Anacetrapib Reduces (V)LDL Cholesterol by Inhibition of CETP Activity and Reduction of Plasma PCSK9

Supplemental Material can be found at: http://www.jlr.org/content/suppl/2015/09/04/jlr.M057794.DC1 .html

Anacetrapib reduces (V)LDL cholesterol by inhibition of CETP activity and reduction of plasma PCSK9

Sam J. L. van der Tuin ,1, *,† Susan Kühnast ,1,†,§, ** Jimmy F. P. Berbée , *,† Lars Verschuren ,†† Elsbet J. Pieterman ,** Louis M. Havekes , *,†, ** José W. A. van der Hoorn, †,§, ** Patrick C. N. Rensen , *,† J. Wouter Jukema ,†,§ Hans M. G. Princen , ** Ko Willems van Dijk, *,†,§§ and Yanan Wang2, *,†,§§ Department of Medicine, Division of Endocrinology,* Department of Human Genetics,§§ Department of Cardiology,§ and Einthoven Laboratory for Experimental Vascular Medicine,† Leiden University Medical Center, Leiden, The Netherlands; The Netherlands Organization for Applied Scientiﬁ c Research (TNO), Metabolic Health Research, Gaubius Laboratory,** Leiden , The Netherlands; and TNO, Microbiology and ††

Systems Biology, Zeist, The Netherlands Downloaded from

Abstract Recently, we showed in APOE*3-Leiden choles- clearance. —van der Tuin, S. J. L., S. Kühnast, J. F. P. Berbée, teryl ester transfer protein (E3L.CETP) mice that anacetra- L. Verschuren, E. J. Pieterman, L. M. Havekes, J. W. A. van pib attenuated atherosclerosis development by reducing (V) der Hoorn, P. C. N. Rensen, J. W. Jukema, H. M. G. Princen, LDL cholesterol [(V)LDL-C] rather than by raising HDL K. Willems van Dijk, and Y. Wang. Anacetrapib reduces (V) cholesterol. Here, we investigated the mechanism by which LDL-cholesterol by inhibition of CETP activity and reduc- www.jlr.org anacetrapib reduces (V)LDL-C and whether this effect was tion of plasma PCSK9. J. Lipid Res. 2015. 56: 2085–2093. dependent on the inhibition of CETP. E3L.CETP mice were fed a Western-type diet alone or supplemented with anace- Supplementary key words cholesteryl ester transfer protein • choles- at Walaeus Library / BIN 299, on November 9, 2015 trapib (30 mg/kg body weight per day). Microarray analyses terol/metabolism • drug therapy/hypolipidemic drugs • low density of livers revealed downregulation of the cholesterol biosyn- lipoprotein/metabolism • lipids • lipoproteins/metabolism • propro- thesis pathway (P < 0.001) and predicted downregulation of tein convertase subtilisin/kexin type 9 • very low density lipoprotein pathways controlled by sterol regulatory element-binding ;proteins 1 and 2 (z -scores ؊ 2.56 and ؊ 2.90, respectively both P < 0.001). These data suggest increased supply of High plasma levels of (V)LDL cholesterol [(V)LDL-C] cholesterol to the liver. We found that hepatic proprotein and TGs, as well as low levels of HDL cholesterol (HDL-C), convertase subtilisin/kexin type 9 (Pcsk9 ) expression was decreased (؊ 28%, P < 0.01), accompanied by decreased are important risk factors for cardiovascular diseases. The plasma PCSK9 levels (؊ 47%, P < 0.001) and increased he- standard treatment for the reduction of cardiovascular patic LDL receptor (LDLr) content (+64%, P < 0.01). Con- disease risk is statin therapy aiming to reduce plasma (V) sistent with this, anacetrapib increased the clearance and LDL-C. However, a substantial residual risk remains de- hepatic uptake (+25%, P < 0.001) of [14 C]cholesteryl oleate- spite statin treatment. This has prompted the search for labeled VLDL-mimicking particles. In E3L mice that do not secondary treatment targets ( 1, 2 ). Prospective epidemio- express CETP, anacetrapib still decreased (V)LDL-C and logical studies indicate HDL-C as a potential target ( 3 ). plasma PCSK9 levels, indicating that these effects were in- The ratio of plasma (V)LDL-C to HDL-C is to a great ex- dependent of CETP inhibition. We conclude that anacetra- tent affected by cholesteryl ester transfer protein (CETP). pib reduces (V)LDL-C by two mechanisms: 1 ) inhibition of CETP activity, resulting in remodeled VLDL particles CETP facilitates the transfer of cholesteryl esters from that are more susceptible to hepatic uptake; and 2 ) a CETP- HDL to (V)LDL in exchange for TG ( 4 ). In several mouse independent reduction of plasma PCSK9 levels that has the potential to increase LDLr-mediated hepatic remnant

