1521-0103/347/3/635–644$25.00 http://dx.doi.org/10.1124/jpet.113.208595THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 347:635–644, December 2013Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics

Combined Analysis of Pharmacokinetic and Efficacy Dataof Preclinical Studies with Statins Markedly ImprovesTranslation of Drug Efficacy to Human Trials s

E. van de Steeg, R. Kleemann, H. T. Jansen, W. van Duyvenvoorde, E. H. Offerman,H. M. Wortelboer, and J. DeGrootTNO (The Netherlands Organization for Applied Scientific Research), Delft, The Netherlands

Received August 16, 2013; accepted September 17, 2013

ABSTRACTCorrect prediction of human pharmacokinetics (PK) and thesafety and efficacy of novel compounds based on preclinicaldata, is essential but often fails. In the current study, we aimedto improve the predictive value of ApoE*3Leiden (E3L) trans-genic mice regarding the cholesterol-lowering efficacy ofvarious statins in humans by combining pharmacokinetic withefficacy data. The efficacy of five currently marketed statins(atorvastatin, simvastatin, lovastatin, pravastatin, and rosu-vastatin) in hypercholesterolemic patients (low-density lipo-protein $ 160 mg/dl) was ranked based on meta-analysis ofpublished human trials. Additionally, a preclinical combinedPK efficacy data set for these five statins was established inE3L mice that were fed a high-cholesterol diet for 4 weeks,followed by 6 weeks of drug intervention in which statins weresupplemented to the diet. Plasma and tissue levels of the

statins were determined on administration of (radiolabeled)drugs (10 mg/kg p.o.). As expected, all statins reduced plasmacholesterol in the preclinical model, but a direct correlationbetween cholesterol lowering efficacy of the different statinsin mice and in humans did not reach statistical significance(R2 5 0.11, P , 0.57). It is noteworthy that, when murine datawere corrected for effective liver uptake of the differentstatins, the correlation markedly increased (R2 5 0.89, P ,0.05). Here we show for the first time that hepatic uptake ofstatins is related to their cholesterol-lowering efficacy andprovide evidence that combined PK and efficacy studies cansubstantially improve the translational value of the E3L mousemodel in the case of statin treatment. This strategy may alsobe applicable for other classes of drugs and other preclinicalmodels.

IntroductionThe pharmaceutical industry is facing a huge challenge

in marketing new medicines, mainly because of decreasedresearch and development productivity and the decreasingnumber of truly innovative new medicines approved by theUS Food and Drug Administration (FDA) (Booth and Zemmel,2004). For example, the number of new molecular entities(NMEs) that were approved by the FDA during 2000–2005was 50% lower compared with the preceding 5 years. Althoughactual costs may vary between different classes of medicines,the average investment needed to bring an NME to themarket is estimated to be approximately $1.8 billion and isrising rapidly (Paul et al., 2010). Interestingly, the number ofdrug candidates successfully reaching phase 1 has increasedin recent years, partly a result of better preclinical charac-terization and improved absorption, distribution, metabolism

excretion, and toxicity properties. Nevertheless, attrition inlate-stage drug development (phases 2 and 3) is still high andtherefore remains the most important determinant of theoverall research and development efficiency. For example,the phase 2 success rates for NMEs have fallen from 28%(2006–2007) to 18% (2008–2009), and the prospect ofsuccessfully progressing through phase 2 is currently 50%.Importantly, attrition in phase 2 is caused mainly by insuf-ficient efficacy of the newly developed drug, accounting for51% of the drug failures (Kola and Landis, 2004; Arrowsmith,2011, 2012). The importance of correctly predicting the efficacyof novel drug candidates and demonstrating the disease-modifying potency of those candidates, especially in the earlystage preclinical phases, are therefore crucial. Although thetranslation from mouse to human proves to be difficult as aresult of interspecies differences in efficacy or pharmacokinet-ics, humanized mouse models are being developed to improvesuch translation.The apolipoprotein E (ApoE)*3Leiden (E3L) transgenic

mousemodel is awell-established humanizedmodel for (familial)hyperlipidemia and cholesterol-induced atherosclerosis (van

This work was supported by a grant of the Centre for Medical SystemsBiology.

dx.doi.org/10.1124/jpet.113.208595.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: ApoE, apolipoprotein E; AUC, area under the curve; CI, confidence interval; E3L, ApoE*3Leiden; FDA, US Food and DrugAdministration; HFC diet, high -fat cholesterol diet; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; HPLC, high-performance liquid chromatography;LDL, low-density lipoprotein; LDL-C, LDL-cholesterol; MS, mass spectrometry; NME, new molecular entity; PK, pharmacokinetics; TC, total plasmacholesterol; TG, total plasma glyceride; TNO, The Netherlands Organisation for Applied Scientific Research.

