent, has undergone a thorough preclinical toxicology evaluation. This review describes preclinical tosicology findings associated with simvastatin administration in animals and provides the rationale for our conclusion that these changes are not indicative of potential human toxicity. Although it was not surprising to find that a potent inhibitor of this key biochemical pathway produces tosicity at high dosages in animals, none of the observed changes poses a significant risk to humans at clinical dosages. Many of the tosicities produced by high dosage levels of simvastatin in animals are directly related to the drug’s biochemical mechanism of action and are the result of a profound, sustained inhibition of the target enzyme that is not anticipated at clinical dosages. Furthermore, several of the simvastatin-induced changes are species-specific responses to this agent and are not relevant to human risk assessment. Of the treatment-related changes reported for simvastatin, the development of cataracts in dogs has received considerable attention. The available data demonstrate a wide margin of safety in terms of dosage levels required to elicit this response as well as the plasma concentrations associated with the development of these ocular lesions. The data suggest that the development of lenticular opacities at clinical doses of simvastatin is highly improbable. Overall, simvastatin was well-tolerated by animals in preclinical toxicology studies, and no findings contraindicating its use in humans were identified.

From the Departments of Safety Assessment and Drug Metaboksm. Merck Sharp & Dohme Research Laboratones, West Pomt. PennsylvanIa; the Department of BIOchemical Regulation. Merck Sharp & Dohme Research Laboratones, Rahway. New Jersey; and Rarer Pharmaceuticals Corporation, Fort Washington, PennsylvanIa. Requests for reprints should be addressed to Dr. Ronald J. Gerson. Department of Safety Assessment, Merck Sharp & Dohme Research Laboratories, West Point, Penn. sylvania 19486. 4-20s

October 16, 1989

The American Journal of Medicine

imvastatin is a hyclrosy-methylglutaryl-coenzyme A reductase inhibitor intended for clinical use as S an agent for lowering serum cholesterol. By inhibiting hepatic cholesterol biosynthesis at the level of hyclrosy-methylglutaryl-coenzyme A recluctase, this drug (as well as other members of this class) produces compensatory increases in hepatic low-density lipoprotein receptors, resulting in an increased uptake of low-density lipoprotein cholesterol from the blood and the subsequent lowering of circulating cholesterol levels [1,2]. Simvastatin is structurally similar to lovastatin (currently approved for use within the United States, Canada, and several other countries), except for the presence of an additional methyl group on the ester side chain (Figure 1). Simvastatin exists as a lactone (the prodrug form) and is hydrolyzed i,r viva to the biologically active beta-hyclrosy acid form designated L-654,969 (Figure 1). A similar hydrolysis occurs with lovastatin, which also is administered as the lactone prodrug form. The basis for administering the lactone proclrug forms of these inhibitors has been addressed by Germershausen el crl [3] and Duggan et al [43. The lactone forms have been observed to undergo a greater degree of hepatic extraction from the circulation than do their respective beta-hyclrosy acid derivatives, rendering them more liver specific [3]. As a consequence of these higher hepatic extraction ratios, systemic exposure to circulating drug substance is decreasecl. Simvastatin is a potent inhibitor of hydroxy-methylglutaryl-coenzyme A reductase, with a 50 percent inhibiting concentration for isolated rat hydrosy-methylglutaryl-coenzyme A reductase in the low nanomolar range. Inhibition of hyclrosy-methylglutaryl-coenzyme A reductase results in a decreased synthesis of mevalonic acid, which serves as a precursor not only for biosynthesis of sterol, but also for biosynthesis of ubiquinone (involved in electron transport), dolichols (involved in glycoprotein synthesis), isopentenyl tRNA (involved in DNA translation) [5], and other unidentified isoprenoicl-containing proteins [Gl (Figure 2). It was not unespectecl, therefore, that the administration of high dosages of simvastatin to animals in preclinical tosicology studies resultecl in a spectrum of treatment-related changes. Many of these changes, however, either were shown to be the product of a profound, prolonged inhibition of the target enzyme not anticipated at clinical dosages (esaggerated biochemical effect) or were directly related to a substantial systemic exposure to circulating drug achieved as a consequence of the high dosages of test compound administered.

TOXICOLOGIC PERSPECTIVES In general, preclinical toxicology studies conducted

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Hydroxy-Acid

Lactone Form:

Form:

“O\yyO

3

MK-733 (Simvastatin)

Figure 1. Structures of principal hydroxy-meth. ylglutaryl-coenzyme A reductase inhibitors and their respective beta.hydroxy acid forms.

L-154,819

MK-803 (Lovastatin)

ACETYL CoA 1 HM; I

Figure 2. Cholesterol synthetic pathway display ing isoprenoid products derived from mevalonic acid. CoA = coenzyme A; HMG = hydroxy methyl glutarate.

I

CoA

1HMG CoA reductase] t Mevalonate : Mevalonate pyrophosphate I t lsopentenyl pyrophosphate I

: Ubiquinone

Squalene T T Cholesterol

with any drug candidate are specifically designed to describe the toxicologic liabilities of a compound after administration to animal species for a finite period of time. To maximize the ability of these tests to detect potential toxicity, high dosage levels of the test article are routinely used. These dosages are commonly many times higher than the intended clinical dosage. A common approach in preclinical toxicology studies is the cletermination of a “no-effect dose” for toxicity in animal species. By demonstrating that toxic effects in animals are elicited only at high dosages, but not at lower dosages (which are still substantially above the intendecl clinical dose), a margin of safety for toxicity is clemonstrated. This risk assessment can be further strengthened by demonstrating that the toxic effects produced at these high dosage levels are the product of the intencled biochemical mechanism of action of the

JL-

Dimethylallyl pyrophosphate

--*

lsopentenyl tRNA

I v Dolic\ol

test article and not clue to an intrinsic toxic property of the drug molecule itself. By clemonstrating a relationship to the biochemical mechanism of action of the test article, the definition of the threshold close is based on not only circumstantial evidence (effect versus no effect), but on exaggerated biochemical/pharmacologic effects not anticipated at lower (clinical) closes. This approach has been used in the safety assessment of simvastatin and other hyclroxy-methylglutaryl-coenzyme A reductase inhibitors in this laboratory. Many of the toxicities attributed to simvastatin have been reversed by the coaclministration of mevaionic acid, the product of the enzyme inhibited by simvastatin. When giving animals mevalonic acid supplements, the biochemical inhibition of hyclroxy-methylglutaryl-coenzyme A recluctase is essentially by-

