increased hdl cholesterol and apoa-i in humans and...

Increased HDL Cholesterol and ApoA-I in Humans andMice Treated With a Novel SR-BI Inhibitor

David Masson, Masahiro Koseki, Minako Ishibashi, Christopher J. Larson, Stephen G. Miller,Bernard D. King, Alan R. Tall

Objectives—Increasing HDL levels is a potential strategy for the treatment of atherosclerosis.Methods and Results—ITX5061, a molecule initially characterized as a p38 MAPK inhibitor, increased HDL-C levels by

20% in a human population of hypertriglyceridemic subjects with low HDL levels. ITX5061 also moderately increasedapoA-I but did not affect VLDL/LDL cholesterol or plasma triglyceride concentrations. ITX5061 increased HDL-C inWT and human apoA-I transgenic mice, and kinetic experiments showed that ITX5061 decreased the fractionalcatabolic rate of HDL-CE and reduced its hepatic uptake. In transfected cells, ITX5061 inhibited SR-BI–dependentuptake of HDL-CE. Moreover, ITX5061 failed to increase HDL-C levels in SR-BI�/� mice. To assess effects onatherosclerosis, ITX5061 was given to atherogenic diet–fed Ldlr�/� mice with or without CETP expression for 18weeks. In both the control and CETP-expressing groups, ITX5061-treated mice displayed reductions of earlyatherosclerotic lesions in the aortic arch �40%, P�0.05), and a nonsignificant trend to reduced lesion area in theproximal aorta.

Conclusions—Our data indicate that ITX5061 increases HDL-C levels by inhibition of SR-BI activity. This suggests thatpharmacological inhibition of SR-BI has the potential to raise HDL-C and apoA-I levels without adverse effects onVLDL/LDL cholesterol levels in humans. (Arterioscler Thromb Vasc Biol. 2009;29:2054-2060.)

Key Words: scavenger receptor B-I � high-density lipoproteins � inhibitors � p38 MAPK � atherosclerosis

Plasma high-density lipoprotein (HDL) levels are in-versely correlated with atherosclerotic cardiovascular

disease, and raising HDL levels by lifestyle changes orpharmacological interventions is an emerging strategy thatmight help to reduce the residual burden of disease in patientstreated with low-density lipoprotein (LDL)–lowering ap-proaches.1,2 Whereas increasing synthesis or infusion ofapolipoprotein A-I (apoA-I) or HDL reduces atherosclerosisin animals and humans, it is not clear that all approaches toraising HDL will reduce atherosclerosis.

The scavenger receptor B-I (SR-BI) is a major factorregulating HDL catabolism. SR-BI binds HDL and mediatesthe selective uptake of HDL-cholesteryl ester (CE) in theliver and steroidogenic tissues.3 Although the role of SR-BI inHDL metabolism in mice and rats has been clearly shown,much less information is available on the function of SR-BIin humans. Primary human hepatocytes do demonstrateselective uptake of HDL-CE,4 but the quantitative importanceof this pathway has not been shown. In humans, cholesterylester transfer protein (CETP), a factor lacking in mice andrats, plays a major role in HDL-CE catabolism. In the presentstudy, we demonstrate that ITX5061, a molecule initiallycharacterized as a p38 mitogen-activated protein kinase

(MAPK) inhibitor, caused an increase in HDL cholesterol(HDL-C) and apoA-I levels in humans. Studies in mice andcultured cells indicated that ITX5061 is an inhibitor of theSR-BI activity. Interestingly, ITX5061 did not cause in-creases in very-low-density lipoprotein (VLDL) and LDLcholesterol and reduced atherosclerosis in the aortic arch inLDL receptor (Ldlr) heterozygous mice fed an atherogenicdiet.

Materials and MethodsA complete Materials and Methods section is available in the supple-mental materials (available online at http://atvb.ahajournals.org).

MoleculeStructure of ITX5061 is shown in supplemental Figure I. Theempirical formula of ITX5061 is C30H38ClN3O7S. The molecularweight of ITX5061 (as an HCl salt) is 620.2.

Human DataKC706-C06 study was conducted from November 6, 2006 to May 8,2007. It was a multicenter, randomized, double-blind, placebo-controlled study. Its primary objective was to evaluate the ability ofITX5061 to increase HDL-C in patients with low HDL-C andelevated triglycerides (TG) at baseline.

Received May 11, 2009; revision accepted September 28, 2009.From the Division of Molecular Medicine, Department of Medicine (D.M., M.K., M.I., A.R.T.), Columbia University, New York; Exelixis Inc (C.J.L.),

San Diego, Calif; Array Biopharma Inc (S.G.M.), Boulder, Colo; iTherX Inc (B.D.K.), San Diego, Calif.Correspondence to David Masson, 630 West 168th Street, P&S 8-401, New York NY 10032. E-mail [email protected]© 2009 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.109.191320

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AnimalsC57Bl/6 wild-type (WT) mice, C57Bl/6 SR-BI– deficient mice(SR-BI�/�),5 and C57Bl/6 transgenic mice expressing humanapoA-I under the control of its natural flanking regions(HuAITg)6 were used in the present study. Atherosclerosis studieswere conducted in F1 hybrid C57BL/6�DBA/1 Ldlr�/� mice fedthe Paigen diet, which is high-fat/cholesterol/bile salt diet con-taining 1.25% cholesterol, 7.5% cocoa butter, and 0.5% cholic acid(TD 88051; Harlan Teklad). Animal protocols were approved bythe Institutional Animal Care and Use Committee ofColumbia University.