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Vlijmen et al., 1994; Zadelaar et al., 2007). E3L mice developa human-like lipoprotein profile upon feeding a fat- and/orcholesterol-containing diet, with elevated plasma cholesteroland triglyceride levels mainly present in very-low-densitylipoproteins and low-density lipoproteins (LDLs). After long-term treatment (∼14 weeks) with a fat- and/or cholesterol-containing diet, E3L mice develop atherosclerotic lesions withall the characteristics of human vascular pathology (Zadelaaret al., 2007). The E3L mouse model is therefore a widely usedmodel for studying the differential effects of cholesterol-loweringdrugs (e.g., statins, fibrates, niacin) (van Vlijmen et al., 1998;Kleemann et al., 2003; Zadelaar et al., 2007; Verschuren et al.,2005, 2012).Statins are lipid-lowering drugs that are widely prescribed

to reduce the risk of primary and secondary coronary heartdisease. Statins reduce plasma cholesterol levels by inhibitingthe enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and by enhancing hepatic uptake of LDLcholesterol via upregulation of LDL receptor expression (Liaoand Laufs, 2005). It is estimated that about 50 million peopletake statins everyday worldwide. The currently marketedstatins used in this study belong to the second-generation(lovastatin, simvastatin, and pravastatin) and third-generation(atorvastatin and rosuvastatin) statins, with the latter havinga higher affinity to inhibit HMG-CoA reductase and inhibitthe enzyme for a longer duration (Kleemann and Kooistra,2005). This is also obvious from clinical studies that demon-strate that only atorvastatin and rosuvastatin can reduceplasma LDL-cholesterol (LDL-C) by more than 40% in patientswith hypercholesterolemia (Weng et al., 2010). The therapeuticequivalence of statins to reduce LDL-C in humans was recentlyreviewed in this meta-analysis by Weng et al. (2010), providinga ranking order from most to least potent of rosuvastatin .atorvastatin . simvastatin . lovastatin . pravastatin.Importantly, the intensity of a drug’s effect, besides its affinityfor the drug target (e.g., receptor or enzyme), is determinedmainly by the drug concentration at the site of action. SinceHMG-CoA reductase is expressed mainly in the liver (hep-atocytes), the efficacy of statins to reduce plasma cholesterollevels is largely dependent on the pharmacokinetics and localconcentration of these drugs in the liver. For example, it hasbeen demonstrated that patients carrying a variant of thehepatic uptake transporter, OATP1B1*15 (Asn130Asp andVal174Ala), which is known to have a strongly reducedtransport activity, show markedly increased plasma levels,decreased pharmacological response, and even increasedextrahepatic toxicity after rosuvastatin or pravastatin treat-ment (Niemi et al., 2011).The aim of this study was to test the hypothesis that the

intelligent combination of pharmacokinetic and efficacy datamay increase the predictability of drug efficacy outcomes inpreclinical models. To this end, we set up a preclinical studywith E3L transgenic mice in which we determined both thePK as well as the efficacy profile of the five currentlymarketed statins and compared them with their efficacy inhumans based on data from a literature meta-analysis. Here,we report for the first time a systematic link between thehepatic uptake of statins and their cholesterol-loweringefficacy in a valuable mouse model of hyperlipidemia andprovide evidence that PK studies combined with efficacystudies could substantially improve the translational value ofthe E3L mouse model in case of statin treatment.

Materials and MethodsSystematic Review of the Cholesterol-Lowering Abilities of

Statins in Humans. To review systematically the abilities of fivecommonly used statins (atorvastatin, pravastatin, simvastatin,lovastatin, and rosuvastatin) known to reduce plasma cholesterollevels in patients with hyperlipidemia, we selected all clinical trialspublished between 1966 and 2009 that were previously also reviewedby Weng et al. (2010) and the Oregon Health Resources Commission(Beth Smith et al., 2009). These meta-analyses used the followingselection criteria to include the studies: 1) only randomized controlledtrials with 2) head-to-head comparisons of at least two statins wereincluded. Trials needed to be based on 3) human subjects older than18 years 4) who used statins as monotherapy to treat hyperlipidemia.The studies had to 5) report the primary data, 6) needed to last for atleast 4 weeks, 7) and had to be published in English. As additionalselection criteria, we included only trials in which patients withbaseline plasma LDL-C levels .160 mg/dl (i.e., cutoff for hypercho-lesterolemia) (National Cholesterol Education Program, 2002) wereselected, resulting in 77 selected studies (see Supplemental MaterialNote 1).

Animals. E3L mice, bred by The Netherlands Organisation forApplied Scientific Research (TNO), were housed and handledaccording to institutional guidelines complying with Dutch andEuropean legislation. In this study, we used female heterozygousE3L transgenic mice (8–12 weeks of age), characterized by enzyme-linked immunosorbent assay for the presence of human apoE*3 inthe serum (van Vlijmen et al., 1998). All animal experiments wereapproved by the Institutional Animal Care and Use Committee ofTNO.