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40-mg dose administered to a 50-kg patient) has been shown to result in the development of a low incidence Simvastatin: Target Organs Observed in Animal Studies of subcapsular lenticular opacities in dogs (Table II). These ocular lesionsare characterized by an increased Monkey Organ MOWS Rat Rabbit Dog prominence of the posterior and and/or anterior suture lines. With continu.ed drug administration, they t t t Lrver progress to involve the cortical region and may event Kidney tuall:y develop into complete cataracts. Not all dngs Ni Gallbladder t Ni4 Stomach (nonglandular) + NtA NiA receiving high dosages of simvastatin develop catat Eye (lens) racts; ocular changesare observed only in a small pert Testes centage (approximately 10 percent). Furthermore, + = hwe affected in some way by drug treatment; - = no effecl observed m this organ simvastatin has been administered at dosagesof up to in this species: NA = not apphcable [twe does no1 exrst In this specres). 10 mglkg per day (more than 12 times the maximal clinical close)for up to two years without producing ocular abnormalities (Table II). TABLE II Considerable attention has been given to the geneIncidence of Cataracts in Dogs Treated with Hydroxy sis of these ocular lesions, especially in view of past MethylglutarylGoenzyme A Reductase Inhibitors clinical esperience with triparanol (MER-29). Triparano1 is a late-stage inhibitor of cholesterol synthesis Total Incidence of that was marketed for human use (and subsequently Cataracts Maximal withdrawn) in the 1960sby Wm. Merrell PharmaceuDuration of ’ Dose ticals. This hypocholesterolemic agent was associated Level Period of Exposure with the production of similar lenticular lesionsin both Sensitivity* (weeks) Drug hgikglday) humans and animals at clinical dosages173,as well as 90 5140 9-13 26 Simv3stahn the production of other toxicities in tissues of ectoderi: 105 50 2120 ma1origin (skin and hair) [8]. Several other late-stage 105 O/28 inhibitors of cholesterol synthesis have also been asso105 Lovastalin 1: 3142 11-28 ciated with the nroduction of cataracts in animal modo/to 1:: 60 1116 :: els [9,10]. * 79 LG4,969t ii 8128 IO-28 Simvastatin and hydrosy-methylglutaryl-coenzyme IO I/20 A reductase inhibitors in general are biochemically Iti : O/20 distinct from previous hgocholesterolemic agents, L-645,164$ 5: 618 :: 10 O/8 rfA most of which were associatedwith an accumulation of I late-stage cholesterol biosynthesis intermediates (i.e., NtR = not applicable (no cataracts produced). Necks during which lens changes first detected. desmosterol) clue to their site of inhibition in the cho;: ntemal designabon for acid form of slmvastalin , the betahydroxy lesterol synthetic pathway (conversion of desmnsterol tlnremal oesrgnanon Ior a SlNCtUrally Olsslmllar hydroxy-methylglularyl+zoenzyme A re. ductase Inhibitor that is a Ruorina(ed brphenyi. to cholesterol). Although the role of cholesterol precursor accumulation in the development of cataracts passed.Any toxicities prevented or reversed by mev- incluced by inhibition of cholesterol synthesis is conalonic acid supplementation are then attributable to troversial [ill, an analogous build-up of precursors the biochemical mechanism of action of simvastatin does not occur in the lens (or any organ) after treat(provided drug uptake or distribution is not altered) ment with simvastatin or other hydrosy-methylglutid not due to an intrinsic toxic property of the drug taryl-coenzyme A reductase inhibitors [ 121. Excess molecule itself. hydroxy-methylglutaryl-coenzyme A is shunted into This article describes the chug-induced changes other metabolic pathways. identified during the preclinical toxicology studies of The development of cataracts in clogsis not limited simvastatin and discusseshow these changesrelate to to simvastatin, but has also occurred after the subthe clinical use of this drug. The results of the preclini- chronic/chronic administration of a variety of other cal investigations have clemonstrated that none of the hyclroxy-methylglutaryl-coenzyme A reductase inhibchanges attributecl to simvastatin administration in itors studied in this laboratory [12,13] (Table II). animal modelshave significant clinical implications for These lenticular opacities are morphologically identihuman safety. cal to the opacities produced by simvastatin. However, the incidence of cataract varies from compound RESULTS OF SIMVASTATIN PRECLINICAL TOXICITY to compound, with some producing a relatively low STUDIES incidence of cataracts (approximately 10 percent for Administration of high dosages of simvastatin to simvastatin and lovastatin) and others a relatively animals in preclinical toxicology studies has resulted high incidence (between 28 and 75 percent for Lin a spectrum of toxicities in a variety of organ sys- 654,969 and L-645,164, respectively [L-645-164 is a tems. Table I presents a summary of affectecl organ fluorinated biphenyl hydroxy-methylglutaryl-coensystems, listed by species. Each toxic effect is dis- zyme A recluctase inhibitor]). cussed individually by the affected organ system in Attempts to use these animal data to assessthe pothe following sections. tential risk of cataract development in patients receiving clinical dosagesof simvastatin were greatly faciliCataracts tated by comparisonsamolig hyclroxy-methylglutarylThe long-term administration of simvastatin at dos- coenzyme A reductase inhibitors producing differing agesof at least 50 mg/kg per day (more than 60 times incidences of cataracts in clogsin our laboratory. An the maximal clinical dose of 0.8 mg/kg, based on a initial observation was that the degree of serum choTABLE I

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Figure 3. Comparison of serum cholesterol lowering and cataract incidence produced in dogs receiving various hydroxy-methylglutaryl-coenzyme A reductase inhibitors. Ears represent data obtained from at least eight dogs. Error bars Indicate SD.