ResultsITX5061 Increases HDL Levels in HumansITX5061 was initially characterized as a type II (non com-petitive) inhibitor of p38 MAPK (supplemental Figure II) andwas noted to cause increases in HDL-C levels in humans. Tosystematically investigate effects on plasma lipoprotein lev-els, ITX5061 was administered at 2 different doses in apopulation of subjects with low HDL-C (men �45 mg/dLand women �55 mg/dL) and increased TG concentrations(TG ranging from 150 to 400 mg/dL). Baseline characteristicsare given in supplemental Table I. Subjects received eitherplacebo or ITX5061 at 150 mg or 300 mg daily. ITX5061treatment resulted in an increase in HDL-C concentration thatwas similar in the 2 ITX5061-treated groups (�20% in-creases). The rise in HDL-C was observed throughout thetreatment period and was reversed after cessation of the drug(Figure 1). The mean HDL particle size was significantlyincreased in ITX5061-treated subjects (P�0.05; Figure 2A).No significant changes in other lipid parameters (LDL-C andTG) were observed, although a transient nonsignificant in-crease in TG was observed after 2 weeks of treatment(supplemental Figure III). ApoA-I protein levels were mod-

erately increased in the 150 mg group (�10%, P�0.05) butwere not statistically different in the 300 mg group (Figure2B). Adverse events were similar in all groups except for onepatient in the 150 mg group and 4 patients in the 300 mggroup who had reversible increases in transaminase levels.These findings indicate a selective increase in HDL-C andapoA-I in subjects treated with the 150 mg dose of ITX5061.Further increases in these parameters were not seen at the 300mg dose.

ITX5061 Increases HDL-C and Size inHuAITg MiceTo investigate the mechanism of HDL-C raising byITX5061, further studies were conducted in mice. HuAITgmice were treated with ITX5061 (30 mg/kg/d) or vehiclefor 1 week. This resulted in a 50% increase in HDL-Clevels compared to baseline, but no change in non-HDL-Clevels (Figure 3A). As determined by scanning SDS-PAGEgels, apoA-I levels were moderately (�15%) but signifi-cantly increased in ITX5061-treated HuAITg mice, com-pared to mice received vehicle (Figure 3B). Consistentwith the more prominent increase in HDL-C than apoA-I,ITX5061 treatment induced a shift toward larger sizedHDL as shown by native-PAGE (Figure 3C). To assesswhether ITX5061 could affect expression of genes in-volved in HDL biosynthesis or catabolism in the liver,relative mRNA levels of apoA-I, ABCA1. and SR-BI were

Figure 1. Changes in lipid parameters over time during ITX5061administration. Patients received placebo (n�37), ITX5061 at150 mg (n�38), and ITX5061 at 300 mg (n�40) daily. Lipid pa-rameters were determined at indicated time points. A, Absolutechange in HDL-C concentration. B, Mean percent changes inHDL-C from baseline. Values are mean�SD. **P�0.05, probabil-ity values are against placebo, same time point, Kruskall–Wallis,Mann–Whitney test.

Figure 2. A, HDL size distribution in ITX5061-treated patients.HDL size was determined after 6 weeks of treatment usingNMR. Probability value based on Mann–Whitney test. B, Effectof ITX5061 treatment on apoA-I concentration. ApoA-I concen-tration was determined at the indicated time point. Probabilityvalue based on Mann–Whitney test.

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determined in ITX5061 and vehicle-treated animals. Nochanges in mRNA levels were found for any of thesetranscripts (supplemental Figure IV), suggesting thatITX5061 raised HDL independent of these transcriptlevels.

ITX5061 Decreases HDL-CE Catabolism andHepatic UptakeTo gain more insight into the mechanisms by whichITX5061 increases HDL-C, a kinetic study was performed.HDL labeled with [3H] CE was injected via the tail veininto HuAITg mice pretreated or not with ITX5061. Asshown in Figure 3D, ITX5061 significantly decreasedHDL-CE catabolism with an FCR of 1.86�0.40 pools/dversus 2.47�0.26 pools/d in the control group (P�0.05),whereas calculated production rates were identical in both

groups (129�24 �g/g/d versus 129�16 �g/g/d). More-over, accumulation of [3H] CE in the liver was signifi-cantly lower in ITX5061-treated mice, indicating thatincreased HDL-CE levels were attributable to reduceduptake by the liver (Figure 3E).

ITX5061 Does Not Increase HDL Levels inSR-BI�/� Mice and Reduces Selective Uptake viaSR-BI in Cell CultureBecause SR-BI has a major role in the clearance of HDL-Cand CE in the liver, we determined whether ITX5061 couldstill raise HDL levels in SR-BI�/� mice. WT and SR-BI�/�

mice were treated for 1 week by daily administration ofITX5061 at 30 mg/kg/d. Although ITX5061 increasedHDL-C in WT mice its effects were abolished in SR-BI�/�

mice, suggesting that the mechanism of action was depen-dent on SR-BI (Figure 4B). A significant increase innon-HDL cholesterol levels was also observed inITX5061-treated WT mice. Analysis of apolipoproteins bySDS-PAGE in fractions corresponding to the LDL regionof similar profiles indicated they consisted primarily ofapoB lipoproteins with only trace amounts of apoA-1 (datanot shown). Interestingly, despite increases in HDL-C, no