Chemicals and Reagents. The following drugs were supple-mented to the diet: atorvastatin (Lipitor; atorvastatin-calcium; Pfizer,New York, NY), pravastatin (Selektine; pravastatin-sodium; Bristol-Myers Squibb, NewYork, NY), lovastatin (L472225; Toronto ResearchChemicals, Toronto, ON, Canada), simvastatin [Zocor; simvastatin(lacton); Merck Sharp & Dohme, Whitehouse Station, NJ], androsuvastatin (Crestor; rosuvastatin-calcium; AstraZeneca, London,UK). For the pharmacokinetic experiments, we used atorvastatincalcium and rosuvastatin calcium from Sequoia Research Chemicals(Pangbourne, UK) and simvastatin, lovastatin, and pravastatinsodium from Toronto Research Chemicals). [3H]Atorvastatin (740GBq/mmol) was purchased from American Radiolabeled Chemicals(St. Louis, MO). [3H]Rosuvastatin (40.7 GBq/mmol) and [3H]pravasta-tin (136.9GBq/mmol) were custom synthesized byMoravekBiochemicals(Brea, CA).

Study Design. During a 4-week run-in period, all animals (exceptfor the chow-control group, n 5 12) received a semisynthetic high-fatcholesterol (HFC) diet containing 40.5% sucrose, 15% cacao butter,and 1% cholesterol (all w/w) (4021.04; Abdiets, Woerden, TheNetherlands). After randomization into 11 groups (n 5 8 per group)matched for age, body weight, plasma cholesterol, and triglyceridelevels, the mice received the HFC diet alone (control group) or HFCsupplemented with either atorvastatin (0.003 or 0.008% w/w),pravastatin (0.03 or 0.05% w/w), lovastatin (0.05 or 0.07% w/w),simvastatin (0.03 or 0.06%w/w), or rosuvastatin (0.0025 or 0.005%w/w)for 6 weeks (Table 1). Blood samples were taken after a period of 3 and6 weeks of treatment. Animals were kept in a temperature-controlledenvironment with 12-hour light/12-hour dark cycle and received foodand water ad libitum. Body weight and food intake were monitoredduring the study.

Analysis of Plasma Lipids. Blood was sampled after 4 hours offasting into EDTA-containing cups by tail bleeding, and plasma wasisolated. Immediately after isolation, total plasma cholesterol (TC),and total plasma triglyceride (TG) levels were measured withcommercially available enzymatic kits according to the manufac-turer’s protocols (Roche Diagnostics, No-1489437 for total cholesterol;Roche Diagnostics, No-1488872 for triglyceride; Roche Diagnostics,Indianapolis, IN).

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Plasma and Tissue Pharmacokinetic Experiments. Thegroups of mice that were treated with the highest dose of statin wereused for analysis of pharmacokinetics. The evening before the PKstudy, diets of all mice were switched to the HFC control diet to allowwashout of the statin (half-time of orally dosed statins #5 hours inmice) (Lau et al., 2006; Peng et al., 2009; Tiwari and Pathak, 2011; Zhuet al., 2011; Iusuf et al., 2012). Subsequently, after 2 hours of fasting,mice were dosed with a 10 mg/kg concentration of the different statins[10 ml/g body weight of drug solution; 1 mg/ml in saline finallycontaining 8.3% EtOH:polysorbate 80 (1:1, v/v)] by oral gavage. In caseof [3H]atorvastatin, [3H]pravastatin, and [3H]rosuvastatin, we useda 4 mCi/ml concentration of the final drug solution. Each group of eightmice was split up into two subgroups. Mice of the first subgroup (n5 4)were sacrificed after 30 minutes by CO2, blood was collected by cardiacpuncture, and tissues were isolated. In the other subgroup (n 5 4),blood was sampled by tail bleeding after 60 and 90 minutes, and micewere sacrificed after 120 minutes by CO2, followed by cardiac punctureand isolation of tissues. All blood samples were centrifuged at 2700gfor 5 minutes at 4°C; plasma was collected and stored at 220°C untilanalysis.

Drug Analysis. Levels of [3H]atorvastatin, [3H]pravastatin, and[3H]rosuvastatin in plasma and organs were determined by scintil-lation counting (Tri-Carb 3100 TR; PerkinElmer Life and AnalyticalSciences, Shelton, CT). Amounts of lovastatin in plasma and liverwere determined by high-performance liquid chromatography (HPLC)analysis. Simvastatin in plasma and liver were determined by HPLCand LC-MS analysis, respectively (see below).

