Figure 7 Exogenous aldosterone did not increase blood pressure. Mean arterial blood pressure was measured in pentobarbital (55 mg kg 1, i.p.)-anaesthetized rats that were administered aldos- terone as sequential 30 min infusions at 1 and 10 mg kg 1 or equivalent volumes of vehicle (saline). Fifteen minutes later, both groups of animals were administered torcetrapib (5 mg kg 1) as an i.v. infusion of 30 min duration. Each point represents the mean and vertical lines represent the s.e. for the numbers of animals indicated in parentheses. anacetrapib was without effect at concentrations up to 10 mM. Attempts to measure corticosterone levels by enzyme immunoassay in isolated adrenal cells were not successful using torcetrapib, anacetrapib or angiotensin II as agonists. Discussion Reduction of circulating LDL levels through the use of statins is a mainstay of the pharmacological management of atherosclerosis. However, despite their unequivocal efficacy, there is still considerable room for additional cardiovascular risk reduction in patients. Raising plasma HDL levels is an attractive objective to complement LDL-lowering drugs, particularly in light of the strong epidemiological relation- ship between increased HDL levels and reduced cardiovas- cular risk. There exist many potential strategies by which levels of HDL can be increased. Recently, in phase I and II studies, CETP inhibitors produced robust dose-dependent elevation of HDL levels (Grooth et al., 2002; Clark et al., 2004). However, early clinical studies with the CETP inhibitor torcetrapib, either alone or in combination with atorvastatin, demonstrated mild increases in systolic and diastolic blood pressure in humans (Davidson et al., 2006; McKenney et al., 2006). More recently, a phase III study of torcetrapib in combination with atorvastatin was terminated prematurely after approximately 18 months when the incidence of serious cardiovascular events, including death, was greater in the torcetrapib–atorvastatin group compared with subjects receiving atorvastatin alone (Barter et al., 2007). In this phase III study, there was also reported an increase in blood pressure and hypertensive adverse events in patients treated with torcetrapib. An evaluation of serum electrolytes revealed a decrease in serum potassium and increases in serum sodium and bicarbonate. The biochemical changes, along with the increase in blood pressure, suggested a possible mineralocorticoid excess that was further sub- stantiated by determining increases in serum aldosterone. In the present study, we report an acute increase in blood Aldosterone and pressor responses to torcetrapib MJ Forrest et al 1471 pressure following torcetrapib administration in a variety of preclinical animal species. Furthermore, we showed that torcetrapib administration caused an acute increase in plasma aldosterone and corticosterone levels in rats and that torcetrapib released aldosterone via a direct action on rat adrenocortical cells. The blood pressure response to torcetrapib is independent of inhibition of CETP. This conclusion is derived from two lines of evidence. The first is from data obtained with normal mice that do not express CETP and a line of transgenic mice expressing simian CETP. Torcetrapib increases HDL levels in the CETP transgenic mice but not in normal mice. However, torcetrapib increased blood pressure to an equivalent extent in both normal and CETP transgenic mice. The second line of evidence is that anacetrapib raises HDL to an equivalent extent as torcetrapib in CETP transgenic mice and at similar exposure levels as torcetrapib but, in contrast to torcetrapib, anacetrapib has no effect on blood pressure in either strain of mouse. Our observation that anacetrapib does not increase plasma adrenal steroids, at exposures that increase plasma HDL levels, strongly suggests that the mechanism of torcetrapib- induced adrenal steroid elevation is also not via a mechan- ism dependent on CETP inhibition. This was further substantiated by demonstrating that torcetrapib can directly release aldosterone from cultured primary rat adrenocortical cells in vitro in the absence of serum. Furthermore, anace- trapib had no effect on aldosterone release in this in vitro assay. The biochemical pathway(s) by which torcetrapib evokes aldosterone release are under investigation. Although the first biochemical step in the synthesis of aldosterone is the conversion of cholesterol to pregnenolone, the true rate- limiting step of hormone-stimulated steroidogenesis is the delivery of the substrate, cholesterol, to the inner mitochon- drial membrane (Stoccco and Clark, 1996). Whether torce- trapib is able to influence this transfer is not known but this would be a pathway worthy of further investigation. We have demonstrated that administration of torcetrapib to rats evokes an acute increase in blood pressure accom- panied by a rise in plasma adrenal steroids. We believe it is unlikely that the acute increase in blood pressure is mediated via aldosterone or corticosterone for the following reasons. First, as shown in Figure 7, acute administration of aldosterone (1 or 10 mg kg 1, i.v.) in rats had no effect on blood pressure under conditions where there was an acute blood pressure response to torcetrapib. Second, administra- tion of the mineralocorticoid receptor antagonist eplerenone (20 mg kg 1, p.o.) did not reduce the pressor effect of torcetrapib (data not shown). Finally, administration of the 3b-hydroxysteroid dehydrogenase inhibitor, trilostane, completely prevented the torcetrapib-induced increase in plasma aldosterone and corticosterone but did not reduce the torcetrapib-induced increase in acute blood pressure (Figure 6). It remains to be determined whether chronic administration of torcetrapib to rats would have a sustained effect on increasing blood pressure or adrenal steroid levels. It is conceivable, that chronic elevation of aldosterone or corticosterone in response to torcetrapib administration would provide additional pathways for increasing blood pressure via either genomic or non-genomic pathways (Ullian, 1999; Schiffrin, 2006). British Journal of Pharmacology (2008) 154 1465–1473