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Figure 3. A, Plasma lipid levels and cholesterol distribution inHuAITg mice with or without ITX5061. HuAITg mice weretreated for 1 week with ITX5061 or vehicle at 30 mg/kg/d.100 �L of pooled plasma from 5 distinct mouse were usedfor FPLC analysis. Plasma HDL-C was determined after ultra-centrifugation. * indicates a significant difference from micereceiving vehicle. (P�0.05 in both cases; Mann–Whitney test).B, ApoAI concentrations in HuAITg mice with or withoutITX5061. * indicates a significant difference from mice receiv-ing vehicle. (P�0.05; Mann–Whitney test). C, HDL size distri-bution in HuAITg mice with or without ITX5061. HDL size wasdetermined by native-PAGE. Profiles were determined in indi-vidual plasma samples. One presentative profile in eachgroup is shown. D, Plasma kinetic of HDL labeled with [3H]CEin HuAITg mice with or without ITX5061. Values are the frac-tion of the injected dose remaining at each time point. Thecurves were fitted using a biexponential equation. Data aregiven as mean�SD; n�4 mice per group for each point.*P�0.05 vs vehicle, same time point, Mann–Whitney test. E,HDL-C uptake. Values are expressed as the percentage oftotal injected radioactivity retrieved in the liver. *P�0.05 vsvehicle, Mann–Whitney test.

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Figure 4. Plasma lipids, apoA-I concentrations, and cholesteroldistribution in WT and SR-BI�/� mice with or without ITX5061.WT (A) and SR-BI�/� mice (B) were treated for 1 week withITX5061 or vehicle at 30 mg/kg/d. 100 �L of a pooled plasmafrom 4 distinct mouse were used for FPLC analysis. * indicatesa significant difference from mice before treatment (P�0.05 inboth cases; Wilcoxon test). C, Effect of ITX5061 on SR-BI–medi-ated cholesterol uptake. HEK 293 cells were transfected withSR-BI cDNA or a mock vector, and HDL-C uptake was deter-mined as describe in Materials and Methods (*P�0.05 vs0 �mol/L; Mann–Whitney test). Values obtained at 4°C weresubtracted.

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increase in apoA-I was observed in ITX5061-treated WTmice (Figure 4A). We also confirmed by Western blot thatITX5061 did not affect the protein levels of apoA-I, ABCA1,and SR-BI in the liver (supplemental Figure IVB).

To test the SR-BI inhibitory potential of ITX5061 in vitro,HEK 293 cells overexpressing SR-BI were incubated with[3H]CE-labeled HDL. As shown in Figure 4C, ITX5061significantly decreased HDL uptake at 1 �mol/L concentra-tion, whereas a structurally related p38 MAPK inhibitor(KR-004515) had no effect at the same concentration.

ITX5061 Increases HDL-C and ReducesAtherosclerosis in Ldlr�/� Mice Fed thePaigen DietMost prior studies of the effects of SR-BI deficiency orinhibition on atherogenesis have used Ldlr�/� or apoE�/�

mice.7–9 We wished to determine effects of ITX5061 onatherogenesis in a setting where the clearance of VLDL/LDL particles by apoE- or Ldlr-dependent pathways is notcompletely impaired and with the potential availability ofa compensatory pathway of reverse cholesterol transportvia CETP. Indeed, it has been shown recently that CETPexpression was able to reverse the atherogenic phenotypein SR-BI�/� mice.10 Thus, to determine the effects ofITX5061 on atherogenesis, Ldlr�/� mice, with or withoutexpression of CETP, were fed the Paigen diet containingITX5061 (0.037%) for 18 weeks. CETP expression wasinduced by injection of AAV-CETP11 and was monitored 2weeks later. Plasma CETP levels were slightly lower thanobtained in a human plasma sample in mice injected withAAV-CETP but within the physiological range for humans(supplemental Figure V). Plasma lipid parameters weremonitored after 2 and 18 weeks of treatment. No differ-ences in total cholesterol or TG levels were observedbetween ITX5061-treated or control mice. However, a30% increase in HDL-C concentrations was observed inITX5061-treated groups as determined by FPLC (Figure5A) and ultracentrifugation (supplemental Table II). Athuman-like levels, CETP expression has only little effectson plasma lipid levels in WT mice as originally reported.12

As expected, animals fed the Paigen diet developed mod-erately elevated transaminase levels,13 but there was nohyperbilirubinemia and there were no differences betweenthe 4 groups (supplemental Table II).

After 18 weeks of ITX5061 administration, atheroscleroticlesion areas were first quantified by en face analysis of aorticarches. Data are reported as the percentage of the atherosc-lerotic lesions stained by Oil Red O on aortic surface. Figure5B shows representative pictures of aortic arches fromfemale mice with or without treatment by ITX5061.Lesions were observed primarily at branch points andsometimes along the lesser curvature. A significant 40%reduction of atherosclerotic lesions was observed inITX5061-treated mice. Similar reductions in atherosclerosiswere seen in ITX5061-treated mice injected with AAV-CETPor AAV-Ctrl (Figure 5C). No difference in lesion area wasobserved when comparing AAV-Ctrl groups and AAV-CETPgroups either in control or ITX-5061 treated mice. Analysisof the lesions was also performed in the aortic valves (Figure

5D). Although there was a trend toward reduced lesion area inboth groups treated with ITX5061, the differences were notstatistically significant.