Abbreviations: [3 H]TO, glycerol tri(9,10(n )[ 3 H])oleate; [14 C]CO, This research was performed within the framework of the Center for Transla- ␣ ␣ 14 tional Molecular Medicine (http://www.ctmm.nl), project PREDICCt Grant [1 ,2 (n )- C]cholesteryl oleate; CETP, cholesteryl ester transfer 01C-104 (K.W.v.D.). P.C.N.R. is an Established Investigator of the Netherlands protein; E3L, APOE*3-Leiden; HDL-C, HDL-cholesterol; (V)LDL-C, Heart Foundation (Grant 2009T038), received a grant from Merck Sharp & (V)LDL-cholesterol; LDLr, LDL receptor; LRP1, low density lipopro- Dohme (MSD), and consults for Astellas. J.W.J. received research grants from and tein receptor-related protein 1; NR1I2, nuclear receptor subfamily 1, was speaker for Continuing Medical Education (CME)-accredited meetings group I, member 2; NR1I3, nuclear receptor subfamily 1, group I, mem- sponsored by Amgen, Astellas, Astra-Zeneca, Biotronik, Boston Scientifi c, Daiichi ber 3; PCSK9, proprotein convertase subtilisin/kexin type 9 ; SREBP, Sankyo, Lilly, Genzyme, Medtronic, Merck-Schering-Plough, Pfi zer, Orbus Neich, sterol regulatory element-binding protein; TC, total cholesterol. Novartis, Roche, Servier, Sanofi Aventis, the Netherlands Heart Foundation, the 1 S. J. L. van der Tuin and S. Kühnast contributed equally to this Interuniversity Cardiology Institute of the Netherlands, and the European Com- munity Framework KP7 Programme. work. 2 To whom correspondence should be addressed. Manuscript received 21 January 2015 and in revised form 2 September 2015. e-mail: [email protected] Published, JLR Papers in Press, September 4, 2015 The online version of this article (available at http://www.jlr.org) DOI 10.1194/jlr.M057794 contains a supplement.

Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 56, 2015 2085 Supplemental Material can be found at: http://www.jlr.org/content/suppl/2015/09/04/jlr.M057794.DC1 .html Ϫ Ϫ models, including C57Bl/6, Ldlr / , and APOE*3-Leiden particles. By using E3L mice with or without CETP expres- (E3L) transgenic mice, CETP expression aggravates the sion ( 7 ), we also determined whether these effects of anac- development of atherosclerosis ( 5–7 ). Although human etrapib were CETP dependent. studies have shown confl icting results with regard to the association between CETP defi ciency and decreased cardiovascular disease risk ( 8, 9), CETP inhibition is actively pursued as a potential strategy to reduce this risk ( 10 ). MATERIALS AND METHODS This has led to the development of pharmacological CETP inhibitors, such as torcetrapib, dalcetrapib, anacetrapib, RNA isolation, microarray, and qPCR validation and evacetrapib. Liver pieces were obtained from a previous experiment per- In clinical trials, torcetrapib, anacetrapib, and evacetra- formed by Kühnast et al. ( 22 ), investigating the effects of anacetrapib on atherosclerosis in female E3L.CETP mice ( 22 ). In this pib have been shown to increase HDL-C [up to +72% ( 11 ), study, mice were treated with a semisynthetic cholesterol-rich +139% ( 12 ), and +129% (13 ), respectively] and to reduce Ϫ Ϫ Ϫ diet, containing 15% (w/w) cacao butter, 1% corn oil, and 0.1% LDL-C [down to 25% (11 ), 40% (12 ), and 36% (13 ), cholesterol (Western-type diet; AB-Diets, Woerden, The Nether- respectively], whereas dalcetrapib only increased HDL-C lands) with or without anacetrapib (30 mg/kg body weight per [up to +40% ( 14 )]. Although torcetrapib showed favor- day) for 21 weeks. Total RNA was extracted from these liver able effects on the lipoprotein profi le, it failed in phase III pieces using the Nucleospin RNAII kit (Macherey-Nagel) accord- clinical development due to increased risk of major car- ing to the manufacturer’s protocol. The microarray, including diovascular events and mortality. These adverse effects quality control, RNA labeling, hybridization, and data extraction, Downloaded from were ascribed to an off-target effect ( 11 ) and proinfl am- was performed by ServiceXS B.V. (Leiden, The Netherlands). To perform real-time quantitative PCR (qPCR) for validation, matory lesions ( 15 ). A large phase III clinical trial with dal- RNA quality was verifi ed by the lab-on-a-chip method using Ex- cetrapib was prematurely terminated due to a lack of perion TM RNA StdSens analyses kit (Bio-Rad). Total RNA was re- clinical benefi t (14 ). Nonetheless, the effects of anacetra- verse transcribed with iScript cDNA synthesis kit (Bio-Rad), and TM pib and evacetrapib on cardiovascular outcomes are cur- qPCR was performed using a CFX96 Touch Real-Time PCR www.jlr.org rently being evaluated in phase III clinical trials ( 16 ). Detection System (Bio-Rad). Gene expression was normalized to Neither compound shows increased blood pressure as ob- beta-2 microglobulin and hypoxanthine-guanine phosphoribos- yltransferase. Relative expression was calculated as compared served with torcetrapib ( 12, 13), and both compounds are TM more potent in increasing HDL-C and reducing LDL-C as with the control group using Bio-Rad CFX Manager software at Walaeus Library / BIN 299, on November 9, 2015 3.0 (Bio-Rad). compared with torcetrapib and dalcetrapib. While regular C57BL/6J mice have a very rapid clear- Microarray data analyses ance of apoB-containing lipoproteins, E3L mice have an The probe-level background subtracted expression values attenuated clearance and increased TG level, thereby were used as input for lumi package ( 24 ) of the R/Bioconductor mimicking the slower clearance observed in humans, par- (http://www.bioconductor.org; http://www.r-project.org) to per- ticularly in patients with familial dyslipoproteinemia ( 17 ). form quality control and a quantile normalization. Unexpressed Similarly as in familial dyslipoproteinemia patients, in E3L probes ( P > 0.01 in all experiments) were removed from fur- and E3L.CETP mice as a model for mixed dyslipoprotein- ther analyses. Differentially expressed probes were identifi ed emia, a major part of plasma cholesterol is contained in using the limma package of R/Bioconductor ( 25 ). The calculated P values <0.05 were used as a threshold for pathway analyses the VLDL (remnant) particles, leading to the formation of using Ingenuity Pathway Analysis suite (http://www.ingenuity. ␤ -VLDL particles, which further increases after cholesterol Ϫ / Ϫ com). Upstream regulator analysis was performed using the Inge- feeding. The E3L.CETP mouse model, unlike Apoe nuity Pathway Analysis software. This analysis determines the ac- Ϫ / Ϫ and Ldlr mice, responds in a human-like way to the tivation state of transcription factors based on the observed lipid-lowering effects of statins ( 18 ), fi brates (19 ), niacin differential gene expression and results in an overlap P value and ( 20, 21), torcetrapib (15 ), anacetrapib (22 ), and anti- activation z -score for each transcription factor in the Ingenuity PCSK9mabs ( 23 ), with respect to both direction and mag- Pathway Analysis knowledgebase. The overlap P value indicates nitude of the change. In conclusion, E3L.CETP mice have the signifi cance of the overlap between the known target genes a more human-like lipoprotein metabolism when com- of a transcription factor and the differentially expressed genes Ϫ / Ϫ Ϫ / Ϫ measured in an experiment. The activation z -score indicates pared with C57Bl/6, Apoe , or Ldlr mice. activation (positive z -score) or inhibition (negative z -score) of a Recently, we have shown that anacetrapib treatment in- particular transcription factor. An activation z -score >2 or <2 creased HDL-C, reduced (V)LDL-C and TG, and dose-de- indicates signifi cant activation or inhibition of a pathway or pendently reduced atherosclerotic lesion size and severity process. in E3L.CETP mice. ANCOVA showed that the effect on lesion size was mainly explained by a reduction in (V)LDL-C Experimental setup (22 ). However, the mechanism by which anacetrapib re- To investigate the effects of anacetrapib on VLDL production duces plasma (V)LDL-C and TG is not fully understood. and clearance, female E3L (26 ) and E3L.CETP (7 ) transgenic mice, 8–10 weeks of age, were fed a Western-type diet for a run-in To elucidate this, we performed microarrays on the livers period of 3–4 weeks. They were then matched based on plasma from this latter study, identifying pathways affected by total cholesterol (TC), HDL-C, TG, body weight, and age into two anacetrapib. To confi rm physiological consequences of groups receiving either no treatment (control) or anacetrapib (30 these identifi ed pathways, we performed a VLDL production mg/kg body weight per day) for 4 weeks, after which VLDL pro- experiment and studied the clearance of VLDL-mimicking duction (E3L.CETP only) or clearance (E3L and E3L.CETP) was