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’ Simvastatin 50 mglkglday

lesterol lowering was not, in itself, a sole cletermination of the production of lens opacities in dogs. In fact, the incidence of cataract formation differed from compound to compound, despite the administration of dosages that produced nearly equivalent decreases in serum cholesterol (Figure 3). Furthermore, examination of individual serum cholesterol values for dogs receiving 50 mg/kg per day of simvastatin (which resulted in the development of lens opacities in two of 20 dogs after 41 weeks of continuous treatment at this dosage level) revealed that some of the unaffected dogs had serum cholesterol values as low or lower than those in the dogs that developed cataracts (Figure 4). However, since this type of lenticular lesion is uncommon in untreated dogs of this age and only occurs in dogs receiving high dosages of simvastatin (and other hydroxy-methylglutaryl-coenzyme A reducatase inhibitors), additional risk factors were apparently involved. Further comparisons among various hydroxymethylglutaryl-coenzyme A reductase inhibitors revealed dramatic differences in circulating plasma lev-

Lovastalin 180 mglkglday

L654969 30 mglkglday

L645164 50 mg/kg/day

els of inhibitor at pharmacologically equipotent doses. For example, after the administration of pharmacologically equipotent doses of simvastatin or L-654,969 (its corresponding beta-hydroxy acid form; see Figure 1) plasma levels of circulating inhibitor were approximately one order of magnitude greater in dogs given the beta-hydroxy acid form, L-654-969 (Figure 5). Long-term administration of L-654,969 also yieldecl a higher incidence of cataracts and produced cataracts at lower closage levels in dogs (Table II). When the remaining hydroxy-methylglutaryl-coenzyme A reductase inhibitors were examined, a clear association was observed between circulating plasma drug levels and the cataract incidence produced by these agents (Figure 6). Agents that produced high drug levels in the plasma (i.e., L-654-969 and L-645,164) were associated with a higher incidence of cataracts, whereas those that produced low plasma levels of&-culating inhibitor (i.e., simvastatin and lovastatin) were associated with a lower incidence. Although higher plasma levels of drug would intuitively be expected to procluce a greater level of drug

Simvastatin (50 mg/kg/day)

-2-l

12

3

4

5

6

7

8

9

10111219232631343642

Drug Week

I ^. .-. c;alaracrs uiagnosea

Figure 4. Relationship between individual serum cholesterol values and the development of cataracts in dogs receiving 50 mgfkg per day of simvastatin. Individual serum cholesterol values are plotted for dogs with clear lenses (boxes) and for dogs that were observed to have lens opacities during Drug Week 4.1 of . this ..- studv ____ hlid ,-____ circles1 _______,_A total of 20 dogs were followed during the course of this 105.week study (data are shown up to Week 42 of drug administration). No other dogs receiving simvastatin in this study developed lens abnormalities after Week 41 of treatment.

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2 14000 $

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deposition in the lens (due to higher levels of drug in the aqueous humor), cataract-containing lenses were not found to have higher levels of associated drug than were clear lenses, and no correlation between lens drug levels and cataract formation could be described (Figure 7). Therefore, the best measure of cataractogenic risk in dogs was fomld to be the degree of systemic exposure to inhibitor, as quantitatecl by circulating inhibitory activity. These data were subsequently used in formulating a risk assessment for cataract formation in humans. Plasma drug level analysis performed on patients receiving simvastatin in clinical trials revealed that very low levels of circulating inhibitor are achieved in humans at clinical dosages when compared with those measurecl in clogs at cataractogenic close levels. The maximal anticipated clinical dose of 40 mg per day of simvastatin (approximately 0.8 mg/kg per day) produced plasma levels approximately 14 times lower than the minimally cataractogenic close (50 mg/kg per 100 r

*Ot

L-645,164

0

10000

20000

3OCOO 40000

50000

60000

Plasma AUC (ng Equiv * hrlml) Figure 6. Relationship between circulating plasma inhibitory activity and cataract incidence in dogs after the administration of various hydroxy-methylglutarylcoenzyme A reductase inhibitors. Dogs received all agents at approximately pharmacologically equipotent doses: lovastatin (180 mg/kg per day); simvastatin (50 mg/kg per day); L-654,969 (30 mg/kg per day); and L-645,164 (50 mg/kg per day). Plasma inhibitory activities are expressed as 24.hour area under the curve (AUC) values. Bars represent at least eight dogs.

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(Hours)

Ill?

24

Figure 5. Trme course of plasma Inhibitory acbvsty in dogs after oral admrnistration of either srmvastatin or its beta-hydroxy acid form (L.654, 969). Dogs received either simvastatin (50 mg/kg per day) or L-654.969 (30 mg/kg per day) for one month. Plasma levels of inhibitory activity were assayed at various bmes after the last dose of test compound. Each pomt represents the mean ? SD of four mdrvrdual dogs.

clay) in clogs and approximately five times lower than the noncataractogenic close (10 mg/kg) in clogs (Figure 8. In addition, the data also indicate that long-term aclministration of hydroxy-methylglutaryl-coenzyme A recluctase inhibitors (including simvastatin) was not associated with an increased risk of cataract development over time. The incidence of opacities dicl not increase as the closing duration increased. In the particular case of simvastatin, although two of 20 clogs receiving 50 mg/kg per clay developed cataracts by Week 41 of drug administration, none of the remaining dogs developed cataracts during the subsequent 64 weeks of this two-year stucly. These data reveal that, despite the clevelopment of cataracts in susceptible animals relatively early in the study, continued closing with simvastatin did not increase the risk of cataract formation in the remaining clogs. Finally, a comparison of clata obtained from laboratory animals given triparanol and simvastatin has revealecl dramatic differences between clinical closes ancl those requirecl to elicit cataracts (Tables III and IV). In a study conducted in this laboratory, clogs receiving triparanol showed evidence of lenticular damage and cataract formation at clinical closes (Table III). These data have permitted strong conclusions regarcling the potential risk of cataract development in patients receiving clinical closes of simvastatin. The low circulating plasma levels of inhibitor obtained at clinical dosages and the apparent lack of an increased risk of cataract development after long-term closing suggest that cataract development is highly unlikely with simvastatin (or lovastatin) therapy in humans. Hepatic Effects As previously mentioned, the liver is the primary site of endogenous cholesterol synthesis ancl is the target organ of simvastatin [141. Studies concluctecl in dogs ancl rats have inclicated, respectively, that simvastatin undergoes a high degree of hepatic extraction (approximately 95 percent) 1151 and is found in high concentrations in the liver. The development of hepatic changes in a variety of species was not surprising, therefore, in view of the central role played by the cholesterol synthetic pathway in cellular biochemistry and the high closages used in preclinical toxicology stuclies. Three distinct changes were observed in