DiscussionOur findings indicate that ITX5061, initially characterizedas a p38 MAPK inhibitor, is able to increase HDL-C levelsin humans and rodents and strongly suggest that this occursat least in part through inhibition of SR-BI activity.Importantly, whereas previous studies have shown thatmodulation of SR-BI activity could affect apoB-containinglipoprotein levels, ITX5061 did not increase VLDL/LDLcholesterol or plasma TG concentrations in humans andLdlr�/� mice. Finally, although many studies have docu-mented markedly increased atherosclerosis in variousmouse models deficient in SR-BI, we observed thatITX5061 had no significant effect on atherosclerotic le-sions in the aortic valves and reduced early atheroscleroticlesions in the aortic arch of Ldlr�/� mice fed the Paigendiet for 18 weeks.

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Figure 5. A, FPLC analysis of plasma cholesterol in maleLdlr�/� mice with or without ITX5061. 100 �L of pooled plasmawere used for FPLC analysis. B, Representative oil red O–stainedaortas Ldlr�/� mice were fed the Paigen diet containing or notITX5061 for 18 weeks. Aortas were stained with oil red O. C,Quantification of atherosclerosis lesions in Ldlr�/� mice. Oil redO-stained lesions were quantified and expressed as a percent oftotal aortic area. Horizontal bars indicate the group medians. Sta-tistical analysis by Kruskall–Wallis Mann–Whitney test. D, Athero-sclerotic lesion development in the proximal aorta. Quantification ofproximal aortic root lesion area was performed by morphometricanalysis of H&E-stained sections. Values represent the average of5 sections per mouse. Horizontal bars indicate the group mediansStatistical analysis by Kruskall–Wallis Mann–Whitney test.




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Several lines of evidence suggest that the primary modeof action of ITX5061 is via inhibition of SR-BI activity.First, similar to what is observed in SR-BI�/� mice,ITX5061 increased HDL-C more than apoA-I and led toformation of large sized HDL enriched in cholesterol.Second, ITX5061 raised HDL-C by decreasing HDL-CEplasma catabolism and uptake by liver. Third, ITX5061effects on HDL-C were abolished in SR-BI�/� mice.Fourth, in vitro, ITX5061 inhibited SR-BI–mediatedHDL-CE uptake whereas other structurally related com-pounds without HDL raising ability did not. AlthoughITX5061 was initially developed as a p38 MAPK inhibitor,it seems unlikely that this property contributed to increasedHDL because structurally similar p38 MAPK inhibitorsdid not increase HDL levels. Nevertheless, the exactmechanism of SR-BI inhibition by ITX5061 remains to bedetermined. Other SR-BI inhibitors described to datedecreased HDL-CE selective uptake and increased thebinding of HDL to SR-BI suggesting the formation ofinactive HDL-SR-BI complexes.14,15

The effects of SR-BI deficiency/inhibition on lipopro-tein profiles are well documented in mice.5,16,17 SR-BI�/�

mice display a marked increase in HDL-C concentrationattributable to accumulation of very large size apoE- andcholesterol- and CE- enriched HDL. In the absence ofapoE or Ldlr, SR-BI deficiency also increases VLDL/LDLconcentration, indicating a role for SR-BI as a back upreceptor for these lipoproteins.16,18 –20 In contrast to mice,much less is known about the effects of SR-BI deficiencyor inhibition on lipoprotein profiles in humans. Severalnonfunctional SR-BI polymorphisms have been associatedwith changes in HDL and LDL cholesterol levels, perhapssuggesting a role of SR-BI in humans.21–24 No geneticdeficiencies for SR-BI have been described to date.25,26

Only one preliminary report on administration of SR-BIinhibitory molecules in humans has been presented.HDL376, a molecule with SR-BI inhibitory properties wasable to increase HDL-C levels in humans (�20%) and alsoin nonhuman primates.27,28 Our results suggest that the useof SR-BI inhibitors in humans has the potential to increaseHDL-C and to a lesser extent apoA-I without detrimentaleffects on apoB-containing lipoprotein concentration,leading to a potentially less atherogenic lipoprotein profile.Interestingly, ITX5061 increased apoA-I as well as HDL-Cin humans and in HuAITg mice, but in wild-type mice onlyincreased HDL-C levels. This suggests that there could bea difference in effects of SR-BI deficiency on HDLparticles containing human apoA-I versus mouse apoA-I.Nevertheless, the increase of apo A-I was only moderate ascompared to HDL-C, consistent with the SR-BI inhibitionmechanism. The lack of increase VLDL and LDL choles-terol in humans treated with ITX5061 likely reflects theeffect of partial SR-BI deficiency in a setting where apoEand Ldlr are still functional.

After the discovery of dramatically increased HDLlevels in SR-BI�/� mice,5 there was considerable initialinterest in the potential to use inhibition of SR-BI as anantiatherogenic strategy. However, this idea was discardedin the face of compelling evidence that SR-BI deficiency

increased atherosclerosis in several different mouse mod-els, sometimes in a dramatic fashion, while SR-BI over-expression had the opposite effects.7–9,16,29 –32 Besides ge-netic models of SR-BI overexpression or deficiency,pharmacological SR-BI inhibitors such as BLTs, HDL376,and R-138329 have been characterized.14,15,27,33,34 Theeffects of the latter compound on atherosclerosis have beeninvestigated in apoE�/� mice, and moderate proatherogeniceffects were observed but only at high concentration.35 Theincrease in atherosclerosis seen in SR-BI�/� mice has beenattributed to the decrease in reverse cholesterol transportthat has been clearly demonstrated in these mice.36 In favorof this hypothesis, CETP expression was able to reversethe atherogenic phenotype in SR-BI�/� mice.10 However,in many of these studies7,8,16 changes in HDL levels wereassociated with parallel changes in levels of VLDL andLDL, tending to confound the analysis of the mechanismsof atherogenesis. We did not find a proatherogenic effectof CETP on atherosclerosis in our model, either with orwithout ITX5061 treatment. This is possibly because of thelow level of CETP expression at which we chose to work.CETP was previously shown to be moderately proathero-genic in Ldlr�/� that displayed much higher plasma CETPlevels.37