2086 Journal of Lipid Research Volume 56, 2015 Supplemental Material can be found at: http://www.jlr.org/content/suppl/2015/09/04/jlr.M057794.DC1 .html determined. After both experiments, the mice were euthanized by particles (mean diameter 80 nm) were prepared as previously 3 14 CO2 asphyxiation. The mice were housed under standard condi- described ( 31 ). In short, [ H]TO (100 µCi) and [ C]CO (10 tions with a 12 h light-dark cycle and had free access to food and µCi) tracers were added to a mixture of TO (70 mg), egg yolk water during the experiment. Body weight and food intake were phosphatidylcholine (22.7 mg), lysophosphatidylcholine (2.3 monitored during the study. The Institutional Ethics Committee mg), CO (3.0 mg), and cholesterol (2.0 mg). Particles were pre- for Animal Procedures from the Leiden University Medical Cen- pared from this mixture at 54°C using a Soniprep 150 (MSE Sci- ter, Leiden, The Netherlands, approved the protocol. entiﬁ c Instruments) at 10 µm output. After two consecutive density gradient ultracentrifugation steps, VLDL-mimicking par- Plasma lipid measurements ticles (average size of 80 nm) were isolated. The particles were Blood was collected after a 4 h fasting period in heparin- stored at 4°C under argon and intravenously injected into mice coated capillaries via tail vein bleeding, and plasma was isolated. within 5 days. These VLDL-mimicking particles acquire a wide TC and TG were determined using enzymatic kits (Roche) ac- range on interchangeable apolipoproteins from serum, among cording to the manufacturer’s protocol. To measure HDL-C, which apoE ( 31, 32) mediates the hepatic uptake of their rem- apoB-containing particles were precipitated from plasma with nants (32 ). 20% polyethylene glycol 6000 (Sigma Aldrich) in 200 mM gly- After a 4 h fast, particles were injected via the tail vein in cine buffer (pH 10), and HDL-C was measured in the superna- conscious mice (n = 8/9 per group). At 2, 5, 10, and 15 min postinjection, blood was taken to determine the plasma decay tant ( 22 ). The distribution of cholesterol over plasma lipoproteins 3 14 was determined by fast-performance liquid chromatography as of [ H]TO and [ C]CO. Plasma volumes were calculated as previously described (7 ). 0.04706 × body weight (g) as previously described ( 33).Mice were euthanized after 15 min, perfused with ice-cold PBS with

