Raising plasma levels of high-density lipoprotein (HDL) cholesterol has been a therapeutic goal ever since the strong inverse association between HDL levels and the risk of coronary heart disease was first observed.1 Nearly two decades ago, the discovery that persons in Japan had extremely high levels of HDL cholesterol because of a genetic deficiency involving the cholesteryl ester transfer protein (CETP) led to the concept that pharmacologic inhibition of CETP could raise HDL cholesterol levels. When this theory proved to be true in humans, it led to great anticipation that CETP inhibition would permit the ultimate test of the "HDL hypothesis" as to whether raising HDL cholesterol levels would reduce the risk of cardiovascular disease.
This hope suffered a severe blow with the surprise announcement in December 2006 that a large phase 3 clinical trial of the leading CETP inhibitor, torcetrapib, had been terminated because of increased mortality in the active treatment group, as compared with the placebo group. This announcement was followed by a presentation of imaging trials showing that torcetrapib had no effect on the progression of atherosclerosis.2,3 Since then, the biomedical community has been anxiously awaiting detailed information on the trial in the hope of achieving a better understanding of the adverse outcomes. In the issue of the Journal, Barter et al. discuss the results of the torcetrapib trial, called the Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events (ILLUMINATE),4 and the results are, well, illuminating.
In the study, more than 15,000 patients at high risk for cardiovascular disease were treated with atorvastatin during a run-in period to reach a target goal for low-density lipoprotein (LDL) cholesterol of less than 100 mg per deciliter. Then patients who met the target were randomly assigned to receive either 60 mg of torcetrapib plus atorvastatin or placebo plus atorvastatin. At the time the trial was terminated, the median follow-up period was only 550 days.
Despite the very favorable lipid changes in the torcetrapib group (an increase in HDL cholesterol of 72.1% and a decrease in LDL cholesterol of 24.9%), the rate of major cardiovascular events was increased by 25% and that of death from cardiovascular causes by 40%. Furthermore, death from noncardiovascular causes was increased by a factor of two. Torcetrapib was also associated with an increase in blood pressure and aldosterone levels and changes in electrolytes consistent with mineralocorticoid excess. These effects of torcetrapib are molecule-specific and are not related to the mechanism of CETP inhibition. Other CETP inhibitors do not elevate blood pressure in CETP-expressing species, including humans.
The ILLUMINATE trial raises several questions that are vitally important for cardiovascular medicine: Was the increase in adverse events and mortality caused by CETP inhibition, off-target effects of torcetrapib, or both? Might a "clean" CETP inhibitor reduce cardiovascular events without increasing noncardiovascular adverse events? What implications does this trial have for the broader issue of HDL cholesterol as a therapeutic target?
It is indeed possible that the increased rate of cardiovascular events and death associated with torcetrapib could be due either to CETP inhibition itself or to off-target effects of the drug — or to some combination of the two factors (Figure 1). Despite the effects of torcetrapib in raising HDL cholesterol levels, there has been concern that CETP inhibition could impair reverse cholesterol transport, the process by which peripheral cholesterol is transported back to the liver for excretion. The transfer of cholesteryl esters from HDL to lipoproteins containing apolipoprotein B is probably the major route in humans by which HDL cholesterol is returned to the liver, completing the pathway of reverse cholesterol transport. Although this pathway is difficult to measure in humans, in a study by Brousseau et al.,5 torcetrapib therapy was associated with a decreased turnover of HDL and no change in the fecal excretion of sterols. In mice, which do not have CETP, introduction of CETP expression actually promoted reverse cholesterol transport from macrophages despite a reduction in HDL levels.6 On the other hand, in mice that do not have effective uptake of LDL cholesterol by the liver, the expression of CETP had the reverse effect, which implies that there could be a potential benefit of CETP inhibition in this setting. Furthermore, HDL obtained from patients receiving has been shown to promote cholesterol efflux from macrophages through the ABCG1 pathway.7 It is tantalizing that in the ILLUMINATE study, a greater increase in HDL cholesterol levels in torcetrapib-treated patients was associated with fewer major cardiovascular events; it will be important to confirm this observation in the atherosclerosis imaging trials of torcetrapib.

Potential Mechanisms of Adverse Outcomes Associated with Torcetrapib Treatment with torcetrapib has both mechanism-based and off-target effects that may have contributed to an increased rate of adverse cardiovascular and noncardiovascular outcomes. The drug inhibits cholesteryl ester transfer protein (CETP), blocking the transfer of cholesteryl esters to lipoproteins containing apolipoprotein B (ApoB), such as low-density lipoprotein (LDL), resulting in increased levels of high-density lipoprotein (HDL) cholesterol and enlarged HDL particles. Although HDL cholesterol can be taken up directly by the liver through the HDL scavenger receptor, class B, type I (SR-BI), inhibition of CETP may reduce the rate of return of HDL cholesterol to the liver, thus impairing reverse cholesterol transport and increasing cardiovascular risk. In addition, the change in HDL composition could conceivably impair immune function associated with HDL, thus increasing noncardiovascular risks such as infection and cancer. On the other hand, the molecule torcetrapib clearly has the off-target effects of elevating levels of aldosterone and blood pressure, changes that probably contributed to the increased cardiovascular risk. The potential that torcetrapib has off-target effects that contributed to an increased risk of noncardiovascular events is possible but speculative. Finally, CETP inhibition has the potentially beneficial effects of increasing cholesterol efflux from macrophages mediated by ATP-binding cassette transporter G1 (ABCG1) (which could increase the rate of physiologically relevant reverse cholesterol transport from macrophages) and of increasing the uptake of LDL cholesterol by the liver (which reduces LDL cholesterol levels), effects that could be important for CETP inhibitors that do not have the off-target effects of torcetrapib. alert20-nejm_2007_357_2180.ppt (119KB)

