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Translational Review |
1 Cardiovascular Research Group, School of Clinical & Laboratory Sciences, Core Technology Facility (3rd Floor), University of Manchester, 46 Grafton Street, Manchester M13 9NT, UK
Abstract
There has been a fascinating interplay between the discovery of some of the key enzymes, receptors and transporters in cholesterol biosynthesis and transfer and the development of drugs for the regulation of cholesterol metabolism. The discovery of the low-density lipoprotein (LDL) receptor led to the realization that circulating LDL cholesterol could be decreased when hepatic LDL receptor expression was stimulated by decreasing intrahepatic cholesterol levels. The first class of drugs which operate in this way were the bile-acid sequestrating agents, which, by interrupting the enterohepatic circulation of bile acids, deplete the liver of cholesterol used to replenish the pool of bile salts. Ezetimibe, which was developed to block cholesterol absorption from the intestine, led to the discovery of the Nieman-Pick C1-Like 1 sterol transporter channel. The statins, which have proved enormously successful in preventing cardiovascular disease, were discovered amongst fungal metabolites which inhibit hydroxyl methyl CoA reductase, the rate-limiting enzyme for hepatic cholesterol biosynthesis. Drugs which block enzymes at other stages of the cholesterol biosynthetic pathway, particularly the squalene synthase inhibitors, are entering the clinical phase of their development. Drugs which interfere with hepatic very low-density lipoprotein assembly in the liver, such as microsomal triglyceride transfer protein inhibitors and apolipoprotein B mRNA antisense oligonucleotides, are currently undergoing evaluation. Cholesteryl ester transfer protein (CETP) inhibitors, which decrease cholesteryl ester heteroexchange within the circulation, have undergone development to the point of clinical evaluation, and this will eventually settle the controversy about whether CETP is pro- or antiatherogenic.
(Received 8 August 2007;
accepted after revision 11 October 2007; first published online 12 October 2007)
Corresponding author P. N. Durrington: Cardiovascular Research Group, School of Clinical & Laboratory Sciences, Core Technology Facility (3rd Floor), University of Manchester, 46 Grafton Street, Manchester M13 9NT, UK. Email: pdurrington{at}manchester.ac.uk
The complexities of the structure and biosynthesis of cholesterol have historically attracted much interest. The discovery of its multiple loop structure and its creation from its simple acetate precursor has made it, in terms of Nobel Prizes, the most decorated small molecule. The most recent Nobel Prize in the field of cholesterol metabolism was that of Goldstein and Brown for the discovery of the low-density lipoprotein (LDL) receptor and the elucidation of its pathophysiology (Brown & Goldstein, 1986).
This review will concentrate on drugs which modify intestinal cholesterol and bile-acid reabsorption, hepatic lipoprotein biosynthesis and catabolism, and cholesteryl ester heteroexchange within the circulation. We shall discuss bile-acid sequestrants (inhibitors of intestinal bile-acid absorption), ezetimibe (an inhibitor of intestinal cholesterol absorption), statin drugs (inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase (an enzyme in the cholesterol biosynthesis pathway), inhibitors of squalene synthase (another enzyme in the cholesterol biosynthetic pathway), apolipoprotein B (Apo B) mRNA antisense oligonucleotides [Apo B is the principal protein of very low-density lipoproteins (VLDL) and LDL], microsomal triglyceride transfer protein (MTP) inhibitors (MTP is essential for the assembly of VLDL) and inhibitors of cholesteryl ester transfer protein (CETP) activity [responsible for the transfer of cholesteryl ester from high-density lipoprotein (HDL) and LDL to triglyceride (TG)-rich lipoproteins].
Drugs principally modifying triglyceride metabolism, such as omega-3 fatty acids (Davidson, 2006), peroxisome proliferator activator receptor 
agonists (fibrates; Rosenson, 2007) and nicotinic acid (Guyton, 2007), will not be considered here, although fibrates and nicotinic acid do lower serum cholesterol, albeit to a lesser extent than triglycerides. In both cases, the mechanism for the decrease in cholesterol remains controversial. Early reports suggested that fibrates decreased cholesterol biosynthesis and increased biliary cholesterol excretion (Myant, 1981). More recently, the predominant mechanism by which they lower cholesterol has, however, been considered to be enhanced LDL receptor-mediated catabolism due to increased affinity of LDL for its receptor secondary to the decrease in the serum triglyceride concentrations (Gaw & Shepherd, 1999). In the case of nicotinic acid, the latter explanation for its cholesterol-lowering actions has also been invoked, but so also has a decrease in LDL production (Walldius & Wahlberg, 1992). Both nicotinic acid and fibrates may paradoxically increase serum cholesterol under certain circumstances, particularly when pretreatment triglycerides are raised and LDL cholesterol is relatively low.