0.1% heparin (v/v), and organs were harvested to determine Downloaded from Hepatic LDL receptor protein and plasma proprotein 3 14 convertase subtilisin/kexin type 9 measurements tissue-specifi c [ H]TO and [ C]CO uptake. Subsequently, organs were dissolved overnight at 56°C in tissue solubilizer (Am- Snap-frozen mouse livers were lysed in ice-cold lysis buffer 3 14 ersham Biosciences) and quantified for H and C activity. containing 50 mM HEPES (pH 7.6), 50 mM NaF, 50 mM KCl, 3 14 Uptake of [ H]TO- and [ C]CO-derived radioactivity by the or- 5 mM NaPPi, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 5 mM gans was calculated as dose per organ after correction for organ ␤ -glycerophosphate, 1 mM sodium vanadate, 1% NP40, and prote- weight. www.jlr.org ase inhibitors cocktail (Roche). Thereafter, protein level was determined using the BCA Protein Assay Kit (Pierce) according to Statistical analyses the manufacturer’s instructions. Laemmli buffer (Sigma-Aldrich) Signifi cance of differences between the groups was calcu- was added to samples containing equal amounts of protein. Sam- lated nonparametrically using a Mann-Whitney U -test. All reported at Walaeus Library / BIN 299, on November 9, 2015 ples were separated on a 10% sodium dodecyl sulfate-polyacryl- P values are two-tailed, and P values of >0.05 were considered amide gel electrophoresis, and blotted to polyvinylidene difl uoride. statistically signifi cant. Blots were incubated with goat-anti-mouse LDL receptor (LDLr; 1:1,000, AF2255, R and D Systems), rabbit-anti-mouse low density lipoprotein receptor-related protein 1 (LRP1; 1:20,000, ab92544, Abcam), and mouse-anti-␣ -tubulin (1:1,000, T5168, Sigma-Aldrich) and subsequently incubated with the appropriate secondary anti- RESULTS body. Bands were visualized by enhanced chemiluminescence with Pierce ECL 2 substrate following the manufacturer’s protocol and Pathway analyses predict downregulation of pathways quantifi ed using Image J software as previously described (27 ). controlled by sterol regulatory element-binding proteins Plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) was 1 and 2 by anacetrapib measured by using ELISA (MCP900, R and D Systems) according To determine the effects of anacetrapib treatment on to the manufacturer’s instructions. hepatic gene expression in E3L.CETP mice (22 ), microarray analyses were performed. A total of 95 genes (false discovery Hepatic VLDL-TG and VLDL-apoB production analyses rate P < 0.05; supplementary Table 1) were differentially ex- Mice (n = 8/9 per group) were anesthetized with 6.25 mg/kg pressed between control and anacetrapib-treated female acepromazine (Alfasan), 6.25 mg/kg midazolam (Roche), and mice of which 46 genes were upregulated and 49 genes were 0.31 mg/kg fentanyl (Janssen-Cilag) after a 4 h fast. A basal blood downregulated. To gain insight into affected biological sample was taken from the tail, and the mice received an intrave- 35 processes, a gene-set enrichment analysis was performed nous injection of 100 µl PBS containing 100 µCi Tran S label (MP Biomedicals) via the tail vein. After 30 min, animals received using the Ingenuity Pathway Analysis suite (as described in an intravenous injection of Tyloxapol (Triton WR-1339, Sigma- Material and Methods). This analysis showed that the choles- Aldrich; 500 mg/kg body weight), as a 10% (w/w) solution in terol biosynthesis pathway was signifi cantly affected ( Table 1 ). sterile saline, to prevent systemic lipolysis of newly secreted he- In silico prediction of transcription factor activity ( Table 2), patic VLDL-TG (28 ). At indicated time points up to 90 min after based on the differentially expressed genes, predicted in- Tyloxapol injection, blood was taken and plasma TG concentra- hibition of genes regulated by SREBP-1 ( P < 0.001; z -score tion was determined. After 120 min, mice were euthanized, and Ϫ 2.90) and SREBP-2 (P < 0.001; z -score Ϫ 2.56), which are blood was collected for isolation of the VLDL fraction by density key regulators of cholesterol synthesis. SREBP-2 target gradient ultracentrifugation ( 29 ). Tran35 S activity was measured in the VLDL fraction, and VLDL-apoB production rate was calcu- genes mevalonate (diphospho) decarboxylase [ Mvd (34 )], lated as dpm/h (30 ). 7-dehydrocholesterol reductase [Dhcr7 ( 35 )], and trans- membrane 7 superfamily member 2 [Tm7sf2 (36 )] were Clearance analysis of VLDL-mimicking particles validated using qPCR. All genes showed a downregula- Glycerol tri(9,10(n)[3 H])oleate ([3 H]TO) and [1␣ ,2␣ (n)-14 C] tion after anacetrapib treatment (supplementary Fig. 2). cholesteryl oleate ([14 C]CO) double-radiolabeled VLDL-mimicking Furthermore, anacetrapib activated genes regulated by

