Improving Assessment of Drug Safety Through Proteomics: Early Detection and Mechanistic Characterization of the Unforeseen Harmful Effects of Torcetrapib

Abstract

Background: Early detection of adverse effects of novel therapies and understanding of their mechanisms could improve the safety and efficiency of drug development. We have retrospectively applied large-scale proteomics to blood samples from ILLUMINATE (Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events), a trial of torcetrapib (a cholesterol ester transfer protein inhibitor), that involved 15 067 participants at high cardiovascular risk. ILLUMINATE was terminated at a median of 550 days because of significant absolute increases of 1.2% in cardiovascular events and 0.4% in mortality with torcetrapib. The aims of our analysis were to determine whether a proteomic analysis might reveal biological mechanisms responsible for these harmful effects and whether harmful effects of torcetrapib could have been detected early in the ILLUMINATE trial with proteomics.

Methods: A nested case-control analysis of paired plasma samples at baseline and at 3 months was performed in 249 participants assigned to torcetrapib plus atorvastatin and 223 participants assigned to atorvastatin only. Within each treatment arm, cases with events were matched to controls 1:1. Main outcomes were a survey of 1129 proteins for discovery of biological pathways altered by torcetrapib and a 9-protein risk score validated to predict myocardial infarction, stroke, heart failure, or death.

Results: Plasma concentrations of 200 proteins changed significantly with torcetrapib. Their pathway analysis revealed unexpected and widespread changes in immune and inflammatory functions, as well as changes in endocrine systems, including in aldosterone function and glycemic control. At baseline, 9-protein risk scores were similar in the 2 treatment arms and higher in participants with subsequent events. At 3 months, the absolute 9-protein derived risk increased in the torcetrapib plus atorvastatin arm compared with the atorvastatin-only arm by 1.08% (P=0.0004). Thirty-seven proteins changed in the direction of increased risk of 49 proteins previously associated with cardiovascular and mortality risk.

Conclusions: Heretofore unknown effects of torcetrapib were revealed in immune and inflammatory functions. A protein-based risk score predicted harm from torcetrapib within just 3 months. A protein-based risk assessment embedded within a large proteomic survey may prove to be useful in the evaluation of therapies to prevent harm to patients.

Clinical trial registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT00134264.

Keywords: aptamers; biomarkers; peptides; precision medicine; prescription drugs; proteins; proteomics.

Figure 1.

Flowchart of samples, study participants, and analytic processes for the evaluation of torcetrapib.

Figure 2.

Proteins significantly altered by torcetrapib associated with aldosterone synthesis or function. Nodes represent gene symbol name, corresponding to protein measured. Degree of intensity of the node color (red) indicates degree of significance of the false discovery rate P value. Dashed lines correspond to the implicated function of the given proteins according to the Ingenuity Pathway Analysis knowledge bank. Node shapes denote cytokine (□), growth factor (□), and other (○). ANGPT2 indicates angiopoietin-2; BMP6, bone morphometric protein 6; DKK3, Dickkopf-related protein 3; NPPB, natriuretic peptide B; POMC, pro-opiomelanocortin; PYY, peptide YY; and TGFB1, transforming growth factor-β1. *Given protein was represented in the modified aptamer assay more than once.

Figure 3.

Proteins significantly altered by torcetrapib associated with insulin sensitivity or pancreatic β-cell function. Nodes represent gene symbol name, corresponding to protein measured. Degree of intensity of the node color (red) indicates degree of significance of false discovery rate P value. Dashed lines correspond to the implicated function of the given proteins according to the Ingenuity Pathway Analysis knowledge bank. Node shapes denote cytokine (□), growth factor (□), kinase (▽), peptidase (), transmembrane receptor (), transporter (), and other (○). ADIPO1 indicates adiponectin; C1Q, and collagen domain containing; APOE, apolipoprotein E, FSTL3, follistatin-like 3; GHR, growth hormone receptor; MAPK8, mitogen-activated protein kinase-8; MMP2, matrix metalloproteinase-2; PRKCQ, protein kinase Cθ type; TGFB1, transforming growth factor-β1; and TNSFSF12, tumor necrosis factor superfamily member 12. *Given protein was represented in the modified aptamer assay more than once.

Figure 4.

Within-participant changes in 9-protein risk score, baseline to 3 months. A, Percent change in risk (3 months minus baseline) by treatment group. B, Percent change in risk (3 months minus baseline) by treatment group and event status. Legend to bar charts: Bar height extends to the median change in risk, and the whiskers represent the 95% confidence interval about the median. Labels at the top of each bar are the median risk change. P values on the sides of the bars are from testing with a Wilcoxon signed-rank test the null hypothesis that the risk change is distributed symmetrically with a median of zero. P values in brackets are from testing with a Wilcoxon rank-sum test the null hypothesis of equal medians for the 2 populations. A indicates atorvastatin; and T, torcetrapib.