Measurement of LDL-C after treatment with the CETP inhibitor anacetrapib

Abstract

Estimation of low-density lipoprotein cholesterol (LDL-C) using the Friedewald (FR) formula is often inaccurate when triglycerides are elevated or VLDL particle composition is altered. We hypothesized that LDL-C estimation by the FR formula and other measurement methods might also be inaccurate in individuals treated with a cholesteryl ester transfer protein (CETP) inhibitor. An assay comparison study was conducted using pre and posttreatment serum samples from 280 of the 811 patients treated with the CETP inhibitor anacetrapib in the DEFINE study (determining the ef ficacy and tolerability of CETP in hibition with anac e trapib). After 24 weeks of treatment with anacetrapib, mean LDL-C values by FR formula, Roche direct method (RDM) and Genzyme direct method (GDM) deviated from that measured by the β-quantification (BQ) reference method by –12.2 ± 7.5, –10.2 ± 6.6, –10.8 ± 8.8 mg/dl, respectively. After treatment with anacetrapib, the FR formula and detergent-based direct methods provided lower LDL-C values than those obtained by the BQ reference method. The bias by the FR formula appeared to be due to an overestimation of VLDL-C by the TG/5 component of the formula. Evaluation of the clinical significance of these findings awaits comprehensive lipid and cardiovascular outcome data from ongoing Phase III clinical studies of anacetrapib. Estimation of low-density lipoprotein cholesterol (LDL-C) using the Friedewald (FR) formula is often inaccurate when triglycerides are elevated or VLDL particle composition is altered. We hypothesized that LDL-C estimation by the FR formula and other measurement methods might also be inaccurate in individuals treated with a cholesteryl ester transfer protein (CETP) inhibitor. An assay comparison study was conducted using pre and posttreatment serum samples from 280 of the 811 patients treated with the CETP inhibitor anacetrapib in the DEFINE study (determining the ef ficacy and tolerability of CETP in hibition with anac e trapib). After 24 weeks of treatment with anacetrapib, mean LDL-C values by FR formula, Roche direct method (RDM) and Genzyme direct method (GDM) deviated from that measured by the β-quantification (BQ) reference method by –12.2 ± 7.5, –10.2 ± 6.6, –10.8 ± 8.8 mg/dl, respectively. After treatment with anacetrapib, the FR formula and detergent-based direct methods provided lower LDL-C values than those obtained by the BQ reference method. The bias by the FR formula appeared to be due to an overestimation of VLDL-C by the TG/5 component of the formula. Evaluation of the clinical significance of these findings awaits comprehensive lipid and cardiovascular outcome data from ongoing Phase III clinical studies of anacetrapib. Evaluation of low density lipoprotein cholesterol (LDL-C) in clinical practice has been largely based upon estimation of LDL-C by the Friedewald (FR) formula (1Friedewald W.T. Levy R.I. Fredrickson D.S. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.Clin. Chem. 1972; 18: 499-502Crossref PubMed Scopus (64) Google Scholar) or by direct measurement of LDL-C using detergent- or antibody-based methods. The FR formula [total cholesterol (TC) – high-density lipoprotein cholesterol (HDL-C) – triglycerides (TG) / 5] uses direct measurements of plasma or serum TC, HDL-C, and TG to estimate LDL-C (1Friedewald W.T. Levy R.I. Fredrickson D.S. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.Clin. Chem. 1972; 18: 499-502Crossref PubMed Scopus (64) Google Scholar). In the FR formula, VLDL-C is estimated by TG/5 and is based upon the assumption that most of the TG in fasting plasma is in the VLDL fraction and that there is a 5:1 ratio of TG to cholesterol in VLDL particles in the fasting state (2Fredrickson D.S. Levy R.I. Lees R.S. Fat transport in lipoproteins–an integrated approach to mechanisms and disorders.. 