Anacetrapib increases VLDL remnant clearance 2087 Supplemental Material can be found at: http://www.jlr.org/content/suppl/2015/09/04/jlr.M057794.DC1 .html TABLE 1. Signiﬁ cantly regulated pathways

Ingenuity Canonical Pathways Ϫ Log (P ) Ratio Superpathway of cholesterol biosynthesis 10.50 0.24 PXR/RXR activation 6.32 0.09 Superpathway of geranylgeranyldiphosphate biosynthesis I (via mevalonate) 6.00 0.22 Bupropion degradation 5.39 0.16 LPS/interleukin-1-mediated inhibition of RXR function 5.28 0.04 Cholesterol biosynthesis I 4.65 0.23 Cholesterol biosynthesis II (via 24,25-dihydrolanosterol) 4.65 0.23 Cholesterol biosynthesis III (via desmosterol) 4.65 0.23 Isoleucine degradation I 4.54 0.21 Mevalonate pathway I 4.54 0.21

Female E3L.CETP mice were fed a Western-type diet with or without anacetrapib (30 mg/kg body weight per day) for 22 weeks. RNA was isolated from liver tissue, and a microarray analysis was performed. Selected differentially expressed genes (95 genes; see supplementary Table 1) were used as input for pathway analysis through ingenuity pathway analysis suite. LPS, lipopolysaccharides; PXR, pregnane X receptor; RXR, retinoid X receptor.

NR1I2 (P < 0.001; z -score +2.75) and NR1I3 (P < 0.001; z -score Anacetrapib does not affect TG metabolism but increases +2.94). Both nuclear receptors function as sensors of lipoprotein remnant clearance by the liver in E3L.CETP endobiotic and xenobiotic substances. These data indicate mice Downloaded from that anacetrapib reduces cholesterol biosynthesis and acti- To further investigate the effects of anacetrapib on lipo- vates a xenobiotic response. protein metabolism, we performed a new experiment with female E3L.CETP mice fed a Western-type diet with or Anacetrapib decreases hepatic and circulating PCSK9 and without anacetrapib for 4 weeks. Plasma lipid and lipopro- increases hepatic LDLr protein in E3L.CETP mice tein levels were decreased to the same extent as shown in

In addition to effects on cholesterol biosynthesis and xe- Fig. 2A (lipoprotein profi le in supplementary Fig. 3A). www.jlr.org nobiotic metabolism, microarray analyses showed a de- Anacetrapib treatment did not affect the VLDL-TG pro- crease in the expression of Pcsk9 mRNA, a downstream duction rate ( Fig. 2B ), the VLDL-35 S-apoB production rate target of the SREBP-2 pathway [supplementary Fig. 1 ( Fig. 2C), or the ratio of VLDL-TG to VLDL-apoB produc- at Walaeus Library / BIN 299, on November 9, 2015 ( 37 )], in the liver of anacetrapib-treated E3L.CETP mice tion rate (Fig. 2D), indicating no changes in number or ( Ϫ 78%, P < 0.05; supplementary Table 1), which was con- composition of newly synthesized VLDL particles. fi rmed by qPCR (Ϫ 27%, P < 0.01; Fig. 1A). In accordance, The effect of anacetrapib on VLDL clearance was as- anacetrapib reduced plasma PCSK9 levels ( Ϫ 47%, P < sessed by an intravenous injection of [ 3 H]TO and [14 C] 0.01; Fig. 1B). Because PCSK9 plays an important role in CO double-radiolabeled VLDL-mimicking particles. At the degradation of intracellular LDLr ( 38–40 ) and LRP1 the indicated time points, blood was taken to determine ( 41 ), hepatic mRNA expression and protein levels were clearance from plasma. After 15 min, mice were eutha- measured. Anacetrapib did not affect the hepatic mRNA nized and organs were harvested to determine tissue-spe- expression of Ldlr (Fig. 1C) or Lrp1 ( Fig. 1E) but did in- cifi c uptake of radioactivity derived from [3 H]TO and crease hepatic LDLr protein expression (+64%, P < 0.05; [ 14 C]CO. We observed no effects on the plasma clearance Fig. 1D). No effect on LRP1 (Fig. 1F) protein levels was ( Fig. 3A) or the tissue-specifi c uptake of [3 H]TO-derived observed. The decrease in plasma PCSK9 levels and in- activity ( Fig. 3B ). However, anacetrapib increased the crease in LDLr suggest an increased capability of the liver plasma clearance of the [ 14 C]CO label of the VLDL-mim- to take up lipoprotein remnants. icking particles ( Fig. 3C), decreased plasma half-life of

TABLE 2. In silico prediction of transcription factor activity based on the expression changes of known target genes

Upstream Regulator Molecule Type Activation State z -Score P of Overlap

Sterol regulatory element-binding Transcription regulator Inhibited Ϫ 2.56 1.65E-13 protein 2 (SREBP-2) Sterol regulatory element-binding Transcription regulator Inhibited Ϫ 2.90 6.56E-12 protein 1 (SREBP-1) Nuclear receptor subfamily 1, group I, Ligand-dependent nuclear receptor Activated 2.94 9.27E-12 member 3 (NR1I3) Peroxisome proliferator-activated Ligand-dependent nuclear receptor — Ϫ 0.24 6.74E-10 receptor ␣ (PPARA) Nuclear receptor subfamily 1, group I, Ligand-dependent nuclear receptor Activated 2.75 6.01E-09 member 2 (NR1I2) PXR ligand-PXR-retinoic acid-RXR␣ Complex Activated 2.42 1.34E-08 CAR ligand-CAR-retinoic acid-RXR␣ Complex Activated 2.22 1.71E-08 Ncoa-Nr1i3-Rxra Complex Activated 2.00 6.83E-07 NAD-dependent deacetylase sirtuin-2 (SIRT2) Transcription regulator Inhibited Ϫ 2.00 6.83E-07 Ncoa-Nr1i2-Rxra Complex Activated 2.00 8.63E-07

To determine the activation status of transcription factors, an upstream regulator analysis was performed. The z -score indicates activation (positive) or inhibition (negative). CAR, constitutive androstane receptor.