HPLC Analysis of Lovastatin and Simvastatin. Levels oflovastatin acid and simvastatin acid in plasma and liver weremeasured by HPLC analysis based on the method previouslydescribed by Zhang and Yang (2007). In brief, 50 ml of methanol and200 ml of acetonitrile were added to 100 ml of plasma or liverhomogenate (homogenized in ice-cold saline using a ULTRA-TURRAX;Sigma-Aldrich, St. Louis, MO). After thoroughly vortexing, thesamples were centrifuged for 10 minutes (14,000 rpm at roomtemperature), and 20 ml of the supernatant was injected into thecolumn (Hypersil BDS column; 250 � 4.6 mm, 5 mm) for analysis.Samples were eluted at flow rate of 1.0 ml/min at 20°C with 0.1%trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acidin acetonitrile (solvent B). The following mobile-phase conditionswere used: 0–10 minute linear from 100 to 60% A and 0 to 40% B;10–30minutes linear from 60 to 30%A and 40 to 70%B; 30–40minutelinear from 30 to 0% A and 70 to 100% B; 35–40 minutes 100% B(isocratic). Ultraviolet chromatograms were obtained at 238 nm. Peakidentification and quantification were performed using LablogicLaura software (South Yorkshire, UK).

Ultraperformance Liquid Chromatography-Mass Spectrom-etry Analysis of Simvastatin. Fractions of the livers (∼200 mg)were sonicated twice in acetonitrile. Lysates were centrifuged 10minutes at 14,000 rpm, and 5 ml of the supernatant was injected intothe ultraperformance liquid chromatography-mass spectrometry(MS) for analysis (BEH Acquity column; C18, 50 � 2.1 mm). Sampleswere eluted at flow rate of 0.5 ml/min at room temperature with 0.1%formic acid in water (solvent A) and 0.1% formic acid in acetonitrile(solvent B). The following mobile-phase conditions were used: 0–1minute 98% A and 2% B (isocratic); 1–4 minutes linear 98 to 20% Aand 2 to 80% B; 4–5 minutes 20% A and 80% B (isocratic); 5–5.1minutes linear 20 to 98% A and 80 to 2% B; 5.1–6 minutes 98% A and2% B (isocratic). The mass spectrometer was operating in selectivereaction mode using turbo spray ionization in positive ion mode,with a capillary voltage of 5.5 kV and a spray temperature of 450°C.The multiple reaction monitoring transitions were determined fromMS spectra and appeared to be m/z 437.5 for the precursor ion ofsimvastatin acid andm/z 285.3 for the product ion. Peak identificationand quantificationwere performed usingMasslynx software version 4.1(Waters Corporation, Milford, MA).

Data and Statistical Analysis. ED30 values of statin efficacy inhuman and mouse were determined by linear regression with a 95%confidence interval (CI) (Liengme, 2010). Unless otherwise specified,the two-sided unpaired Student’s t test was used throughout the studyto assess the statistical significance of differences between two setsof data. Differences were considered statistically significant whenP , 0.05.

ResultsComparison of Cholesterol-Lowering Effects of Sta-

tins in Humans. The cholesterol-lowering effects of thedifferent statins in patients with hypercholesterolemia areshown in Fig. 1 and Supplemental Material Note 1. There wasa clear linear dose-effect relation, with the exceptions ofatorvastatin and rosuvastatin at the highest dose of 80 mg/day, which might be due to saturation of HMG-CoA reductaseinhibition (Supplemental Material Note 2). We also observeda clear and significant correlation between the reduction inplasma levels of TC and LDL-C after statin treatment (R2 50.83, P , 0.001; data not shown). Based on linear regression,we calculated the ED30 [i.e., effective dose (milligrams perday) to reduce plasma TC by 30%] for the individual statinsand found that these were, in order from strong to weak:rosuvastatin [1.79 mg/day (CI: 0.15–3.66)], atorvastatin

TABLE 1Food intake and baseline characteristics of E3L mice after randomization into different groupsTime (t) = 0 after 4-week run-in period.

Group n BW at t = 0 Food Intakea Statin Intake Statin Intakeb Plasma TC at t = 0 Plasma TG at t = 0

g g/day mg/day mg/kg mM

Chow control 12 20.8 6 0.61 3.72 6 0.14 — — 2.83 6 0.21 2.83 6 0.48HFC-control 8 20.3 6 1.11 2.70 6 0.20 — — 15.1 6 3.49 2.41 6 0.77HFC-0.003% Atorvastatin 8 20.9 6 0.82 2.61 6 0.21 0.08 3.76 15.1 6 1.89 2.49 6 0.90HFC-0.008% atorvastatin 8 21.8 6 1.39 2.65 6 0.13 0.21 10.1 15.1 6 3.52 2.52 6 0.41HFC-0.03% pravastatin 8 20.9 6 1.57 2.58 6 0.18 0.77 37.2 14.9 6 2.35 2.28 6 0.58HFC-0.04% pravastatin 8 21.0 6 1.76 2.55 6 0.18 1.02 48.7 15.2 6 2.81 2.45 6 0.58HFC-0.05% lovastatin 8 20.6 6 1.21 2.51 6 0.11* 1.26 60.9 15.0 6 2.21 2.37 6 0.40HFC-0.07% lovastatin 8 20.9 6 1.11 2.53 6 0.28 1.77 84.7 14.8 6 3.03 2.37 6 0.50HFC-0.03% simvastatin 8 20.7 6 1.03 2.60 6 0.13 0.78 37.7 15.1 6 2.01 2.41 6 0.38HFC-0.06% simvastatin 8 21.5 6 0.85 2.60 6 0.19 1.56 72.7 14.9 6 2.71 2.72 6 0.34HFC-0.0025% rosuvastatin 8 21.5 6 0.77 2.59 6 0.09 0.06 3.01 15.0 6 2.50 2.57 6 0.49HFC-0.005% rosuvastatin 8 21.0 6 0.89 2.55 6 0.25 0.13 6.08 15.2 6 2.84 2.71 6 0.63