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Figure 7. Hydroxymethylglutarylcoenzyme A reductase inhibttory activity assocrated with clear and cataract-contaming lenses from dogs treated long-term with simvastatin or L-654,969. Points represent the total inhibitory activity associated with individual clear (solid circles) and cataract-containing (open circles) lenses at various times after the long-term administration of simvastahn (50 mg/kg per day) or its betahydroxy acid form L-654,969 (30 mg/kg per day). Assay weeks represent scheduled Interim necropsies conducted during respective long term toxicity studies, Cataracts were frrst observed during Week 41 of drug administration for dogs receiving 50 mg/kg per day of simvastahn and during Weeks 16 to 28 of drug administration for dogs receiving 30 mg/kg per day of L.654,969.

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5000

I 105

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Figure 8. Mean 24.hour plasma area under the curve (AUC) values In dogs and humans after administration of simvastabn. Bars represent the mean ? SD of at least seven separate sub. jects.

20

30

Dose (mg/kg/day)

three of the species tested: periportal cellular atypia in rats, centrilobular necrosis in rabbits, and transient transaminase elevations (primarily alanine aminotransferase) in clogs. No hepatic changes were identified in mice or rhesus monkeys. HEPATIC CHANGES IN RATS. Inrats, acharacteristic change produced by simvastatin (as well as lovastatin and other hydroxy-methylglutaryl-coenzyme A reductase inhibitors) is periportal hepatocellular atypia. This change, which is limited to the rat liver, is typified by pleomorphic periportal hepatocytes that exhibit varying tinctorial properties. Most commonly, the periportal hepatocytes are hypertrophied and are eosinophilic. A significant amount of evidence indicates that this is a. mechanism-based lesion specific to . . . . rats, with no implications for human risk. Rats appear to adapt to hydroxy-methylglutarylcoenzyme A recluctase inhibition differently than do other species, including humans. In humans! administration of simvastatin is believed to result m the upregulation of hepatic low-density lipoprotein receptors as a principal means of maintaining intracellular cholesterol concentrations. Rats, in contrast, do not experience an analogous increase in hepatocellular lowdensity lipoprotein receptors, but compensate for this inhibition by inducing more of the target enzyme.

TABLE III Incidence of Cataract Development in Dogs Receiving Triparanol* 33+Week Toxicity Study in Dogst

Dose (mg/kg/day)

Percent Decrease in’ Serum Cholesterol

Dogs Affected (n)/ Dogs Tested (n) 014

1: ii

37 50 73

2 313

*Clinical dose, 4 to 25 mglkg per day; based on reported ___

dosages

between

250 an

1.500 mghg.

tStudy conducted at Merck Sham & Dohme Research Laboratories, November 24,1961. *Lens

swelling reported.

They do this to such a degree that the inhibition of hydroxy-methylglutaryl-coenzyme A reductase is essentially overcome, thereby maintaining both intracellular and circulating cholesterol concentrations. Examination of rat hepatocytes via electron microscopy after the subchronic/chronic administration of hydroxy-methylglutaryl-coenzyme A reductase inhibitors revealed a significant proliferation of the smooth

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TABLE IV incidence of Cataract Formation in Dogs Receiving Simvastatin* 10SWeek Toxicity Study in Dogs Cataract Incidence

Decreasein SerumCholesterol

DogsAffected(n)/ DogsTested(n)

15 36 55

0120 0120 0120 2/20

Percent

Dose

hglkdday) 0

Xnrcal dose, 0.1 to 0.8 mg/kg per day; based on anhcrpated I rng per day

clmrcal dose behveen

4 to

TABLE V incidence of Hepatocellular Atypia in Rats after Six Months of Dosing with Lovastatin Lovastatin

Cellular atypra

3

0

3

21

devalonate adminrstered at 0.5 percent rn the dret. den and women combrned. lapted wrth permrssron from Am J Cardrol (131.

TABLE VI Simvastatin-Induced Hepatotoxicity and Its Reversal by Mevalonic Acid Supplementation or Forced Feeding in Rabbits Incidence of Hepatic Necrosis

Dosage Level* Control Srmvastahn Simvastabn plus mevalonate

90 m&/day

Srmvastatin plus forced feedrng

90 mglkglday

90m&/dayt 4O&Il&.iay

Rabbits Aff;,c;$b AST

ALT ALP

Tested(n)

14 967 37

21 397 41

64 132 46

016

35

64

99

116 (I)t

LP = alkakne phosphatase; ACT = alanrne aminotransferase; AST = aspartate amrnoansferase. 4dmmistered for srx days. \verage hrstologrc score for all anrmals wrth hepatic necrosrs based on followrng descnp Ins: 1 = very slight; 2 = slight; 3 = moderate: 4 = marked: and 5 = severe.

endoplasmic reticulum [lGl (hyclroxy-methylglutarylcoenzyme A recluctase is a membrane-bound enzyme). Staining with fluorescent antibody prepared against rat hydroxy-methylglutaryl-coenzyme A reductase [16] and examination by immunoelectron microscopy [1’71demonstrated a substantial induction of the target enzyme in this species in response to hydroxy-methylglutaryl-coenzyme A reductase inhibitor aclministration and showed that the induced enzyme is associated with the proliferated smooth encloplasmic reticulum. The areas of fluorescent staining within the liver lobule correlate with the areas of periportal cellular atypia seen microscopically. The regions of periportal 4A-34s