2088 Journal of Lipid Research Volume 56, 2015 Supplemental Material can be found at: http://www.jlr.org/content/suppl/2015/09/04/jlr.M057794.DC1 .html Downloaded from www.jlr.org at Walaeus Library / BIN 299, on November 9, 2015 Fig. 1. Anacetrapib decreases PCSK9 mRNA expression and plasma levels and increases hepatic LDLr protein expression. Female E3L.CETP MICE were fed a Western-type diet with or without anacetrapib (30 mg/kg body weight per day) for 21 weeks; blood was collected for plasma PCSK9 levels and livers for mRNA expression. Hepatic PCSK9 mRNA expression (A) and plasma levels (B). Hepatic LDLr mRNA (C) and protein (D) expression. Hepatic LRP1 mRNA (E) and protein (F) expression. Data are presented as means ± SD (n = 14/15 per group). ** P < 0.01, *** P < 0.001 when compared with control group.

[14 C]CO ( Ϫ 56%, P < 0.001; Fig. 3C, inlay), and increased DISCUSSION the uptake of [14 C]CO by the liver (+25%, P < 0.001; Fig. 3D). Because these particles refl ect the behavior of In this study, we investigated the mechanism by which VLDL (32, 42), these results indicate that anacetrapib in- anacetrapib reduces plasma (V)LDL-C and whether these creases the uptake of lipoprotein remnants by the liver. effects are dependent on CETP. In E3L.CETP mice, anacetrapib decreased gene expression of cholesterol biosyn- Anacetrapib decreases (V)LDL-C and PCSK9 levels in thesis pathways in the liver, most probably via inhibition of E3L mice Srebp-1 and/or Srebp-2 signaling. In addition, we identifi ed To determine whether the effects of anacetrapib on (V) two important processes by which anacetrapib increases LDL metabolism were dependent on CETP inhibition, simi- cholesterol clearance. First, anacetrapib increased choles- lar experiments were performed in female E3L mice that terol clearance by the liver, without affecting VLDL-TG do not express CETP. Notably, anacetrapib reduced TC production rate and clearance in E3L.CETP mice. Sec- (Ϫ 17%, P < 0.05) and (V)LDL-C (Ϫ 20%, P < 0.05; Fig. 4A ; ond, in a CETP-independent manner, anacetrapib de- lipoprotein profi le in supplementary Fig. 3B) levels in E3L creased hepatic Pcsk9 expression and plasma PCSK9 levels. mice without CETP, concomitantly with a decrease in he- In E3L mice that do not express CETP, anacetrapib de- patic Pcsk9 expression and plasma PCSK9 levels (Ϫ 37%, P < creased (V)LDL-C and plasma PCSK9 levels. However, no 0.05; Fig. 4B). These data clearly show that anacetrapib has effects on cholesterol or VLDL-TG clearance were de- a CETP-independent lipid-lowering effect. Comparing E3L tected. These results indicate that CETP inhibition results with E3L.CETP mice, anacetrapib increased the particle in remodeled particles that are more susceptible for he- clearance in E3L.CETP mice to the similar level as observed patic clearance. in E3L mice without anacetrapib (Figs. 3C and 4C). Anace- The observed reduction in plasma PCSK9 levels after trapib showed no additional effects on the plasma clearance anacetrapib treatment is in accordance with recent fi nd- of [3 H]TO and [14 C]CO labels of the VLDL-mimicking par- ings in rhesus macaques ( 43 ). Here, we demonstrate that ticles (Fig. 4C, E) or the tissue-specifi c uptake of [3 H]TO this effect is independent of CETP inhibition as this was and [14 C]CO (Fig. 4D, F) in E3L mice. also observed in E3L mice without CETP. A recent study in

Anacetrapib increases VLDL remnant clearance 2089 Supplemental Material can be found at: http://www.jlr.org/content/suppl/2015/09/04/jlr.M057794.DC1 .html Downloaded from

Fig. 2. Anacetrapib does not affect hepatic VLDL-TG production in E3L.CETP mice. Female E3L.CETP mice were fed a Western-type diet with or without anacetrapib (30 mg/kg body weight per day) for 4 weeks, blood was collected by tail bleeding after 4 h fasting, and plasma TC, TG, non-HDL-C, and HDL-C were determined (A). After treatment, hepatic VLDL production was assessed (B). 35 S-apoB production was determined by scintillation counting of the isolated VLDL fraction (C), and the VLDL-TG production rate to

VLDL-apoB production rate ratio was calculated (D). Data are presented as means ± SD (n = 8/9 per group). www.jlr.org * P < 0.05, *** P < 0.001 when compared with control group.