In the present study we observed that ITX5061 treatmentresulted in a 40% reduction in atherosclerosis in the aorticarch after 18 weeks of treatment in Ldlr�/� mice fed thePaigen diet. The antiatherogenic effect of the molecule wasobserved in the control and in the CETP expressing group.Our results suggest therefore that partial SR-BI inhibitioncould have some beneficial effects per se whether an alter-native pathway for HDL cholesterol uptake or transfer isprovided by CETP or not.

Several points might account for differences betweenour results and those from previous studies. First, we useda model of Ldlr�/� mice where the clearance of apoBlipoproteins is not completely impaired, leading only tomoderate hypercholesterolemia. Secondly, SR-BI inhibi-tion was not complete in our experimental conditions, onlya 30% to 40% increase in HDL levels was observed ascompared with typical �125% increases in SR-BI�/�

animals. Because it has been shown that SR-BI deficiencyhas different effects in liver and in vascular wall, selectiveinhibition of SR-BI by ITX5061 in different tissues couldalso explain some of the differences.38 Third, whereasmost studies used Western-type diet, we used a cholicacid– containing diet, which have different effects onatherogenesis. Although this diet has been extensivelyused in atherosclerosis studies,39 it is also recognized tohave significant limitations. Indeed, cholate that is presentin the Paigen diet induces liver inflammation and fibrosisand these effects contribute to the peculiar atherogenicityof the diet.40 These concerns limit the usefulness of thismodel in extrapolating results to other settings such as inhumans. Nonetheless, in the same dietary model markedoverexpression of SR-BI was antiatherogenic in conjunc-tion with both HDL and VLDL/LDL lowering. Fourth,because ITX5061 is also a potent p38 MAPK inhibitor, itis possible that this off-target effect contributes, at least




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partially, to the reduction of atherosclerotic lesions inITX5061-treated mice. However, recent data do not sup-port a role for p38 MAPK in regulating atheroscleroticlesion size. A p38 MAPK inhibitor decreased inflamma-tion in atherosclerotic plaques but did not affect lesion sizein apoE�/� mice infused with angiotensin II.41 Similarly,p38 MAPK deficiency in macrophages increased necroticcores in advanced lesions in Western-type diet–fedapoE�/� mice but did not change lesion size.42 Therefore,although it seems unlikely that p38 MAPK inhibitioncontributed significantly to the reduction of atherosclerosisin our model, we cannot rule this out. In conclusion,inhibition of hepatic SR-BI in a setting that leads toincreases in HDL without concomitant increases in VLDLand LDL cholesterol levels could potentially have anantiatherogenic effect.

AcknowledgmentsWe thank Carrie Welch and Rong Li for determination of the lesionsin aortic roots.

Sources of FundingD.M. was supported by a grant form the Philippe foundation.

DisclosuresC.J.L., S.G.M., and B.D.K. were formerly employed by Kemia Inc.

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atherosclerotic cardiovascular disease. Nat Rev Drug Discov. 2005;4:193–205.

3. Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M.Identification of scavenger receptor SR-BI as a high density lipoproteinreceptor. Science. 1996;271:518–520.

4. Rinninger F, Brundert M, Jackle S, Galle PR, Busch C, Izbicki JR,Rogiers X, Henne-Bruns D, Kremer B, Broelsch CE, Greten H. Selectiveuptake of high-density lipoprotein-associated cholesteryl esters by humanhepatocytes in primary culture. Hepatology. 1994;19:1100–1114.

5. Rigotti A, Trigatti BL, Penman M, Rayburn H, Herz J, Krieger M. Atargeted mutation in the murine gene encoding the high densitylipoprotein (HDL) receptor scavenger receptor class B type I reveals itskey role in HDL metabolism. Proc Natl Acad Sci U S A. 1997;94:12610–12615.

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Bernard D. King and Alan R. TallDavid Masson, Masahiro Koseki, Minako Ishibashi, Christopher J. Larson, Stephen G. Miller,

InhibitorIncreased HDL Cholesterol and ApoA-I in Humans and Mice Treated With a Novel SR-BI

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Supplementary files

Increased HDL cholesterol and apo A-I in humans and mice treated with a novel SR-BI

inhibitor

Masson et al.