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cellular atypia have, therefore, been shown to represent areas of markecl smooth endoplasmic reticulum proliferation, which contain greatly increased quantities of the target enzyme hydroxy-methylglutarylcoenzyme A reductase. A final experiment confirming the mechanismbased origin of this hepatic change in rats was performecl by supplementing their diet with mevalonic acid. Mevalonic acid is the procluct of hydroxy-methylglutaryl-coenzyme A recluctase; by supplementing the diet of rats with sufficient amounts of the enzyme product, the biochemical effects of enzyme inhibition are essentially overcome, despite the continued administration of inhibitor. Administration of 0.5 percent mevalonic acid in the rat diet completely preventecl the periportal cellular atypia induced by simvastatin (data not shown) and lovastatin (Prahalacla S, personal communication) [131 (Table VI. These experiments demonstrated that this hepatic change is a product of the biochemical mechanism of action of this class of compounds, coupled with the unique compensatory response of rats to inhibition of hyclroxymethylglutaryl-coenzyme A reductase. Since a similar compensatory mechanism does not occur in other species (including humans), similar hepatic lesions are not expected to occur. HEPATIC NECROSIS IN RABBITS. The administration of high dosages of simvastatin (at least 50 mglkg per day), which are well-tolerated by other species, produces dramatic toxicity in rabbits. Body weight losses, profound decreases in food consumption, morbidity, and mortality occurred in approximately 50 percent of the rabbits receiving at least 50 mg/kg per clay of simvastatin [18]. Histologic examination of necropsy tissue samples revealed varying clegrees of centrilobular hepatic necrosis in the affectecl animals, and corresponding elevations in serum transaminase activity were observecl in serum samples analyzed cluring these studies (Table VI). Renal tubular necrosis and necrosis of the gallbladder epithelial mucosa were also observecl. Since hepatic necrosis (as well as renal ancl gallbladcler necrosis) was not observed in any other species receiving comparable closes of simvastatin, further studies were undertaken to determine what factors distinguish rabbits from other species. Data on plasma levels revealecl that rabbits experienced plasma levels of clrug that were far greater than those in any other species. For example, at a dose of 50 mg/kg per clay of simvastatin, plasma levels in rabbits were more than seven times higher than those in dogs (Figure 9). These plasma drug levels were also more than two orders of magnitude higher than those achieved in human subjects at maximal clinical dosages. Another factor that apparently contributed to the sensitivity of the rabbit to simvastatin was the decreased food consumption in the affected animals. Starvation has been associated with a down-regulation in both hepatic low-density lipoprotein receptors [191 and enclogenous hydroxy-methylglutaryl-coenzyme A recluctase enzyme levels [20]. These factors may have further recluced. the intracellular mevalonate ancl cholesterol pools that were already depressed due to the inhibition of hyclroxy-methylglutaryl-coenzyme A reductase by simvastatin. Forced feeding of rabbits receiving hepatotoxic closes of simvastatin ameliorated this toxicity (Table VI) [El], demonstrat-

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6000

Figure 9. Time course of mean plasma inhibitory activity in dogs (solid circles) and rabbits [solid squares) after a single oral dose of sim. vastatin (50 mg/kg). Points represent the mean ?I SD of at least three separate determinations.

12

4

6

8 Time

ing a contributory role of anorexia in the development of simvastatin-induced toxicity in this species. The available data demonstrate that the production of hepatic necrosis (ancl renal as well as gallbladcler necrosis) in rabbits is entirely related to an exaggerated biochemical/pharmacologic effect. First, a similar spectrum of toxicity was produced in rabbits by every inhibitor of hydroxy-methylglutaryl-coenzyme A reductase tested in our laboratory 1211, although the doses necessary to elicit toxicity varied for different compounds. Furthermore, supplementation of rabbits with mevalonic acid completely prevented the clevelopment of toxicity, despite the simultaneous aclministration of toxic doses of simvastatin [18] (Table VI) or other hydroxy-methylglutaryl-coenzyme A reductase inhibitors [21]. Since mevalonic acid had no effect on simvastatin uptake by the liver (data not shown), the results indicate that by antagonizing simvastatin’s biochemical effect one coulcl completely prevent toxicity in rabbits, thereby implicating the biochemical mechanism of action of simvastatin in the genesis of the hepato-, nephro-, and gallblaclcler toxicity in this species. These data have supplied an important perspective regarding the relevance of these changes to human safety. Rabbits are uniquely sensitive to simvastatin. This may be due to a number of cooperative factors, such as’high circulating levels of inhibitor and the synergistic effect producecl by decreases in food consumption (simvastatin does not produce decreases in food consumption in any other species). Simvastatin induced toxicity in the rabbit only at high dosage levels (at least 50 mg/kg); closes of 30 mg/kg were tolerated without similar effects. Finally, simvastatin-induced toxicity in rabbits is entirely mechanism based. Simvastatin has produced toxicity in this species via an exaggeration of its desired pharmacologic effect produced at high dosage levels (at least 60 times greater than the maximal Clinical dose of 0.8 mg/kg per day). Since a clear threshold for toxicity (30 mg/kg)-well above the maximal anticipated clinical dose-exists in rabbits, the development of toxicity in this species has little relevance for human safety. TRANSAMINAiE ELEVATIONS IN DOGS. Transient increases in serum transaminase activities (primarily serum alanine aminotransferase) are observed in a small percentage of dogs receiving simvastatin. As with all the previously discussed toxicities, these ele-

12 (Hours)