C57Bl/6 mice also conﬁ rmed a CETP-independent de- Accumulating evidence shows that inhibiting PCSK9 is at Walaeus Library / BIN 299, on November 9, 2015 crease in plasma PCSK9 levels by anacetrapib ( 44 ). Path- an effective strategy to reduce LDL-C both in preclinical way analyses of the gene expression data predicted that and clinical studies ( 45, 46). This effect is attributed to a anacetrapib decreases liver cholesterol synthesis by reduc- reduction of hepatic LDLr degradation and a subsequent ing SREBP-2 regulated pathways. qPCR analysis of four increase of LDL remnant clearance. Our results showed SREBP-2 target genes (i.e., Mvd , Dhcr7 , Tm7sf2 , and Pcsk9 ) an increase in hepatic LDLr protein levels in E3L.CETP conﬁ rmed downregulation of genes in the cholesterol syn- mice after 21 weeks of anacetrapib in parallel with a de- thetic pathway. It is known that SREBP-2 is the principal crease in plasma PCSK9 levels. Partially in contrast, we nuclear transcription factor for the regulation of hepatic found after 4 weeks of anacetrapib treatment in E3L mice Pcsk9 expression (37 ). Therefore, the reduction of plasma with or without CETP expression, anacetrapib did not PCSK9 levels by anacetrapib may be attributed to the re- change hepatic LDLr protein levels (data not shown) de- duction of SREBP-2 pathway. spite a clear reduction of plasma PCSK9 levels ( Fig. 4B).

Fig. 3. Anacetrapib increases lipoprotein remnant clearance by the liver in E3L.CETP mice. Female E3L.CETP mice were fed a Western-type diet with or without anacetrapib (30 mg/kg body weight per day) for 4 weeks. Mice received an injection with glycerol [3 H]TO and [14 C]CO double-labeled VLDL-mimicking particles. Blood was drawn at the indicated time points, and 3 H and 14 C plasma decay (A, C) and tissue-speciﬁ c activity (B, D) were determined. The inlays in A and C show plasma half-life. Data are presented as means ± SD (n = 8/9 per group). *** P < 0.001 when compared with control group.

2090 Journal of Lipid Research Volume 56, 2015 Supplemental Material can be found at: http://www.jlr.org/content/suppl/2015/09/04/jlr.M057794.DC1 .html Downloaded from www.jlr.org at Walaeus Library / BIN 299, on November 9, 2015

Fig. 4. Anacetrapib decreases non-HDL-C and plasma PCSK9 levels, without affecting lipoprotein remnant clearance in E3L mice. Female E3L mice were fed a Western-type diet with or without anacetrapib (30 mg/kg per day) for 4 weeks, blood was collected by tail bleeding after 4 h fasting, and plasma TC, TG, non-HDL-C, and HDL-C (A) and plasma PCSK9 levels (B) were determined. After treatment mice received an injection with [3 H]TO and [14 C]CO double-labeled VLDL-mimicking particles. Blood was drawn at the indicated time points, and 3 H and 14 C plasma decay (C, E) and tissue-speciﬁ c activity (D, F) were determined. The inlays in C and E show plasma half-life. Data are presented as means ± SD (n = 8/9 per group). * P < 0.05 when compared with control group.

This discrepancy of anacetrapib affecting plasma PCSK9 21 weeks of anacetrapib treatment, suggesting that anace- levels but not hepatic LDLr protein levels might be due to trapib does not affect hepatic LRP1 level. Although the the duration of the treatment (i.e., 4 weeks vs. 21 weeks of effects of anacetrapib in E3L.CETP mice closely resemble treatment). Also, in C57BL/6 mice receiving anacetrapib the effects in humans ( 12, 22 ), the effects of anacetrapib for 1 week, anacetrapib reduced plasma PCSK9 without on plasma PCSK9 and hepatic LDLr protein levels in hu- affecting hepatic LDLr protein levels (44 ), suggesting mans remains to be determined. a time-dependent treatment effect of anacetrapib on The comparison of the clearance rates of VLDL rem- PCSK9 and hepatic LDLr protein. The mRNA expression of nants in E3L mice with and without CETP ( Figs. 3C and Ldlr , despite being a SREBP-2 target gene (47 ), was not 4C ) indicates that the presence of CETP results in a de- downregulated after anacetrapib treatment. Apparently, creased remnant particle clearance. This implies that in- SREBP-2 is not dominant for regulation of Ldlr mRNA ex- hibiting the activity of CETP alone is sufﬁ cient to increase pression under these conditions. In line with this conten- lipoprotein remnant clearance in E3L.CETP mice. The tion, it is known that mouse liver cholesterol homeostasis main activity of CETP is to transfer cholesteryl esters from is mainly dependent on endogenous cholesterol biosyn- HDL to (V)LDL in exchange for TG. Apparently, this lipo- thesis and to a lesser extent to on LDLr-mediated uptake protein remodeling activity of CETP renders the (V)LDL of cholesterol ( 48 ). Because PCSK9 has been reported to less susceptible to clearance. In E3L.CETP mice, anacetra- modulate LRP1 expression ( 41 ), a backup receptor for pib treatment results in an increase of plasma apoE levels (V)LDL clearance, we also measured LRP1 expression af- (+59%, P < 0.001; data not shown), which is indicative for ter anacetrapib treatment. No effect on either mRNA or lipoprotein remodeling. Previously, we have shown the protein expression of LRP1 was observed after 4 weeks or importance of a functional apoE-LDLr pathway in the