—, not done; BW, body weight; HFC, high-fat cholesterol diet.aAverage over the 6-week treatment period.bBased on body weight at t = 0.*P , 0.01 compared with HFC-control group; data are presented as mean or mean 6 S.D.

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[16.3 mg/day (CI: 14.0–18.9)], simvastatin [48.5 mg/day (CI:40.9–58.1)], lovastatin [75.7 mg/day (CI: 69.9–81.6)], andpravastatin [80.8mg/day (CI: 77.3–84.2)] (SupplementalMaterialNote 2).Comparison of Lipid-Lowering Effects of Statins in

E3L Mice. E3L mice treated with a HFC diet containing 1%cholesterol for 4 weeks showed on average a 5.3-fold increasein plasma total cholesterol levels compared with the chow-control reference mice. After this 4-week run-in period toachieve steady-state conditions, mice were randomized into11 groups (n5 8 per group) based on age, body weight, plasmacholesterol levels, and triglyceride levels, and the micereceived the HFC diet (control group) or supplemented withdifferent doses of statin during an intervention period of 6weeks (Table 1). Body weights and food intake of the E3Lmiceduring the intervention period are presented in Table 1 andFig. 2. The average body weights of the mice did not differbetween the different groups. The food intake was comparablebetween all treatment groups (∼2.6 g/day on average), exceptthe group receiving 1.26 mg of lovastatin per day, of whichthe food intake was 2.5 g/day (P , 0.05; one-way analysisof variance, followed by Dunnett’s multiple comparison test;Table 1). The average food intake in the chow-control groupwas 3.7 g/day. The effects of the different statins on plasmaTC levels after 6 weeks of treatment in E3Lmice are presentedin Table 2 (individual data in Supplemental Material Note 3).With the exception of simvastatin, all statins decreased plasmaTC levels by at least 30% at the highest tested dose. Based onthe food intake, we calculated the statin intake (in milligramsper day) per group and plotted these against the percentage

reduction in TC compared with the HFC-control group (Fig. 3).Linear regression was used to calculate the ED30 for plasmaTCreduction in E3L mice for the individual statins (extrapolationof the data was used to estimate the ED30 for simvastatin).ED30 values (in milligrams per day) were in order from strongto weak: rosuvastatin [0.11 mg/day (CI: 0.10–0.12)], atorvasta-tin [0.14 mg/day (CI: 0.12–0.16)], pravastatin [0.97 mg/day(CI: 0.90–1.03)], lovastatin [1.53 mg/day (CI: 1.51–1.54)], andsimvastatin [3.34 mg/day (CI: 2.84–4.05)] (Fig. 3). The effectsof the different statins on plasma TG levels after 6 weeks oftreatment in E3L mice are presented in Table 2 (individualdata are presented in Supplemental Material Note 4). Rosuvas-tatin, pravastatin, and lovastatin decreased plasma TG levels inE3L mice (62, 63, and 59% upon treatment of E3L mice with0.13 mg of rosuvastatin per day, 1.02 mg of pravastatin per day,and 1.77 mg of lovastatin per day, respectively), as has beenobserved before (Kleemann et al., 2003; Delsing et al., 2005; vander Hoorn et al., 2007). Atorvastatin and simvastatin, however,did not statistically change plasma TG levels in E3Lmice, whichis also in agreement with previous observations (van De Pollet al., 2001; Wang et al., 2002; Delsing et al., 2003).Pharmacokinetic Analysis of Different Statins in

E3L Mice. To study putative differences in pharmacokinet-ics and tissue distribution between the various statins used inthis study, E3L mice were orally dosed with a bolus injectionof 10 mg/kg of the (radiolabeled) drug. Concentrations of thestatin in plasma and liver are presented in Figs. 4 and 5,respectively. Plasma exposure as determined by the areaunder the curce (AUC) was 373 (pravastatin), 101 (simvasta-tin), 36.6 (atorvastatin), 9.77 (rosuvastatin), and 4.78 minutes

Fig. 1. Comparison of the cholesterol-lowering effect of different statins in patients with hypercholesterolemia (plasma LDL-C . 160 mg/dl). Data arepresented as the percentage reduction in TC or LDL-C after treatment compared with baseline levels (mean 6 S.D.). The different statins and the dosestudied are presented on the x-axis (A, P, L, S, and R represent atorvastatin, pravastatin, simvastatin, lovastatin, and rosuvastatin, respectively, withthe number indicating the dose in milligrams per day). The value below the drug label indicates the number of head-to-head comparisons involving thatspecific statin and dose. LDL-C, LDL cholesterol.