24

vations are a class effect seen with every hyclroxymethylglutaryl-coenzyme A recluctase inhibitor tested in clogs thus far [13]. In contrast to the hepatotoxicity observed after simvastatin administration in rabbits, however, no evidence of any hepatic damage has been observed after the administration of high dosages of simvastatin to dogs. The transaminase elevations are close dependent; they are of greater magnitude and occur at a higher frequency with increasing closages of simvastatin. The elevations can be anywhere from slightly above baseline to levels approximately 10 times the normal baseline level (Figure 10). Although the duration of increasecl serum transaminase activity varies, all the elevations have been transient, resolving spontaneously to baseline levels clespite continued administration of simvastatin (Figure 10). . None has progressed to levels inclicative of frank hepatotoxicity, ancl not all clogs have experienced these elevations. Furthermore, affectecl dogs were otherwise asymptomatic; there are no corroborating physical signs, decreases in body weight gain, or food consumption. A thorough retrospective analysis of hepatic tissue taken at necropsy from clogs receiving simvastatin or high dosages of other hydroxy-methylglutaryl-coenzyme A reductase inhibitors after extendecl treatment periods (up to two years) has shown no changes in these livers on light microscopy, despite the occurrence of serum transaminase elevations during the course of these studies (data no! shown). In addition, no other extrahepatic source of serum alanine aminotransferase activity (i.e., cardiac or skeletal muscle) has been identified as a potential source of these elevations. There appear to be some similarities between the transient transaminase elevations seen in clogs receiving simvastatin in preclinical toxicology studies and the transaminase elevations seen in a small percentage of patients receiving simvastatin in clinical trials [221. Patients experiencing these elevations are also completely asymptomatic, and preliminary evidence suggests that these elevations also resolve spontaneously, despite continued dosing with the drug. Although the leakage of hepatocellular enzymes into the circulation is usually considered evidence of hepatotoxicity, the spontaneous resolution of these transaminase elevations is not characteristic of continued closing with a classic hepatotoxicant. The finding

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SYMPOSIUM

ON SlMVASTATtN / GERSON ET AL

210 190 170 150 -g

110 130 i

2::

-1 210 190 170 150 130 F 110 22 90 70

4

8

12 19 23 26 31 34 38 42 46 Drug Week

210 190 170 150 $- 130 ; 110 2 90 70 50 30 10

E * 2 3, 3Q

1 -

-1

84-0527

4

8

12 19 23 26 31 34 38 42 46 Drug Week

-1

4

8

12 19 23 26 31 34 38 42 46

-1

4

8

12 19 23 26 31 34 38 42 46

210 190 170 150 130 110 90 70 50 30 10

Drug Week

Figure 10. Elevations in serum alanrne aminotransferase (ALT; serum glutamic-pyruvic transaminase) activity in dogs treated long-term with simvastatin (50 mg/kg per day). Panels represent the individual serum ALT activities of four dogs receiving simvastatin (50 mg/kg per day]. Points represent individual determinations that were within (open circles) or outside (solid circles) the 95 percent age-adjusted historical control values for this laboratory.

glutaryl-coenzyme A recluctase inhibitors stucliecl in this laboratory. These changes include acanthosis (hyperplasia of the squamous epithelium) and hyperkeratosis, often accompanied by submucosal edema and cellular inflammatory infiltrate. The development of acanthosis and hyperkeratosis has been found to occur only in the rodent forestomach. No treatment-related changed have been observed in the glandular portion of the rodent stomach, nor has any evidence of this change been observecl in any other squamous epithelium of the roclent gastroin- _ testinal tract (i.e., esophagus or anorectal junction). Furthermore, similar changes have not been observed in the gastrointestinal squamous epithelium of any other species tested (dogs or monkeys). In particular, dogs received doses of simvastatin of up to 50 mg/kg per clay for as long as two years without any eviclence of gastrointestinal squamous epithelial changes. Similarly, monkeys receivecl closes of simvastatin of up to 25 mg/kg per clay for 12 weeks without the production of any changes in the squamous gastrointestinal epithelium. The doses used in these clog ancl monkey bizdular Stomach(forestomach)Changesin studies were 31 to 63 times the maximal anticipate<1 clinical dose of 0.8 mg/kg per day. Comparable closes A spectrum of changes confined to the nonglandular of simvastatin, when aclministerecl to roclents, progastric epithelium (forestomach) of rats and mice (a duced a high incidence of squamous epithelial hyperstructure specific to rodents, not found in humans) plasia in the forestomach. In aclclition, no effects of have been described in association with the adminissimvastatin were observed in the glandular stomach tration of simvastatin and all other hydroxy-methylor any of the other gastrointestinal epithelia in any of

that these elevations occur with hydroxy-methylglutaryl-coenzyme A recluctase inhibitors, regardless of molecular structure, implicates the biochemical mechanism of action of this class of compound in the development of this change. In preliminary experiments conducted with lovastatin, mevalonic acid supplementation appearecl to protect against the development of transaminase elevations in clogs [13], thereby suggesting that interference with mevalonate synthesis might be responsible for these elevations. In terms of implications for clinical safety, it is important to emphasize that no microscopic evidence of hepatic damage has ever been observecl in clogs receiving daily simvastatin closes GO to 100 times the maximal clinical dose for three months to two years. Although these findings suggest that hepatocellular damage is unlikely in the clinical setting, it is recommended that patients receiving simvastatin be closely monitored and that the clrug be discontinued if transaminase elevations three times over baseline levels are detected.

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SYMPOSIUM

these species. Therefore, it appears that this lesion occurs only in rodents and is limited to an anatomical structure not found in dogs, monkeys, or, importantly, humans. The production of these forestomach lesions in rodents appears to be directly linked to the biochemical mechanism of action of simvastatin and hydroxymethylglutaryl-coenzyme A reductase inhibitors in general. The evidence for this is described next. Forestomach acanthosis and hyperkeratosis are class effects, produced by every inhibitor of hydroxymethylglutaryl-coenzyme A reductase tested in this laboratory. Some of these inhibitors (e.g., L-645-164, a fluorinated biphenyl) are structurally different from simvastatin, thereby implicating the biochemical mechanism of action of this class of compouncls (rather than an intrinsic structural property) in the genesis of rodent forestomach hyperplasia. In addition, the aclministration of a number of biochemically inactive enantiomers of potent hydroxy-methylglutaryl-coenzyme A recluctase inhibitors (including the inactive enantiomer of the beta-hydroxy acid form of simvastatin) failed to produce any gross or histologic evidence of forestomach thickening (Table VII). These data indicated that hydroxy-methylglutaryl-coenzyme A reductase inhibitory activity was requirecl to produce these forestomach lesions in roclents. The development of forestomach thickening in rodents also requires a direct exposure of the nonglandular gastric epithelium to high concentrations of inhibitor. The parenteral administration of L-654,969 (the beta-hydroxy acid form of simvastatin) produced no changes in the forestomach epithelium of rodents at doses that, when administered by gavage, produced a significant incidence of thickening (Table VII). Subsequent studies may have identified, in part, the basis for the sensitivity of the rodent nonglandular stomach epithelium to this class of compounds. Drug levels attained in this epithelium are approximately five- to lo-fold greater than those achieved in the esophageal squamous epithelium or in glanclular epithelium of the stomach after the oral administration of simvastatin to rodents, and are also higher than those attained in any other tissue (data not shown). Although the forestomach contains a higher burden of simvastatin than does any other tissue in rodents, the physiologic basis for the observed hyperplasia is unknown. In summary, the development of nonglandular stomach changes is limitecl to a unique anatomic structure in rodents that is not found in dogs, monkeys, or humans. This change appears to be directly linked to the biochemical mechanism of action of this class of compounds and requires the direct contact of the forestomach squamous epithelium with high concentrations of inhibitor for prolonged periods of time. This level of exposure subsequently results in very high, localized concentrations of inhibitor in this tissue. Based on these findings, rodent forestomach hyperplasia is not considered to be inclicative of human risk. TESTICULAR DEGENERATION IN DOGS Varying degrees of testicular degeneration have been sporadically observed in dogs receiving simvastatin. Of the target organs identified, the changes in the testes are the least well understood. Testicular