Anacetrapib increases VLDL remnant clearance 2091 Supplemental Material can be found at: http://www.jlr.org/content/suppl/2015/09/04/jlr.M057794.DC1 .html clearance of remnants from plasma and, subsequently, in 3 . Di Angelantonio, E. , N. Sarwar , P. Perry , S. Kaptoge, K. K. Ray , A. the development of atherosclerosis ( 23, 42 ). Interestingly, Thompson, A. M. Wood , S. Lewington , N. Sattar , C. J. Packard , et al. 2009 . Major lipids, apolipoproteins, and risk of vascular disease. anacetrapib treatment in humans has also been shown to J. Am. Med. Assoc. 302 : 1993 – 2000 . increase plasma apoE levels (12 ). 4 . Tall , A. R. 1993 . Plasma cholesteryl ester transfer protein. J. Lipid Although the direct effects of CETP inhibition in hu- Res. 34 : 1255 – 1274 . 5 . Marotti , K. R., C. K. Castle , T. P. Boyle, A. H. Lin , R. W. Murray , mans on lipoprotein remnant clearance have not been de- and G. W. Melchior . 1993 . Severe atherosclerosis in transgenic scribed, treatment with the CETP-inhibitor torcetrapib mice expressing simian cholesteryl ester transfer protein. Nature . has been shown to increase the fractional catabolic rate of 364 : 73 – 75 . 6 . Plump , A. S. , L. Masucci-Magoulas , C. Bruce , C. L. Bisgaier , J. L. both VLDL-apoE ( 49 ) and VLDL-apoB100 (50 ). However, Breslow , and A. R. Tall . 1999 . Increased atherosclerosis in ApoE the potentially increased catabolism of (V)LDL and thus and LDL receptor gene knock-out mice as a result of human cho- antiatherogenic properties were clearly not sufﬁ cient to lesteryl ester transfer protein transgene expression. Arterioscler. Thromb. Vasc. Biol. 19 : 1105 – 1110 . offset or overrule the adverse side effects of torcetrapib. 7 . Westerterp , M. , C. C. van der Hoogt, W. de Haan, E. H. Offerman , Microarray analyses also revealed that anacetrapib acti- G. M. Dallinga-Thie , J. W. Jukema , L. M. Havekes , and P. C. Rensen . vates genes regulated by NR1I2 and NR1I3. These transcrip- 2006 . Cholesteryl ester transfer protein decreases high-density lipo- tion factors function as sensors of both toxic and xenobiotic protein and severely aggravates atherosclerosis in APOE*3-Leiden mice. Arterioscler. Thromb. Vasc. Biol. 26 : 2552 – 2559 . exogenous compounds and toxic products derived from 8 . de Grooth, G. J., K. E. Zerba , S. P. Huang , Z. Tsuchihashi, T. endogenous metabolism and activate pathways to eliminate Kirchgessner, R. Belder , P. Vishnupad , B. Hu , A. H. Klerkx , A. these products (51 ). In our study, activation of these recep- H. Zwinderman, et al. 2004 . The cholesteryl ester transfer pro-

tein (CETP) TaqIB polymorphism in the cholesterol and recur- Downloaded from tors indicated that anacetrapib was recognized as a xenobi- rent events study: no interaction with the response to pravastatin otic product by the liver. It is not yet known whether this has therapy and no effects on cardiovascular outcome: a prospective consequences beyond activation of elimination pathways analysis of the CETP TaqIB polymorphism on cardiovascular outcome and interaction with cholesterol-lowering therapy. J. Am. Coll. and whether this will have clinical implications. Phase I/II Cardiol. 43 : 854 – 857 . clinical studies evaluating the effects of anacetrapib re- 9 . Ridker , P. M., G. Pare , A. N. Parker , R. Y. Zee , J. P. Miletich , and D. I. Chasman. 2009 . Polymorphism in the CETP gene region, HDL ported an acceptable side-effect profi le (12, 52–54). How- www.jlr.org ever, it should be noted that a nonsignifi cant increase in cholesterol, and risk of future myocardial infarction: genomewide analysis among 18 245 initially healthy women from the Women’s C-reactive protein was found after anacetrapib treatment Genome Health Study. Circ. Cardiovasc. Genet. 2 : 26 – 33 . (52 ). We also found elevated serum amyloid A levels in 10 . Karalis , I. , P. C. Rensen , and J. W. Jukema . 2013 . Journey through anacetrapib-treated E3L.CETP mice (22 ). cholesteryl ester transfer protein inhibition: from bench to bed- at Walaeus Library / BIN 299, on November 9, 2015 side. Circ. Cardiovasc. Qual. Outcomes. 6 : 360 – 366 . In the present study, we present evidence that anacetra- 11 . Barter , P. J., M. Caulfi eld , M. Eriksson, S. M. Grundy, J. J. Kastelein, pib reduces (V)LDL-C by two mechanisms: 1 ) inhibition of M. Komajda , J. Lopez-Sendon , L. Mosca , J. C. Tardif , D. D. 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Anacetrapib increases VLDL remnant clearance 2093