1967; 276 (94–103, 148–156, 215–225, 273–281): 34-44Google Scholar, 3Hatch F.T. Practical methods for plasma lipoprotein analysis.Adv. Lipid Res. 1968; 6: 1-68Crossref PubMed Google Scholar). In clinical settings in which plasma TG is markedly elevated (such as fasting or postprandial chylomicronemia) or in which particle composition of VLDL is altered (such as with fasting TG > 400 mg/dl, and type III hyperlipidemia), the FR formula is known to be inaccurate. In these situations, alternative methods of monitoring LDL-C or levels of atherogenic lipids are recommended (4National Heart Lung and Blood Institute. 2002. Third Report of the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Accessed September 12, 1012, at: http://www.nhlbi.nih.gov/guidelines/cholesterol/index.htmGoogle Scholar, 5Reiner Z. Catapano A.L. De Backer G. Graham I. Taskinen M.R. Wiklund O. Agewall S. Alegria E. Chapman M.J. Durrington P. et al.ESC/EAS guidelines for the management of dyslipidaemias: the task force for the management of dyslipidaemias of the European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS).Eur. Heart J. 2011; 32: 1769-1818Crossref PubMed Scopus (2540) Google Scholar), such as direct LDL-C measurements by detergent or antibody or monitoring non-HDL-C or apolipoprotein B levels. Cholesteryl ester transfer protein (CETP) mediates the exchange of cholesteryl esters (CE) and TG between HDL and apoB-containing lipoprotein particles. Anacetrapib (MK-0859) is an orally active, potent, and selective CETP inhibitor in Phase III development (6Gutstein D.E. Krishna R. Johns D. Surks H.K. Dansky H.M. Shah S. Mitchel Y.B. Arena J. Wagner J.A. Anacetrapib, a novel CETP inhibitor: pursuing a new approach to cardiovascular risk reduction.Clin. Pharmacol. Ther. 2012; 91: 109-122Crossref PubMed Scopus (58) Google Scholar). Previous studies have shown that anacetrapib treatment increases HDL-C by up to ∼139% and decreases LDL-C by up to ∼40% when calculated by the FR formula (7Bloomfield D. Carlson G.L. Sapre A. Tribble D. McKenney J.M. Littlejohn III, T.W. Sisk C.M. Mitchel Y. Pasternak R.C. Efficacy and safety of the cholesteryl ester transfer protein inhibitor anacetrapib as monotherapy and coadministered with atorvastatin in dyslipidemic patients.Am. Heart J. 2009; 157: 352-360Crossref PubMed Scopus (230) Google Scholar, 8Krishna R. Anderson M.S. Bergman A.J. Jin B. Fallon M. Cote J. Rosko K. Chavez-Eng C. Lutz R. Bloomfield D.M. et al.Effect of the cholesteryl ester transfer protein inhibitor, anacetrapib, on lipoproteins in patients with dyslipidaemia and on 24-h ambulatory blood pressure in healthy individuals: two double-blind, randomised placebo-controlled phase I studies.Lancet. 2007; 370: 1907-1914Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). These effects on LDL-C and HDL-C were observed when anacetrapib was coadministered with statins for up to 1.5 years of treatment in the DEFINE (determining the ef ficacy and tolerability of CETP in hibition with anac e trapib) trial (clinicaltrials.gov NCT00685776) (9Cannon C.P. Dansky H.M. Davidson M. Gotto Jr, A.M. Brinton E.A. Gould A.L. Stepanavage M. Liu S.X. Shah S. Rubino J. et al.Design of the DEFINE trial: determining the EFficacy and tolerability of CETP INhibition with AnacEtrapib.Am. Heart J. 2009; 158: 513-519Crossref PubMed Scopus (79) Google Scholar, 10Cannon C.P. Shah S. Dansky H.M. Davidson M. Brinton E.A. Gotto A.M. Stepanavage M. Liu S.X. Gibbons P. Ashraf T.B. et al.Safety of anacetrapib in patients with or at high risk for coronary heart disease.N. Engl. J. Med. 2010; 363: 2406-2415Crossref PubMed Scopus (681) Google Scholar). Recently, Krauss et al. reported an enrichment of TG and reduction of CE in VLDL, IDL, and the smallest LDL fraction in healthy subjects after 2 weeks of treatment with anacetrapib 150 mg daily (11Krauss R.M. Wojnooski K. Orr J. Geaney J.C. Pinto C.A. Liu Y. Wagner J.A. Luk J.M. Johnson-Levonas A.O. Anderson M.S. et al.Changes in lipoprotein subfraction concentration and composition in healthy individuals treated with the cholesteryl ester transfer protein inhibitor anacetrapib.J. Lipid Res. 2012; 53: 540-547Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In that study, the ratio of TG/CE in VLDL particles was increased approximately 3-fold after