The "off-target" effects of torcetrapib probably contributed in important ways to the increased rate of cardiovascular events and death in this trial. Indeed, aldosterone, through activation of the mineralocorticoid receptor, not only elevates blood pressure but has direct vascular effects, including impaired endothelial function, increased inflammation, and increased vascular smooth-muscle migration.8 Death from cardiovascular events was higher in patients with greater changes in serum electrolytes, consistent with the hypothesis that this mechanism played a causal role.
It is much harder to explain the increased rate of death from noncardiovascular causes by either CETP inhibition or off-target effects of torcetrapib (Figure 1). The doubling in the rate of death from noncardiovascular events in patients receiving torcetrapib, as compared with those receiving placebo, was largely due to more deaths from cancer (24 to 14) and infection (9 to 0). Could CETP inhibition have led to the excess noncardiovascular mortality, or are the off-target effects of the drug to be blamed? In many ways, this question is one of the most important ones raised by the ILLUMINATE study. It appears that HDL evolved as a component of the innate immune system and that its composition is critically important to its function. For example, HDL binds endotoxin and protects mice from endotoxin-induced death. In addition, in primates HDL serves as a synergistic platform for the assembly of a complex consisting of apolipoprotein L-I and haptoglobin-related protein that has the unique ability to lyse a species of trypanosome.9 Studies of the human HDL proteome have identified a large number of HDL-associated proteins that are involved in innate immunity, complement regulation, and inflammation.10 CETP inhibition increases the size and alters the lipid and protein composition of HDL particles. Could these changes in composition alter the immune or inflammatory function of HDL in such a way as to increase the risk of death from cancer or infection? Alternatively, off-target effects of torcetrapib could potentially account for the excessive rate of death from noncardiovascular causes through an unknown mechanism.
It is still possible that a CETP inhibitor without the off-target effects of torcetrapib could be viable as a therapy for cardiovascular disease. Mechanistic studies of torcetrapib's effects on the function of HDL cholesterol and the renin–angiotensin–aldosterone system and the investigation of a wide array of candidate genes to search for association with adverse outcomes in the ILLUMINATE study should be performed, along with a systematic study of outcomes in persons with homozygous CETP deficiency. It is possible that CETP inhibition might be better suited to certain subgroups of patients, such as those with impaired clearance of LDL cholesterol or low levels of HDL cholesterol at baseline. In any case, it is premature to announce the death of CETP inhibitors on the basis of the torcetrapib experience alone.
The results of the ILLUMINATE trial have led some observers to question the entire concept of targeting HDL cholesterol therapeutically. However, torcetrapib therapy is just one mechanism for raising levels of HDL cholesterol — and with a flawed molecule to boot. There remains substantial reason for cautious optimism regarding the therapeutic targeting of the metabolism of HDL and reverse cholesterol transport.1 Liver-X–receptor agonists promote reverse cholesterol transport and reduce atherosclerosis in animal models. Endothelial lipase inhibition is an approach that may raise HDL cholesterol levels through a mechanism that promotes reverse cholesterol transport and improves the function of HDL cholesterol. Apolipoprotein A-I mimetic peptides have been shown to improve HDL function and reduce atherosclerosis in animals. Up-regulation of apolipoprotein A-I transcription remains in many ways the "holy grail" of HDL-based therapies. New validated targets are certainly still needed in the HDL therapeutics arena and will probably come in part from genetic studies in humans with defined phenotypes.
In any event, the ILLUMINATE trial will undoubtedly stand as a watershed event in the field of HDL-targeted therapies. It may ultimately be seen as the study that brought about the rejection of the "HDL hypothesis." At a minimum, it will have been responsible for shifting the focus from HDL concentration to HDL function and raising the bar for approval of new HDL-targeted therapies. Despite the light that the ILLUMINATE study has shed onto part of the HDL journey, many poorly lit paths remain to be explored.


References
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2) Nissen SE, Tardif JC, Nicholls SJ, et al. Effect of torcetrapib on the progression of coronary atherosclerosis. N Engl J Med 2007;356:1304-1316. [Erratum, N Engl J Med 2007;357:835.]
3) Kastelein JJ, van Leuven SI, Burgess L, et al. Effect of torcetrapib on carotid atherosclerosis in familial hypercholesterolemia. N Engl J Med 2007;356:1620-1630.
4) Barter PJ, Caulfield M, Eriksson M, et al. Effects of torcetrapib in patients at high risk for coronary events. N Engl J Med 2007;357.
5) Brousseau ME, Diffenderfer MR, Millar JS, et al. Effects of cholesteryl ester transfer protein inhibition on high-density lipoprotein subspecies, apolipoprotein A-I metabolism, and fecal sterol excretion. Arterioscler Thromb Vasc Biol 2005;25:1057-1064.
6) 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.
7) Yvan-Charvet L, Matsuura F, Wang N, et al. Inhibition of cholesteryl ester transfer protein by torcetrapib modestly increases macrophage cholesterol efflux to HDL. Arterioscler Thromb Vasc Biol 2007;27:1132-1138.
8) Marney AM, Brown NJ. Aldosterone and end-organ damage. Clin Sci (Lond) 2007;113:267-278.
9) Shiflett AM, Bishop JR, Pahwa A, Hajduk SL. Human high density lipoproteins are platforms for the assembly of multi-component innate immune complexes. J Biol Chem 2005;280:32578-32585.
10) Vaisar T, Pennathur S, Green PS, et al. Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL. J Clin Invest 2007;117:746-756.