Human data

KC706-C06 study was conducted from 6 November 2006 to 8 May 2007. It was a

multicenter, randomized, double-blind, placebo-controlled study. Its primary objective was to

evaluate the ability of ITX5061 to increase HDL-C in patients with low HDL-C and elevated

triglycerides (TG) at Baseline. The study was conducted under a United States (US)

Investigational New Drug Application and in compliance with US Food and Drug

Administration (FDA) Code of Federal Regulations. All principles of the Declaration of

Helsinki (1964) were followed. Patients were randomized in a 1:1:1 ratio to placebo, 150 mg

ITX5061, or 300 mg ITX5061 for oral administration once daily for 6 weeks. A fasting lipid

panel (total cholesterol, HDL-C, LDL-C, and TG), and general laboratory parameters were

assessed at the Baseline Visit, every 2 weeks during dosing, and 4 weeks after the end of

dosing (Follow-up Visit). The main inclusion criteria were 18 to 70 years of age, inclusive;

decreased HDL-C (≤ 45 mg/dL in men or ≤ 55 mg/dL in women); elevated TG (150 to

400 mg/dL); LDL-C ≤ 250 mg/dL; on a stable dose of all medications that might have altered

lipid levels or for diabetes management for at least 1 month before screening. Treatment-

emergent adverse effects (TEAEs) were reported for 56.8%, 65.8%, and 67.5% of patients in

the placebo, 150-mg, and 300-mg groups, respectively. Only one patient (300 mg)

experienced severe adverse effects (unstable angina, atrial fibrillation); these effects resolved

while continuing on study medication and were considered to be unrelated to the study drug.

All other TEAEs were reported as mild or moderate. Increased indirect bilirubin was reported

in 7.5% of patients in the 300-mg group; this effect is related to inhibition of the UGT1A1

enzyme responsible for bilirubin glucuronidation. Total cholesterol, LDL-C, HDL-C and TG

were assayed via reagents from Roche Diagnostics and run on a Roche Modular PPP

Analyzer. ApoA-I was assayed via an immunoturbidimetric assay using reagent from Roche

Diagnostics run on a Roche Modular PPP Analyzer. HDL particle size distribution was

determined using NMR at Liposcience (Raleigh, NC).

Animals: C57Bl/6 Wild Type (WT) mice, C57Bl/6 SR-BI deficient mice (SR-BI-/-) 1, and

C57Bl/6 transgenic mice expressing human apoA-I under the control of its natural flanking

regions (HuAITg) 2 were used in the present study. Animal protocols were approved by the

Institutional Animal Care and Use Committee of Columbia University. Animals had free

access to both food and water and were fed regular chow diet. ITX5061 was prepared as a 6

mg/ml suspension in water containing Methyl cellulose 0.5% and Polysorbate 0.1%, and

given daily by oral gavage at 30mg/ body weight kg.

Atherosclerosis studies were conducted in F1 hybrid C57BL/6 × DBA/1 Ldlr+/- mice fed the

Paigen diet, which is high-fat/cholesterol/bile salt diet containing 1.25% cholesterol, 7.5%

cocoa butter, and 0.5% cholic acid (TD 88051; Harlan Teklad, Madison, WI). This model has

been described previously 3, and it has been shown that moderate increases in VLDL/LDL

levels and early atherosclerotic lesions can be readily induced in Ldlr+/- mice fed with this diet 4. For 18 weeks, the mice were put on the Paigen diet with or without ITX5061 at 0.037 %.

CETP expression was induced by injection of adeno-associated virus (AAV)8 TBG human

CETP vector (AAV-CETP, 3x1010 viral particles) two weeks before starting the Paigen diet as

described previously 5. AAV was a kind gift of Dr. Daniel J. Rader (University of

Pennsylvania School of Medicine). Doses of virus were given by IP injection.

Quantification of atherosclerosis lesions: To quantify the extent of the atherosclerotic

lesions, the aortic arch and the thoracic aorta was opened longitudinally, stained with Oil Red

O, and pinned out on a black wax surface. The amount of Oil Red O staining was measured

using color thresholding to delimit the area of staining with Photoshop® software and the

percentage of the plaque area stained by Oil Red O to the total surface area was determined 6

Proximal aortas were serially paraffin sectioned and stained with Haematoxylin and eosin as

previously described3. Aortic lesion size of each animal was calculated as the mean of lesion

areas in 5 sections from the same mouse.

Plasma and liver tissue sampling: On the day of sample collection, the mice were fasted for

4 h. No differences in body weight were observed between treated and untreated mice over

the period studied. Animals were euthanized with isofurane, and blood samples were

collected by intracardiac puncture in heparin-containing tubes that were centrifuged at 5,000

rpm for 10 min. Plasma was harvested and stored at –80°C. Livers were excised and

immediately snap-frozen in liquid nitrogen, and stored at –80°C before mRNA isolation.

Plasma Cholesterol, Triglyceride, and Lipoprotein Measurements: Total plasma

cholesterol and TG were determined enzymatically using kits from Wako and Thermo

scientific respectively. Pooled plasma from fasted mice was used for fast protein liquid

chromatography (FPLC) analysis using one Superose 6 column with a flow rate of 0.3

ml/min. FPLC buffer contains 0.1 mol/L Tris-HCL and 0.4% NaN3 and pH was adjusted at

7.4. HDL-C and non-HDL-C were assayed in d > 1.07 and d < 1.07 g/ml plasma fractions,

respectively.

Bilirubin and transaminase determinations:

Total bilirubin and transaminase were determined by enzymatic methods on a Dimension

Vista analyser (Siemens, Marburg, Germany). Reference values are those observed in the

same mouse line under standard diet 7

Native polyacrylamide gradient gel electrophoresis (native-PAGE): Total lipoproteins

were separated by ultracentrifugation as the d < 1.21 g/ml plasma fraction with one 5.5 h,

100,000 rpm spin in a TLA100 rotor in a TLX ultracentrifuge (Beckman Instruments; Palo

Alto, CA). Lipoproteins were then applied to a 50–200 g/l polyacrylamide gradient gel and

electrophoresis was conducted as recommended by the manufacturer. Gels were subsequently

subjected to Coomassie brilliant blue staining. The mean apparent diameters of HDL were

determined by comparison with globular protein standards subjected to electrophoresis

together with the samples.