treatment with anacetrapib. This increase in the VLDL TG/CE ratio would be expected to impair the accuracy of the TG/5 estimation of VLDL-C used to calculate LDL-C with the FR formula. Furthermore, the change in TG/CE ratio may also affect the sensitivity and specificity of the detergent-based direct methods due to changes in lipoprotein composition. Careful evaluation of seven direct LDL-C quantitation methods has demonstrated that measurement bias is greater when measuring LDL-C in individuals being treated for cardiovascular disease or who had other conditions that might be expected to affect lipoprotein composition compared with measurement of LDL-C from individuals with no known diseases (12Miller W.G. Myers G.L. Sakurabayashi I. Bachmann L.M. Caudill S.P. Dziekonski A. Edwards S. Kimberly M.M. Korzun W.J. Leary E.T. et al.Seven direct methods for measuring HDL and LDL cholesterol compared with ultracentrifugation reference measurement procedures.Clin. Chem. 2010; 56: 977-986Crossref PubMed Scopus (233) Google Scholar). In the current study, LDL-C and HDL-C were remeasured by several different assay methods using stored frozen serum samples obtained from 280 individuals in a patient subset at baseline and after 24 weeks of treatment with anacetrapib 100 mg daily in the DEFINE trial. To assess the accuracy of the LDL-C methods, the methods were compared with a modified Centers for Disease Control and Prevention (CDC) LDL-C reference measurement procedure (RMP) that involves ultracentrifugation β-quantification (BQ) (13Bachorik P.S. Ross J.W. National Cholesterol Education Program recommendations for measurement of low-density lipoprotein cholesterol: executive summary. The National Cholesterol Education Program Working Group on Lipoprotein Measurement.Clin. Chem. 1995; 41: 1414-1420Crossref PubMed Scopus (305) Google Scholar, 14Lipid Research Clinics Program, National Heart and Lung Institute. 1974. Manual of Laboratory Operations: Lipid and Lipoprotein Analysis. National Institutes of Health, Washington, D.C.Google Scholar). The modified BQ procedure involves subtraction of the HDL-C (measured in the supernatant after dextran sulfate precipitation of serum) from the cholesterol measured in the ultracentrifugation infranate (density > 1.006 g/ml) to obtain the LDL-C. We compared the results obtained with BQ with those obtained by three methods commonly used in clinical practice. Values for HDL-C concentration using direct methods were also compared with those obtained after dextran sulfate precipitation of serum. Lipid measurements in the current study were performed using stored frozen serum aliquots from patients who participated in the DEFINE study, a randomized, double-blind, placebo-controlled trial to assess the efficacy and safety profile of anacetrapib in patients with coronary heart disease or at high risk for coronary heart disease (9Cannon C.P. Dansky H.M. Davidson M. Gotto Jr, A.M. Brinton E.A. Gould A.L. Stepanavage M. Liu S.X. Shah S. Rubino J. et al.Design of the DEFINE trial: determining the EFficacy and tolerability of CETP INhibition with AnacEtrapib.Am. Heart J. 2009; 158: 513-519Crossref PubMed Scopus (79) Google Scholar, 10Cannon C.P. Shah S. Dansky H.M. Davidson M. Brinton E.A. Gotto A.M. Stepanavage M. Liu S.X. Gibbons P. Ashraf T.B. et al.Safety of anacetrapib in patients with or at high risk for coronary heart disease.N. Engl. J. Med. 2010; 363: 2406-2415Crossref PubMed Scopus (681) Google Scholar).Two sets of serum samples were used to perform these measurements. The first set, Study 1, consisted of paired patient samples taken at baseline and Week 24. These samples were selected according to patient baseline LDL-C. Samples from 25 anacetrapib-treated subjects were selected from each of the following three baseline LDL-C intervals of 25 ≤ LDL < 60, 60 ≤ LDL ≤ 90, and > 90 mg/dl (as estimated by the FR formula). Study 1 also included paired samples from 25 additional subjects randomly selected from among patients with LDL-C values < 25 mg/dl as estimated by the FR formula at Week 24 or occurring prior to Week 24, for