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× mg/ml (lovastatin). It is noteworthy that liver levels did notcorrelate with the plasma levels of the different statins andwere, from high to low, 14.7 (atorvastatin), 9.81 (pravastatin),4.58 (rosuvastatin), 3.49 (lovastatin), and 1.56 mg/g (simvas-tatin) 30 minutes after administration. Liver-to-plasma ratiosof the individual statins 30 and 120 minutes after oraladministration are presented in Table 3, demonstrating thehighest liver-to-plasma ratios for rosuvastatin and the lowestfor simvastatin.For the three statins that were analyzed by liquid

scintillation counting ([3H]atorvastatin, [3H]pravastatin,and [3H]rosuvastatin), we determined drug levels in thestomach, intestine (tissue plus contents), colon (tissue pluscontents), and kidney as well (Supplemental Material Note 5).Drug levels in the stomach were comparable between thethree statins (∼50 mg/g 30 minutes after dosage). Comparedwith rosuvastatin and atorvastatin, pravastatin showed thelowest level of drug in the small intestine (including contents),concomitantly with the highest plasma AUC and highestlevel of drug present in the kidneys. These data suggest thatpravastatin has the highest oral bioavailability of these statinsin E3L mice.Comparison of the Efficacy of Different Statins

between Humans and E3L Mice. The cholesterol-lowering

efficacy of the different statins used in this study was comparedbetween humans and E3L mice. To do so, we plotted thecalculated ED30 values of the statins for humans against theED30 values obtained for E3L mice, before and after correctingfor pharmacokinetic differences (plasma AUC or average liverconcentration) between the statins in mice (Fig. 6). Impor-tantly, after correcting for the average liver concentrations inE3L mice, we found a significant correlation between theefficacy of statins in humans and E3L mice (R2 5 0.89, P ,0.05; Fig. 6C). The hepatic extraction of statins in humans is80% for simvastatin, 70% for atorvastatin and lovastatin, 65%for rosuvastatin, and 45% for pravastatin (Neuvonen et al.,2006). When correspondingly taking these differences intoaccount, the correlation between human and mouse studieswas even further increased (R2 5 0.99, P , 0.001; Fig. 6D).

DiscussionThe E3L transgenic mouse model exhibits a human-like

plasma lipid profile in response to high-fat diet feeding anddevelops atherosclerotic lesions that resemble their humancounterparts (van Vlijmen et al., 1994; Zadelaar et al., 2007;Verschuren et al., 2012). This mouse model is a valid and isstill widely used mouse model for studying the cholesterol-

Fig. 2. Body weights and food intake of E3L mice during the 6-week drug treatment period (t = 0 is the time point of randomization after 4 weeks of run-in on HFC diet). A, P, L, S, and R represent atorvastatin, pravastatin, lovastatin, simvastatin, and rosuvastatin, respectively.

TABLE 2Plasma TC and TG levels in E3L mice fed a HFC diet after 6 weeks of treatment with different statinsData are presented as mean 6 S.D. Time (t) = 6 after 6-week intervention period.

Treatment n Plasma TC at t = 6 (mM) Reductiona Plasma TG at t = 6 Reductiona

mM % mM %

Chow 12 2.03 6 0.26 2.59 6 0.35 —HFC 8 16.4 6 3.03 — 2.28 6 0.62 —HFC + 0.08 mg/day atorvastatin 8 11.6 6 1.04*** 29.4 6 6.39 2.52 6 0.56 —

HFC + 0.21 mg/day atorvastatin 8 8.55 6 1.81*** 47.8 6 11.1 2.35 6 0.68 —HFC + 0.77 mg/day pravastatin 8 13.1 6 1.35** 20.1 6 8.27 1.25 6 0.18*** 45.2 6 8.07HFC + 1.02 mg/day pravastatin 8 10.9 6 1.13*** 33.2 6 6.87 0.86 6 0.14*** 62.5 6 6.20HFC + 1.26 mg/day lovastatin 8 12.9 6 1.39** 21.1 6 8.48 2.13 6 0.55 6.60 6 24.2HFC + 1.77 mg/day lovastatin 8 10.5 6 1.65*** 36.0 6 10.1 0.94 6 0.18*** 59.0 6 7.82HFC + 0.78 mg/day simvastatin 8 16.1 6 2.59 7.06 6 10.3 2.55 6 0.34 —HFC + 1.56 mg/day simvastatin 8 14.7 6 2.87 14.1 6 12.3 2.40 6 0.96 —HFC + 0.06 mg/day rosuvastatin 8 12.2 6 0.95** 25.5 6 5.77 1.41 6 0.18** 38.1 6 7.94HFC + 0.13 mg/day rosuvastatin 8 10.5 6 0.58*** 36.1 6 3.56 0.86 6 0.12*** 62.4 6 5.23

—, no difference.aCompared with average TC or TG levels in the HFC-control group at t = 6.**P , 0.01 compared with HFC control group.***P , 0.001 compared with HFC control group.