Cholesterol metabolism
Cholesterol is an important component of cell membranes, where it occupies the spaces between the polar head-groups of the phospholipid molecular bilayer, reducing its fluidity (Myant, 1981). Cholesterol is also the precursor molecule for the synthesis of steroid hormones, vitamin D and bile salts. It is derived from the diet or synthesized within the body. The typical human diet contains 200–500 mg of cholesterol. Cholesterol also enters the intestine in bile (800–1200 mg day–1) and as desquamated intestinal epithelial cells (300 mg day–1). Some 30–60% of intestinal cholesterol is absorbed. The principal sites of cholesterol biosynthesis are the liver and the CNS. Cholesterol can be lost from the body as the fractions of bile salts and intestinal cholesterol which are not absorbed, and in sebum. The daily faecal loss of cholesterol from bile and desquamated cells is 550 mg and as unabsorbed bile salts 250 mg. Daily losses in sebum are 100 mg. A total of some 900 mg must therefore be derived from the diet or synthesized each day (Levy et al. 2007).
Cholesterol circulates as a component of lipoproteins. Its concentration in humans is typically in the range 100–300 mg dl–1 (2.5–7.5 mmol l–1). In many Asian countries, adult levels are often less than 200 mg dl–1 (5 mmol l–1), whereas in Europe and the USA they are generally greater than 200 mg dl–1 (5 mmol l–1; Mackay & Mensah, 2004). At birth, serum cholesterol levels are similar throughout the world, typically around 80 mg dl–1 (2 mmol l–1; Bansal et al. 2005).
The principal plasma lipoproteins are the chylomicrons, VLDL, LDL and HDL; Durrington, 2007; Fig. 1). The evidence that chylomicron remnants, VLDL and LDL cause atherosclerosis and that HDL opposes this is the subject of many reviews and will not be further discussed here (Lusis, 2000; Williams et al. 2007). Chylomicrons are secreted by enterocytes into the lacteals of the intestine. They enter the blood circulation from lymph via the thoracic duct. They are rich in triglyceride. Triglyceride, the principal fat in the diet, is absorbed from mixed micelles (see intestinal cholesterol absorption) formed in the intestinal lumen as fatty acids and monoglycerides after its hydrolysis by intestinal and pancreatic lipases. In the enterocyte, triglyceride is resynthesized and complexed with Apo B48, a process involving MTP (Olofsson et al. 2000), to form chylomicrons. Fatty acids of shorter chain length (< 8–10 carbons) escape this process and enter the portal blood directly. Free cholesterol is also absorbed from mixed micelles by the enterocytes from the gut lumen. It is largely re-esterified and is packaged with triglyceride to form the core of chylomicrons.
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The secretion of VLDL by the liver involves the complexing of triglyceride with Apo B100 during its translation in the endoplasmic reticulum under the agency of MTP (Olofsson et al. 2000). Homozygous mutations of the latter are the basis of abetalipoproteinaemia, in which VLDL and LDL are virtually absent from the circulation, there is little or no chylomicron formation and intestinal fat malabsorption and fat-soluble vitamin deficiencies occur (Hooper & van Bockxmeer, 2005; Durrington, 2007). In a more common disorder, familial hypobetalipoproteinaemia, resulting from mutations of the APOB gene which cause premature truncation during its translation, heterozygotes are often asymptomatic despite having LDL-C levels of one-quarter to one-third of normal (Schonfeld et al. 2005). In the much rarer homozygotes or compound heterozygotes for this condition, the clinical features are similar to abetalipoproteinaemia (Hooper & van Bockxmeer, 2005; Durrington, 2007). From the endoplasmic reticulum, the newly synthesized VLDL progresses via the Golgi complex to secretory vesicles, acquiring in the process unesterified cholesterol and additional triglycerides. When hepatic triglyceride synthetic rates are high, as in hypertriglyceridaemia associated with central obesity and insulin resistance, whether or not frank diabetes has developed, VLDL will be large and loaded with triglycerides. Triglycerides, which cannot be accommodated in VLDL, accumulate in the liver, giving rise to fatty liver disease (non-alcoholic steatohepatitis). Once in the circulation, large VLDL is a particularly avid acceptor of cholesteryl ester from HDL and LDL. This transfer occurs because of the presence of CETP in human plasma (Barter et al. 2003). This protein is not present in species, such as the rat, which have low levels of circulating LDL. Human cholesterol metabolism is also different from the rat because cholesterol is largely secreted from the human liver as unesterified cholesterol, whereas in the rat it is esterified before secretion. Unesterified cholesterol can only be accommodated in lipoproteins in limited quantities. It is confined to their outer shells, because unesterified cholesterol has a charged hydroxyl group which interacts with water. Esterification of this group creates cholesteryl ester, which is lipophilic and can thus enter the hydrophobic core of lipoproteins. The enzyme which allows this esterification to occur in plasma is lecithin:cholesterol acyl transferase (LCAT). This enzyme is largely located on HDL. Unesterified cholesterol can reach HDL from VLDL along a concentration gradient, but to return, once it has been esterified, requires CETP (Barter et al. 2003). Activity of CETP is a major determinant of HDL cholesterol levels.