VII Incidence and Rats Receiving Administration Enantiomer of

ON SIMVASTATIN I GERSON ET AL

’ TABLE

Severity of Forestomach Acanthosis in Female L-654,969* by Different Routes of or after Receiving the Biochemically Inactive L-654,969t Number of Rats

Control (oral) C$$~;bcutaneous)

20 IO

20 &kg/day (oral) 50 mg/kg/day (oral) 50 mg/kg/day (subcutaneous) L.654,969 inactive enanhomer 20 mg/kg/day [oral)

IO 15 25

*L654.969

vastatin.

Percent with Acanthosis 0 0

Range of Severity Normal Normal Very skght to slight ztysi,.t to moderate

ii 0

10

0

Nonal

I is the internal deslgnalion number for the beta-hydroxy acid form of sim-

tAll lest compounds were administered for 15 days.

degeneration is a poorly reproducible lesion that occurs at a very low incidence. No relationship to the administered closage level of simvastatin has been demonstrated. The type of testicular lesion observecl is not an uncommon reaction of the testes to xenobiotics or physiologic stress. The degeneration is generally characterizecl by an increased incidence of spermatidic giant cells and a loss of the normal spermatocyte maturation sequence. In a few rare cases, a loss of the germinal epithelium in scattered seminiferous tubules has been seen, these structures being lined only by Sertoli’s cells. No damage to Sertoli’s cells or Leydig’s cells has been observed. This lesion has also been observed in clogs as a consequence of lovastatin administration [13] and occurs with a similar low sporadic inciclence. Although it is clearly plausible that interruption of mevalonate and/or cholesterol biosynthesis in the spermatogenic epithelium could produce the alterations observed in the testes, the lack of a closeresponse relationship and the poor reproducibility of this lesion have made this change difficult to study. Table VIII summarizes the histologic findings in the testes of dogs from all studies conducted with simvastatin. Treatment-related testicular degeneration has been convincingly demonstratecl only during six-, 14-, and 2%week studies at closes of 10, 30, and 90 mg/kg per clay. The most pronounced change, a moclerate degeneration, was observed in one dog during the 28-week stucly at a dose of 30 mg/kg per clay and in one dog during the 16week study at a close of 90 mg/kg per day. The poor reproducibility of this change is apparent when one considers that simvastation was aclministerecl to dogs at doses of up to 50 mg/kg per day for up to two years without effect. Furthermore, of three short-term stuclies (one of two weeks’ duration ancl two of 14 weeks’ duration), which were specifically conducted in an attempt to reproduce this change, only one study produced an incidence or degree of testicular degeneration that was judged to be different from that observed in concurrent controls (Table VIII). Added to these findings are the facts that: (1) a no-effect level of at least 3 mg/kg per clay has been observed in dogs in all simvastatin studies (including studies in which degeneration has been produced); (2) no evidence of similar change has been observed in

October 16, 1989

The American Journal of Medicine

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SYMPOSIUM

ON SlMVASThlN

I GERSON ET AL

TABLE VIII Grades of Testicular Degeneration Present in Groups of Dogs Administered Various Doses of Simvastatin DoseGroup(mglkglday) Weeksin Study