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lowering and pleiotropic effects of (newly developed) drugs(e.g., statins, fibrates, ezetimibe, olmesartan). There is contin-uously increasing need by the pharmaceutical industry topredict correctly the efficacy of novel drug candidates in earlystage preclinical phases. We therefore aimed to map thepredictive value of the E3L mouse model with respect to thecholesterol-lowering efficacy of a set of statins. Importantly,since the pharmacokinetics, and especially the concentrationat the target site, codetermines the efficacy of drugs, wehypothesized that combining pharmacokinetic with efficacydata can increase the predictability of drug efficacy inpreclinical models. To this end, the efficacy profile of fivecurrently marketed statins was determined in E3L transgenicmice and compared with their efficacy in humans based ondata obtained from a comprehensive meta-analysis of humantrials. Besides efficacy, we also determined the pharmacoki-netics and tissue distribution of these drugs in E3L mice.

These results show that oral bioavailability and target organconcentrations varied markedly between the statins. Wedemonstrate that consideration of these differences improvesthe prediction of the efficacy of the drugs in preclinical modeland facilitates the translation of preclinical data to situationin humans.Statins have been widely prescribed to reduce circulating

cholesterol levels and lower the risk of cardiovascularevents for more than two decades now. The various effectsof atorvastatin, rosuvastatin, and pravastatin have pre-viously been studied in E3L transgenic mice (Delsing et al.,2003, 2005; Kleemann et al., 2003; van de Poll et al., 2003;Verschuren et al., 2005; van der Hoorn et al., 2007). Thesestudies revealed that E3L mice, unlike other mouse modelsof cardiovascular disease, respond to these hypolipidemicdrugs by a reduction of plasma cholesterol levels and thatstatins can reduce atherosclerotic plaque formation in these

Fig. 3. Comparison of the cholesterol-lowering effect of different statins after 6 weeks of treatment in E3L mice fed with a HFC diet. ED30 valuesrepresent the effective dose (milligram per day) to reduce plasma TC by 30% (in case of simvastatin, the value had to be extrapolated). Data arepresented as the percentage reduction of plasma TC compared with the HFC control group (mean 6 S.D.). CI, 95% confidence interval.

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mice, partly even beyond and independent of the cholesterol-lowering effect. This latter effect might be explained bythe more pleiotropic effects of statin drugs (e.g., anti-inflammatory effects) in the vasculature and the liver and is

in line with observations in humans (Liao and Laufs, 2005).In the current study, we concentrated on the cholesterol-lowering effects of the statins in the preclinical E3L model.The observed ED30 values of atorvastatin and rosuvastatin

Fig. 4. Plasma levels of atorvastatin, pravastatin, lovastatin, simvastatin, and rosuvastatin in E3L mice orally dosed with 10 mg/kg. All data arepresented as means 6 S.D. (n = 4). LLQ, lower limit of quantification (i.e., 0.1 mg/ml for lovastatin).

Fig. 5. Liver levels of atorvastatin, pravastatin, lovastatin,simvastatin, and rosuvastatin in E3L mice 30 and 120minutes after receiving an oral dose of 10 mg/kg. All dataare presented as means 6 S.D. (n = 4). For very low bars,the measured value is also presented above the bar.

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(0.14 and 0.11 mg/day, respectively) in this study are highlycomparable to those found in studies by Verschuren et al.(atorvastatin: ∼0.13 mg/day) and Delsing et al. (rosuvastatin:∼0.09 mg/day) that were carried out more than 7 years agousing the samemousemodel and comparable dietary conditions(Delsing et al., 2005; Verschuren et al., 2005). This clearly demon-strates the validity and reproducibility of the E3L transgenicmouse model for these types of research questions.We determined plasma exposure and liver uptake of the

different statins in E3L mice that were exposed to statintherapy for 6 weeks after an oral bolus injection of 10mg/kg. Ithas been shown before that rosuvastatin is more efficientlytaken up by hepatic cells than are pravastatin and simvas-tatin (Nezasa et al., 2002). Here, we also observed morethan 30-fold increased liver-to-plasma ratios of rosuvastatincompared with simvastatin and pravastatin, albeit inthe study by Nezasa et al. (2002), the statins were dosed at

5mg/kg i.v. Furthermore, in linewith results fromDeGorter et al.(2012), liver-to-plasma ratios of rosuvastatin were at the sameorder of magnitude as that of atorvastatin, with higher liverconcentrations of atorvastatin compared with rosuvastatin,but more than 2-fold lower plasma exposure of rosuvastatincompared with atorvastatin. Again, in the referred study,the statins were dosed differently (1 mg/kg i.v.) than in thecurrent study, indicating that comparable studies are thelacking. In the present study, we clearly demonstrate a highbioavailability (i.e., plasma exposure after oral administra-tion) of pravastatin in mice compared with the other statindrugs. To the best of our knowledge, this has never beenpublished before. Notably, this finding is substantiatedby the relative low levels of remaining pravastatin in thesmall intestine and colon 30 and 120 minutes after dosing(Supplemental Material Note 5). The high plasma exposure,but more importantly the relatively high liver uptake of

TABLE 3Liver-to-plasma ratios of different statins 30 and 120 minutes after oral dosage (10 mg/kg)Data are presented as means 6 S.D., n = 4.