which the protocol called for discontinuation from the study. The second set of samples, Study 2, consisted of baseline and Week 24 serum samples from two groups of 110 subjects; one randomly selected among patients with baseline LDL-C ≤ 65 mg/dl and the 2nd with baseline LDL-C >65 mg/dl. Across the 2 studies, there were 20 patients in which there was either a missing baseline or on treatment LDL-C value. Since the results of study 1 (n = 100 subjects) and study 2 (n = 220 subjects) were similar to each other, data from both studies were combined for a total of 280 unique patients with complete datasets. In the 20 cases where a patient's serum was analyzed in both studies, only data from Study 2 were used. Assays were run by Pharmaceutical Product Development (PPD Global Central Labs, Highland Heights, KY) for study 1 and Pacific Biomarkers (Seattle, WA) for study 2. Serum samples were stored at clinical sites at −20°C or at −70°C for 1–5 months and then at a central at −70°C for sets and 24 were analyzed in the Lipid were analyzed on a Roche was measured by an based on cholesterol and TG was measured by an method with to overestimation of TG from or LDL-C was measured by different BQ C.A. of methods for Chem. PubMed Scopus Google the Roche direct method Roche by the Genzyme direct method Genzyme by and calculated using the FR formula (1Friedewald W.T. Levy R.I. Fredrickson D.S. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge.Clin. Chem. 1972; 18: 499-502Crossref PubMed Scopus (64) Google Scholar). The and were performed according to BQ was performed as were for at density After the was to obtain the and (density < 1.006 and density > 1.006 The cholesterol in the fraction g/ml) the cholesterol of and VLDL, the cholesterol in the fraction g/ml) LDL and with from and LDL-C concentration was calculated as the cholesterol in the total density > 1.006 fraction the HDL-C concentration that was measured in the supernatant after dextran sulfate / precipitation of lipoproteins in serum. HDL-C was also measured by Roche and Genzyme direct methods as by the of precipitation and of in the supernatant were by treatment The ratio of in the VLDL of each patient in study 2 was calculated using VLDL-C and values obtained by the cholesterol or TG concentration in the (density > 1.006 g/ml) fraction from the cholesterol and TG values in the serum. To calculate LDL-C by the FR formula, and TG were measured in serum and HDL-C was measured by dextran sulfate / precipitation as LDL-C was calculated as – (HDL-C) – The baseline and 24 of LDL-C were for each of the the between methods were performed to the assay bias to the BQ the of the and changes in LDL-C from baseline were for each method. and were to estimate the bias between each of the methods the BQ were to the of between each of the comparison of methods. The of the current study was to different methods for measuring the concentration of LDL-C in patients and after treatment with the CETP inhibitor anacetrapib. The patient in the current study was a subset of the patients to 100 mg anacetrapib in the DEFINE trial. To LDL-C measurement methods a of LDL-C the patient subset was selected according to baseline plasma LDL-C as calculated by the FR formula using the plasma lipid measurements performed the of the study C.P. Shah S. Dansky H.M. Davidson M. Brinton E.A. Gotto A.M. Stepanavage M. Liu S.X. Gibbons P. Ashraf T.B. et al.Safety of anacetrapib in patients with or at high risk for coronary heart disease.N. Engl. J. Med. 2010; 363: 2406-2415Crossref PubMed Scopus (681) Google Scholar). The patient subset was with a greater of patients with lower baseline LDL-C values baseline plasma LDL-C by the FR in the patient subset was approximately lower than the to anacetrapib ± ± the profile and baseline lipids of the selected patient subset were similar to that of the in the DEFINE trial Anacetrapib Treatment = = ± ± or LDL-C HDL-C TG in a new frozen than plasma that was used in the study, was used to lipids and lipoproteins and assay methods in the current study of the and of stored frozen serum serum LDL-C