Control VQ'SNNNNNNNN 1I I , I I I I N,N,N,N VSNNNNNNNNNNNNNNN I I I I, I I I I I I1 !, 1 NNNN N,N.N,N NNN N,N,N

i ii* 53 1: = moderate:‘N

2to3

10

30

N,N,N.N

VS,S,N,N

N,N,N,NN1NININ1NIN VS,S,N,N

NNNN N,N,N N/W

N,N,N,N N,N,N,N N,N,N NNN

30

90 S,N,N,N,N,N,N,N,N,N VS,S,N,N,N,N,N,N,N,N,N,N,VS,VS,S,M VS,S,N,N

VS,M,N,N NAN

N,NN,N N/W N,N,N

= normal: S = skaht: VS = verv skaht. studres. - -

*I qepresents results of two rndependent

rats, mice, or monkeys at comparable closes; (3) no effect on the fertility of male rats treated with high dosages of simvastatin has been observecl; (4) no effects on testosterone or luteinizing hormone levels have been -observed in clogs receiving up to 90 mg/kg per day of simvastatin; and (5) no effects on sperm counts, morphology, or motility have been observed (luring clinical trials concluctecl with simvastatin or the related hydroxy-methylglutaryl-coenzyme A recluctase inhibitor lovastatin. All these points suggest that this finding is not indicative of a potential problem in humans. CONCLUSIONS The aclministration of high closages of simvastatin, a potent inhibitor of hyclroxy-methylglutaryl-coenzyme A reductase, has resulted in the production of toxicity in a variety of tissues and in a number of species. It is interesting to note, however, that a broader spectrum of toxicity was not producecl in these species, in view of the potency of this drug against hyclroxy-methylglutaryl-coenzyme A reductase, the central role played by this enzyme in cellular biochemistry, and the magnitucle of the closes employecl in these studies. In particular, the completed preclinical toxicity stuclies have demonstrated that simvastatin is clevoicl of the central nervous system toxicity associated with other members of this pharmacologic class. The investigative toxicology studies conductecl to characterize the basis and/or mechanisms of simvastatin-induced changes have indicated that many are mechanism-based phenomena occurring only at high dosage levels of simvastatin ancl are the product of a profound, prolonged inhibition of the target enzyme that is not achieved at clinical dosages. In all cases, a significant margin of safety exists, clue to either dramatic differences in systemic exposure to inhibitor or the lack of an analogous physiologic mechanism in humans. The results of preclinical toxicology studies concluctecl on simvastatin support the use of this agent in humans for the treatment of hypercholesterolemia. ACKNOWLEDGMENT We gratefully acknowledge Mrs. M.E. Hunsberger for her assistance in the preparation of this manuscript.

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JL, Jaramrllo JJ. Brown MS: Regulatory role for in vrvo rn the dog. Proc Nab Acad Sci USA 1981;

778: 1194-1198. - _ _ DW, _ , Grundy _ _ _, SM, _ , Brown _ _ MS,_, Goldstein _ 2. Brlheimer JL: Mevrnolin and colestipol shmulate receptormediated clearance of low-densrty Irpoprotern from plasma rn familial hypercho. lesterolemra heteroz-ygotes. Proc Nab Acar Acad Scr USA 1983; 80: 4124-4128. 3. Germershausen JI. Hunt VM. Eostedor RG. I Barlev PJ. Karkas JD. Alberts AW Tissue selectivity of the cholesterol lowermg agents lovastabn. srmvastatrn and pravastatrn rn rats m vrvo. Brochem Biophys Res Commun 1989; 158: 667-675. 4. Duggan DE, Chen IW, Halprn RA, et al: The physrologrcal disposrhon of lovastatm. Drug Metab Drspos 1989; 17: 166-173. 5. Brown MS, Goldstern JL: Multivalent feedback regulatton of HMGCoA reductase, a control mechamsm coordrnahnn _rsoorenord svnthesrs and cell growth. J Lrprd Res 1980; 21: 505-517. 6. Maltese WA, Sheridan KM lsoprenylated proterns rn cultured cells: subcellular distrrbu tron and changes related to altered morphology and growth arrest Induced by mevalonate deprivation, J Cell Physrol 1987: 133: 471-481. 7. Krrby TJ: Cataracts produced by trrparanol (MER/29). Trans An I Dphthal Sot 1967: 65:

493443. 8. Roe DA: The effects of hvoocholesterolemrc agents on the skrn. Geriatrrcs 1966; Octo. ber: 174-182. 9. Peter JB. Andrman RM. Bowman RL. Nagatomo T: Myotoma induced by diazcholesterol: Increased (Na’ + K’).ATPase acbvrty of erythrocyte ghosts and devel lopment of cata. racts. Exp Neural 1973; 41: 738-744. 10. Sakuwawa N. Sakuaragawa M. Kuwabara T, Pentchev PG. Earrager JA, Brady RO: Niemann.Prck disease experimental model: sphrngomyelinase reduchon Induced by AY. 9944. Science 19R 196: 317-319. 11. Cenedella RI, Bierkamper GG: Mechamsm of cataract productron of U1866A an rnhibrtor of cholesterol biosynthesrs. Exp Eye Res 1979; 28: 673-688. 12. Gerson RI, MacDonald JS. Alberts AW, et al: On the ebology of subcapsular lentrcular opacrtres produced in dogs recervrng HMGCoA reductase inhibitors. Exp Eye Res 1989 (in press). 13. MacDonald JS. Gerson RI. Kombmst DJ. ef al: Preclinical evaluahon of the potentral toxrcdy of lovastahn. Am J Cardrol 1988; 62: 16J-27J. 14. Germershausen JI, Hunt VM, Bostedor RG. Bailey PJ, Karkas JD. Alberts AW Tissue selectivity of the cholesterol lowerrng agents lovastatin. simvastatin and pravastatm in rats in wvo. FASEB J 1988: 6: A1752. 15. Vrckers S, Duncan CA Chen IW, Duggan DE: Absorption, distribution, metabolism and excretion studres on srmvastatin. a cholesteroltowerrng prodrug (abstr). FASEB J 1988; 2: A1060. 16. Singer II, Kawka DW, Kazazis DM, ef a/: Hydroxy-methylglutarylcoeruyme A reductase-contaming hepatocytes are distributed periportally in normal and mevinolin treated rat Ikvers. Proc Natl Acad Scr USA 1984; 81: 5556-5560. 17. Singer IL Scott S, Kazazrs DM. Huff JW: Lovastatrn, an rnhrbitor of cholesterol synthe. srs. Induces hydroxymethylglutarylcoemyme A reductase directly on membranes of ex. panded smooth endoplasmic reticulum rn rat hepatocytes. Proc Nab Acad Sci USA 1988; 85: 5264-5268. 18. Kornbrust DJ, Peter CP, MacDonald JS: Mechanism.based toxicity of HMG-CoA reductase inhibrtors in rabbrts (abstr). Toxrcologist 1988; 8(l): 261. 1 19. Stoudemire JD. Renaud G. Shames DM, Have1 RI: lmparfed receptormediated catabolrsm of low denstty lipoproteins in fasted rabbits. J Lipid Res 1984: 25: 33-39. 20. White LW, Rudney H: Regulation of 3.hydrox.y3-methylglutarate and mevalonate bio. synthesis by rat liver homogenates. Effects of fasting cholesterol feeding and Triton ad. mrnrstratron. Brochemistry 1970; 9: 2725-2731. 21. Kombrust DJ, MacDonald JS. Peter CP, et at Toxicity of the HMG-CoA reductase inhrbrtor. lovastatm, to rabbrts. J Pharmacol Exp Ther 1989; 248: 498-505. 22. Walker JF: Simvastatin: the clinical profrle. Am J Med 1989; 87 (suppl 4A): 4A44S4k.465.

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