During its circulation, VLDL undergoes a similar sequence of events to chylomicrons, namely the progressive removal of triglyceride from its core by lipoprotein lipase. In the case of VLDL, however, the smaller cholesteryl ester-rich lipoprotein particle formed is LDL, which is small enough to cross the vascular endothelium to enter the tissue fluid where, in the human, it supplies tissues with cholesterol. One exception is the CNS, because LDL does not cross the blood–brain barrier. The CNS produces its own lipoprotein, which is an Apo E-rich HDL (Ladu et al. 2000). Cholesteryl ester from this can enter nerve cells and parenchymal cells of the CNS via LDL receptors, because these recognize Apo E as well as Apo B100 as a ligand. This type of lipoprotein metabolism, in which LDL levels are low (or in the case of CNS absent) and HDL supplies cholesterol to tissues, is typical of many animals, such as the rat and the human fetus. In the adult human, HDL is involved in the transfer of excess cholesterol arriving at the tissues in LDL back to the liver (reverse cholesterol transport). It can receive this cholesterol via the ABCA1 receptors of extrahepatic tissues as well as the liver and then transfer it to VLDL and thence LDL from where it can complete its passage back to the liver when LDL is removed by hepatic LDL receptors. Alternatively, it can leave HDL to enter hepatocytes during the circulation of HDL through the liver via the scavenger receptor B1 (SRB1; Krieger, 2001). The SRB1, unlike the LDL receptor, does not degrade the lipoprotein particles binding to it, but returns them to the circulation after it has acted as a portal for the entry of cholesterol to the liver.
Bile salt metabolism
Bile salts are detergents synthesized by the liver from cholesterol and released into the duodenum with bile. Regulation of this pathway is principally at the 7-hydroxylation of cholesterol. The primary bile salt acids are cholic acid and chenodeoxycholic acid (Gibbons et al. 1982). They are usually conjugated with glycine or taurine. In the lower small intestine and colon, as a consequence of the activity of anaerobic bacteria (principally Bacteroides), they undergo a variety of transformations, including deconjugation and conversion of cholic acid to deoxycholic acid and of chenodeoxycholic acid to lithocholic acid. These are the secondary bile acids. The primary bile acids and some of the secondary ones, deoxycholic acid in particular, are absorbed through the terminal ileum. They are extracted from the blood circulation by the liver, conjugated and secreted back into bile. Bile salts emulsify fats by virtue of their polar carboxyl and hydroxyl groups, which are hydrophilic, and their lipophilic hydrocarbon rings, which interface with lipids.
Intestinal cholesterol absorption
Cholesteryl ester is converted into unesterified cholesterol in the intestine by cholesterol esterase and, together with unesterified cholesterol from the diet and bile, joins plant sterols from the diet in mixed micelles (Fig. 3) which, in addition to cholesterol, are composed of bile salts, monoglycerides, phospholipids, lysophospholipids and fatty acids. Cholesterol and other sterols from the smallest of these (40 Å diameter) enter the enterocytes through the recently discovered Nieman-Pick C1-Like 1 (NPC1L1) channel (Huff et al. 2006; Wang, 2007; Levy et al. 2007). Sterols other than cholesterol entering the enterocytes via NPC1L1 are prevented from entering the body to any significant extent because they are transported back into the intestinal lumen by the ATP binding cassette G5/G8 (Kusuhara & Sugiyama, 2007). This transporter comprises the two half-transporters ABCG5 and ABCG8. Mutations of these leads to the excessive accumulation of plant sterols (phytosterols), a disorder called sitosterolaemia, in which phytosterols accumulate in tendons and arteries (Durrington, 2007).