Determination of apoA-I concentration: Relative levels of apoA-I were determined as

described previously 8 by a nonimmunologic method. Briefly, plasma samples (0.5 µl) were

subjected to electrophoresis on 50–200 g/l polyacrylamide gradient gel as recommended by

the manufacturer. Gels were subsequently stained by Coomassie brilliant blue and scanned on

a GS-800 densitometer (Biorad, Hercules, CA).

RNA isolation and quantitative-PCR: Total RNA were extracted from liver by using the

RNeasy Mini kit (Qiagen, Valencia, CA.) according to the manufacturer protocol. Real-time

quantitative PCR assays were performed by using the Mx4000 Quantitative PCR System

(Stratagene, La Jolla, CA.). Briefly, 1 µg of total RNA was reverse-transcribed into cDNA

using MuMLV reverse transcriptase and oligo-dT (Invitrogen, Carlsbad, CA). Fifty

nanograms of the cDNA mixture was used for real time PCR analysis using the SYBR Green

PCR Master Mix® (Applied Biosystems, Carlsbad, CA). Values were normalized to GAPDH

levels. Relative mRNA levels were evaluated using the ΔΔCt method.

Western blot analysis. Protein levels of apoA-I, ABCA1, and SR-BI were measured in the

liver by Western blot analysis. Primary antibodies for mouse apoA-I, ABCA1, and SR-BI

were purchased from Santa Cruz Biotechnology Inc., Abcam, and Biodesign, respectively.

In vivo turnover studies: Mouse HDL was labelled with [3H]CE by the following procedure 8: Briefly, cholesteryl-1,2-[3H]hexadecylether in toluene (Perkin Elmer, Boston, MA) was

dried down under a stream of nitrogen and resuspended in 100 µl of absolute ethanol. The

solution was added dropwise to mouse HDL (1mg of total protein) that were subsequently

incubated for 18 h at 37°C in the presence of d > 1.21 infranatant from human serum as CETP

source. Labelled HDL were subsequently isolated by sequential ultracentrifugation at 1.07

and 1.21 g/ml density in a TLA-100.2 rotor using a TL-100 ultracentrifuge. Control and

ITX5061-treated mice were injected in the tail vein with [3H]CE-labelled HDL (1.5 x 106

cpm). After injection, blood was taken from the tail vein at different time points for

determination of radioactivity. The fractional catabolic rates (FCRs) were calculated from the

decay curves of [3H]CE radioactivity in plasma by fitting the data to a biexponential equation

according to the method of Matthews. The production rates were calculated by multiplying

the FCR by the plasma cholesterol pool and dividing by the body weight. Liver was collected,

weighed, homogenized, and one aliquot was radioassayed after dissolution in NaOH 0.1 M.

Uptake of [3H]CE-labeled HDL by cells in culture

HEK293 cells were plated on 24-well plates, when 80 % confluence was reached, cells were

transfected with mouse SR-BI cDNA expression vector or a mock vector using Lipofectamine

2000 transfection reagent (Invitrogen, Carlsbad, CA). One day after transfection, the cells

were pre-incubated with or without molecule at the indicated concentration. After the pre-

incubation, [3H]CE-labelled HDL were added into the medium and the cells were incubated

for 4 h either at 37°C or at 4°C. The culture medium was removed and the cells were washed

with PBS . Finally, the cells were dissolved in 200 μL of NaOH 100 mM. Radioactivity of the

cell lysate was directly measured by liquid scintillation spectrometry. The radioactivity levels

were normalized to the protein content of the lysates and values at 4°C were substracted

Statistical analysis

Mann Whitney or Wilcoxon tests were used to determine the statistical significance between

two groups.

Figure S1: Structure of ITX5061

Figure S2: Effect of ITX5061 on p38 MAPK activity

Figure S3: Changes in lipid parameters over time during ITX5061 administration.

Patients received placebo (n=37), ITX5061 at 150mg (n=38) and ITX5061 at 300 mg (n=40)

daily. Lipid parameters were determined at indicated time points. (A): mean percent changes

in LDL-C, (B): mean percent changes in triglycerides.

Figure S4: (A) Relative changes in hepatic mRNA levels in HuAITg mice upon ITX5061

treatment. Total RNA was extracted from the liver, and real-time quantitative PCR was

performed as described in Materials and Methods. Data were standardized for GAPDH.

mRNA levels in mice receiving the vehicle were set at 1.00. (B) Western blot analysis of

apoA-I, ABCA1, and SR-BI in the liver. Values are normalized to actin.

Figure S5: Plasma CETP activity in Ldlr+/- mice injected with AAV-CETP or AAV-Ctrl.

Mice were injected IP with AAV at the indicated doses. CETP was determined 2 weeks after

injection using commercially available kit as described by the manufacturer

Table SI: Demographic data and baseline lipid levels in patients.