Statin Liver-to-Plasma Ratio (t = 30 min) Liver-to-Plasma Ratio (t = 120 min)

Atorvastatin 41.7 6 9.76 21.3 6 3.47Pravastatin 1.68 6 0.30 1.70 6 0.08Lovastatin 10.8 6 5.95 naSimvastatin 1.29 6 0.52 0.13 6 0.08Rosuvastatin 49.5 6 6.90 27.4 6 5.27

na, not applicable (i.e., plasma levels were , lower limit of quantification).

Fig. 6. Comparison of cholesterol-lowering effect of different statins between humans and the E3L mouse model, before (A) and after correcting forpharmacokinetic differences in plasma exposure (B) and liver uptake (C) between the different statins in E3L mice and/or hepatic extraction ratio inhumans (D). ED30 values are defined as the effective dose (milligram per day) needed to reduce plasma TC by 30%. A, P, L, S, and R representatorvastatin, pravastatin, lovastatin, simvastatin, and rosuvastatin, respectively.

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pravastatin in E3L mice, might explain the rather highcholesterol-lowering efficacy of pravastatin compared withthe other two second-generation statins, lovastatin andatorvastatin. In contrast, simvastatin, which also has a ratherhigh plasma exposure (101 min×mg/ml) in E3L mice, but thelowest hepatic uptake, was the least effective. These resultsfor the first time systematically link the hepatic uptake ofstatins to their cholesterol-lowering efficacy in a valuablemouse model of hyperlipidemia.The therapeutic equivalence of statins in humans was

recently reviewed in a meta-analysis by Weng et al. (2010),providing a ranking order from most to least potent statinswhen looking at the plasma LDL-cholesterol lowering effect:rosuvastatin . atorvastatin . simvastatin . lovastatin .pravastatin. We added 16 clinical head-to-head trials thatwere published between 2006 and 2009 (references fromBeth Smith et al., 2009) and compared the total cholesterol-lowering properties of the above mentioned statins (since wealso determined the total cholesterol levels in the E3L mice).We found a ranking in potency of statins in humans withhypercholesterolemia that was fully consistent with theanalysis of Weng et al. (2010). Determination of ED30 valuesenabled us to compare directly the efficacy of these statins inE3L mice relative to humans. Interestingly, rosuvastatin andatorvastatin, which belong to the newer third-generationsynthetic statins (McTaggart et al., 2001), are by far themost potent statins in humans as well as in the E3L mousemodel. However, the efficacy of simvastatin, pravastatin, andlovastatin in humans is remarkably less well predicted byjust looking at the potency of these statins in E3L mice. Onlyafter taking into account the differences in liver uptake ofthese statins in E3L mice, we found a significant correlationbetween the cholesterol-lowering efficacy of these statins inE3L mice and patients with hypercholesterolemia. The factthat just correcting for differences in liver accumulation of thevarious statins in E3Lmice is already sufficient to gain a goodcorrelation suggests that the relative differences in liveruptake between these statins in humans are minor comparedwith those in E3L mice. Hepatic extraction of simvastatin,lovastatin, atorvastatin, and rosuvastatin is reported to becomparable in humans (65–80%), whereas that of pravastatinis slightly lower (45%) (Neuvonen et al., 2006, 2008). Whentaking into account these minor differences in hepaticextraction of the studied statins, we retrieve an even bettercorrelation between the efficacy of the drugs in humans andE3L mice (Fig. 6D).In conclusion, in this study, we provide evidence that a

combined PK-efficacy study substantially improves thetranslational value of the E3L mouse model in case of thecholesterol-lowering effect of statin treatment. We hypothe-size that a similar strategy could be used to increase theclinical predictability of drug treatment of other classes ofdrugs in various preclinical (disease) models.

Acknowledgments

The authors thank I. van Scholl (TNO Triskelion, Zeist, TheNetherlands) for the analyses of simvastatin in liver homogenatesusing UPLC-MS.

Authorship Contributions

Participated in research design: Steeg, Kleemann, Wortelboer,DeGroot.

Conducted experiments: Steeg, Duyvenvoorde, Offerman, Jansen.Performed data analysis: Steeg, Duyvenvoorde.Wrote or contributed to the writing of the manuscript: Steeg,

Kleemann, Wortelboer, DeGroot.

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