values were similar for methods, the and were lower when LDL-C was calculated by the FR formula the mean FR formula, and LDL-C values deviated from the BQ reference by ± ± 6.6, and ± mg/dl respectively. After 24 weeks of treatment with 100 mg anacetrapib, the mean LDL-C concentration calculated by FR formula and the two direct methods were similar to each LDL-C by three methods formula, and was lower than the mean LDL-C by the BQ method with an bias of –12.2 ± 7.5, –10.2 ± 6.6, and –10.8 ± 8.8 mg/dl, LDL-C measurements at baseline and after 24 weeks of treatment with of (n = 24 (n = from in a new of patient LDL-C data for a bias after there was a at both baseline = and at 24 = between the FR and BQ methods, the FR formula LDL-C values that were lower than those measured by BQ and were obtained when LDL-C levels by either of the direct methods were compared with the values obtained by the BQ method I and The between patient FR formula and BQ LDL-C values were also in patients with and without and according to TG values and the I and III). The of or TG to the mean bias between the methods either at baseline or after treatment with anacetrapib. as by the of in the the of the bias appeared to be the LDL-C and I and with a of greater bias at TG values after treatment The ratio of in VLDL was measured and after treatment with anacetrapib. the VLDL ratio was ± (n = and increased to ± (n = after 24 weeks of treatment with anacetrapib. TG/5 is used as an estimate of VLDL-C in the FR formula, the and bias between TG/5 and the measured VLDL-C were also there was a between TG/5 and VLDL-C at baseline and after anacetrapib treatment the mean bias between TG/5 and VLDL-C increased from ± mg/dl at baseline to ± mg/dl after and HDL-C were measured by by and in serum after dextran sulfate the dextran sulfate as the reference measurement mean values for HDL-C concentration at baseline and on treatment were similar among the three methods, with the mean lower HDL-C values HDL-C measurements at baseline and after 24 weeks of treatment with of (n = 24 (n = from in a new guidelines for the and of cardiovascular disease LDL-C treatment (4National Heart Lung and Blood Institute. 2002. Third Report of the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Accessed September 12, 1012, at: http://www.nhlbi.nih.gov/guidelines/cholesterol/index.htmGoogle Scholar, 5Reiner Z. Catapano A.L. De Backer G. Graham I. Taskinen M.R. Wiklund O. Agewall S. Alegria E. Chapman M.J. Durrington P. et al.ESC/EAS guidelines for the management of dyslipidaemias: the task force for the management of dyslipidaemias of the European Society of Cardiology (ESC) and the European Atherosclerosis Society (EAS).Eur. Heart J. 2011; 32: 1769-1818Crossref PubMed Scopus (2540) Google Scholar, Jr, Pasternak R.C. Jr, of clinical for the National Cholesterol Education Program Treatment Panel III PubMed Scopus Google Scholar). LDL-C estimation in current clinical practice has been based upon either the FR formula of LDL-C or direct LDL-C measurement is known that FR formula of LDL-C in the of in guidelines alternative methods. The BQ reference method is used in clinical practice is a and assay (13Bachorik P.S. Ross J.W. National Cholesterol Education Program recommendations for measurement of low-density lipoprotein cholesterol: executive summary. The National Cholesterol Education Program Working Group on Lipoprotein Measurement.Clin. Chem. 1995; 41: 1414-1420Crossref PubMed Scopus (305) Google Scholar, 14Lipid Research Clinics Program, National Heart and Lung Institute. 