Characteristics at

baseline

Placebo (N=37)

150 mg (N=38)

300 mg (N=40)

Age (years)

54.5±9.81

55.5±8.76

54.2±9.97

Gender

Male Female

23 (62%) 14 (38%)

21(55%) 17(45%)

23 (58%) 17 (43%)

HDL cholesterol

(mg/dl)

38.2+7.39

37.84+7.08

39.33+8.56

Total cholesterol

(mg/dl)

210.73+36.45

209.45+41.21

198.95+41.45

Triglycerides

(mg/dl)

229.30+83.93

218.55+80.89

221.15+105.35

Table SII: plasma lipid parameters after 2 and 18 weeks of Paigen diet in Ldlr+/- treated

or not with ITX5061. a significantly different from AAV-CETP + Vehicle, b: significantly

different form AAV-Ctrl + Vehicle. Lipid parameters are in mg/dl, AST and ALT are

international units/L, TBIL is in µmol/L. Normal values for ALT : 40±20 UI/L, AST: 140±67

UI/L 7)

2 weeks

18 weeks

TC

Non HDL-

C

HDL-

C

TC

Non HDL-

C

HDL-

C

TG

ALT

AST

TBIL

AAV-CETP + ITX5061

(n=10)

456 ±115

359 ±135

97.2a ±30.7

337 ±73.8

228 ±88.1

136a ±19.9

57.9 ±28.2

152 ±55

179 ±87

3.6 ±1.7

AAV-CETP

+ Vehicle (n=10)

509 ±118.

455 ±125

54.1 ±26.5

276 ±85.4

204 ±86.5

90.3 ±13.2

82.4 ±29.3

117 ±70

127 ±53

2.7 ±1.3

AAV-Ctrl + ITX5061 (n=11)

406 ±147

317 ±154

89.7b ±17.2

313 ±131

213 ±156

122 ±29.8

93.6 ±44.8

252 ±196

173 ±96

2.5 ±1.0

AAV-Ctrl +

Vehicle (n=10)

497 ±132

438 ±143

58.9 ±19.9

326 ±105

217 ±82.3

100 ±31.9

64.3 ±22.7

214 ±166

229 ±122

2.2 ±0.70

1. Rigotti A, Trigatti BL, Penman M, Rayburn H, Herz J, Krieger M. A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism. Proc Natl Acad Sci U S A. 1997;94:12610-12615.

2. Rubin EM, Ishida BY, Clift SM, Krauss RM. Expression of human apolipoprotein A-I in transgenic mice results in reduced plasma levels of murine apolipoprotein A-I and the appearance of two new high density lipoprotein size subclasses. Proc Natl Acad Sci U S A. 1991;88:434-438.

3. Yvan-Charvet L, Ranalletta M, Wang N, Han S, Terasaka N, Li R, Welch C, Tall AR. Combined deficiency of ABCA1 and ABCG1 promotes foam cell accumulation and accelerates atherosclerosis in mice. J Clin Invest. 2007;117:3900-3908.

4. Arai T, Wang N, Bezouevski M, Welch C, Tall AR. Decreased atherosclerosis in heterozygous low density lipoprotein receptor-deficient mice expressing the scavenger receptor BI transgene. J Biol Chem. 1999;274:2366-2371.

5. Tanigawa H, Billheimer JT, Tohyama J, Zhang Y, Rothblat G, Rader DJ. Expression of cholesteryl ester transfer protein in mice promotes macrophage reverse cholesterol transport. Circulation. 2007;116:1267-1273.

6. Ishibashi M, Egashira K, Zhao Q, Hiasa K, Ohtani K, Ihara Y, Charo IF, Kura S, Tsuzuki T, Takeshita A, Sunagawa K. Bone marrow-derived monocyte chemoattractant protein-1 receptor CCR2 is critical in angiotensin II-induced acceleration of atherosclerosis and aneurysm formation in hypercholesterolemic mice. Arterioscler Thromb Vasc Biol. 2004;24:e174-178.

7. Harrison SD, Jr., Burdeshaw JA, Crosby RG, Cusic AM, Denine EP. Hematology and clinical chemistry reference values for C57BL/6 X DBA/2 F1 mice. Cancer Res. 1978;38:2636-2639.

8. Masson D, Qatanani M, Sberna AL, Xiao R, Pais de Barros JP, Grober J, Deckert V, Athias A, Gambert P, Lagrost L, Moore DD, Assem M. Activation of the constitutive androstane receptor decreases HDL in wild-type and human apoA-I transgenic mice. J Lipid Res. 2008;49:1682-1691.

ONH

NH

SO

O

O

O

ON

O•HCl

Figure S1

PreincubationTime

IC50(nM)

0 302

30 38

60 14

120 8

Figure S2

10-9 10-8 10-7 10-6 10-5

0

25

50

75

100

t = 0 mint = 30 mint = 60 mint = 120 min

[ITX5061] (M)

% In

hibi

tion

0 2 4 6-40

-20

0

20

40

Week

% C

hang

e[L

DL-

C]

A

0 2 4 6

-50

0

50

100

Week

% C

hang

e [T

rigly

cerid

es]

B

Figure S3

VehicleITX5061

0

1

2R

elat

ive

mR

NA

leve

ls

apoA

-I

(human

) apoA

-I

(mou

se)

SR-BI

ABCA1

Figure S40

0.4

0.8

1.2

1.6

apoA-I SR-BI ABCA1

ABCA1

SR-BI

apoA-I

actin

Vehicle ITX5061

A

B

Rel

ativ

e pr

otei

nle

vels

VehicleITX5061

020406080

100120140160180

human plasma mouse plasma + AAV-CETP

mouse plasma+ AAV-Ctrl

CE

TP a

ctiv

ity

(incr

ease

in fl

uore

scen

ce A

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Figure S5

increased hdl cholesterol and apoa-i in humans and...

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