1974. Manual of Laboratory Operations: Lipid and Lipoprotein Analysis. National Institutes of Health, Washington, D.C.Google Scholar). The current study at there is a bias between the serum LDL-C values obtained by the FR formula compared with the BQ reference The bias increases to a mean of mg/dl when LDL-C is measured from samples taken after treatment with the CETP inhibitor anacetrapib. The bias was similar in the or of a clinical of when TG was or the and the bias was similar the of baseline LDL-C. of the between the bias between FR and BQ as a of TG after treatment may a greater bias at TG values the of patients with to was in study patients were from the DEFINE study for TG > 400 mg/dl. studies be to estimate the assay bias after treatment with anacetrapib in the of TG and with baseline LDL-C values > 100 mg/dl. The increase in bias for FR formula be by an overestimation of the VLDL-C when that the ratio of in VLDL is In the ratio in treated with anacetrapib was greater than of The for the ratio of in VLDL is that of CETP decreases both the transfer of TG from VLDL to HDL and of cholesteryl esters from HDL to An increase in the ratio of in the VLDL fraction of patients treated with anacetrapib has also been reported in study (11Krauss R.M. Wojnooski K. Orr J. Geaney J.C. Pinto C.A. Liu Y. Wagner J.A. Luk J.M. Johnson-Levonas A.O. Anderson M.S. et al.Changes in lipoprotein subfraction concentration and composition in healthy individuals treated with the cholesteryl ester transfer protein inhibitor anacetrapib.J. Lipid Res. 2012; 53: 540-547Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The that the bias between VLDL-C and the TG/5 component of the FR was of a similar to the bias between the FR LDL-C BQ method the that overestimation of VLDL-C is most for the of LDL-C by the FR method in anacetrapib-treated the for the when using direct methods for measuring LDL-C is is that the used in the direct methods have a to cholesterol from the LDL fraction in the of an increased to results using the BQ the FR the direct provided lower levels of LDL-C after treatment with anacetrapib. These that use of the FR to calculate LDL-C in a greater LDL-C reduction than when LDL-C is measured by the BQ the to which the treatment was using the FR of the samples, the of assay comparison measurements on samples from and (4National Heart Lung and Blood Institute. 2002. Third Report of the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). Accessed September 12, 1012, at: http://www.nhlbi.nih.gov/guidelines/cholesterol/index.htmGoogle Scholar, Kimberly M.M. Leary E.T. Myers G.L. of measurements for and Med. Scopus Google Scholar) and effects of frozen W.J. P.S. J.A. Myers G.L. E.A. the effects of at on and measurements in stored Chem. PubMed Scopus Google may have had an on the of the bias and of the treatment in the current study. is that the bias between FR and BQ might also be by the In a study (7Bloomfield D. Carlson G.L. Sapre A. Tribble D. McKenney J.M. Littlejohn III, T.W. Sisk C.M. Mitchel Y. Pasternak R.C. Efficacy and safety of the cholesteryl ester transfer protein inhibitor anacetrapib as monotherapy and coadministered with atorvastatin in dyslipidemic patients.Am. Heart J. 2009; 157: 352-360Crossref PubMed Scopus (230) Google Scholar), the mean bias between FR and BQ LDL-C was mg/dl after treatment with anacetrapib 150 mg in with atorvastatin 20 mg performed on studies are to the bias clinical settings and In the current study that the LDL-C calculated by the FR formula or measured by direct detergent-based LDL-C after treatment with the CETP inhibitor anacetrapib when compared with LDL-C values obtained by the reference BQ method. This results in an overestimation of the change from baseline the of the overestimation of the treatment is studies are to the effects of anacetrapib on LDL-C using the BQ reference method. is also to of and of which is to the of in LDL-C measurement by BQ or other and both of which have been shown to be of cardiovascular risk than is LDL-C M. A. of cardiovascular disease in the using apolipoprotein the Heart 2007; PubMed Scopus Google Scholar, E. P. S. A. A.M. S. et and risk of 2009; PubMed Scopus Google Scholar). the clinical of the effects of anacetrapib on lipid is being in (clinicaltrials.gov a cardiovascular outcome study of individuals with cardiovascular The Gibbons and A. for with the with β-quantification cholesterol cholesteryl ester cholesteryl ester transfer protein determining the efficacy and tolerability of CETP with anacetrapib Friedewald Genzyme direct method low-density lipoprotein cholesterol Roche direct method total cholesterol