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3-Hydroxy-3-methyl-glutaryl-CoA reductase (EC 1.1.1.3 [EC] 4) is situated on the endoplasmic reticulum and catalyses the conversion of HMG-CoA to mevalonic acid (Fig. 4). 3-Hydroxy-3-methyl-glutaryl-CoA reductase is subject to both short-term and long-term control (Goldstein & Brown, 1990). Long-term effects are mediated through alterations in its rate of synthesis and degradation. Short-term effects involve allosteric effects and alterations in its state of phosphorylation. Cholesterol entering the liver from the diet (from chylomicron remnants, LDL or HDL) suppresses hepatic HMG-CoA reductase and thus cholesterol synthesis. The effect is mediated by an increase in intracellular free cholesterol. If hepatic cholesterol biosynthesis and the supply of endogenous (intestinal) cholesterol to the liver declines to the point where intrahepatic cholesterol levels decrease, hepatic LDL receptor expression is induced. The liver thus extracts more LDL from the circulation, decreasing LDL levels. This is the final common mechanism by which bile-acid sequestrating agents, statins, squalene synthase inhibitors and ezetimibe, reviewed here, lower serum cholesterol, thus enhancing LDL catabolism (Durrington, 2007).
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Squalene synthase (EC 2.5.1.2 [EC] 1) is the enzyme in the cholesterol biosynthetic pathway responsible for conversion of farnesyl pyrophosphate to lanosterol (Gibbons et al. 1982). Its particular importance is that it is the first enzyme in the pathway responsible for the production of a metabolite which is solely committed to cholesterol synthesis.
The LDL receptor
The LDL receptor (Fig. 2) was discovered by J. L. Goldstein & M. S. Brown in 1974, when they found that while LDL would inhibit cholesterol synthesis in cultured fibroblasts, HDL would not, and that the inhibitory effect of LDL was absent in fibroblasts from patients who were homozygotes for familial hypercholesterolaemia (Brown et al. 1974). They and other workers then went on to reveal in detail the fascinating biochemistry of the receptor, which has contributed not only to our knowledge of lipoprotein metabolism, but has also led to advances in our general understanding of receptors and molecular genetics (Brown & Goldstein, 1986).
The gene for the LDL receptor is located on chromosome 19, contains some 45000 base pairs and includes 18 exons and 18 introns (Brown & Goldstein, 1986). The receptor protein itself contains 839 amino acids. Its apparent molecular weight immediately after synthesis is about 120 000 Da, but it subsequently acquires carbohydrate in the Golgi apparatus and undergoes changes in its molecular conformation, altering its electrophoretic mobility, and thus the estimated molecular weight of the mature protein is in the region of 160 000 Da. The receptor migrates to the cell surface (Fig. 5), the interval between synthesis and arrival in the coated pit averaging 45 min. There it enters a cycle in which it enters the cell by invagination of coated pits and closure of their necks to form coated endocytic vesicles. These rapidly lose their clathrin coat and fuse to form larger vesicles (endosomes or receptorsomes). The ATP-dependent proton pumps in their walls lower the pH of the enclosed fluid, and the LDL receptor–LDL complex dissociates. The released LDL receptor leaves the endosome and migrates back to the surface, linking up with other receptors in the coated pit region. The whole cycle is believed to take approximately 10 min.
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The complex nature of the events from the synthesis of the LDL receptor to the successful entry of the receptor into the recycling process and the completion of that process means that the clinical syndrome of familial hypercholesterolaemia (Durrington, 2007) can be produced by a variety of gene mutations.
The receptor has at its amino end (first domain; Fig. 2) the region which binds to Apo B and Apo E. It contains seven repetitive sequences, each of 40 amino acids. Of these, about seven are cysteine residues, which form disulphide bridges retaining a rigidly cross-linked structure in their part of the molecule. Negatively charged clusters of amino acids are displayed which complement the positively charged receptor binding sites of Apo E and Apo B.
The binding site region of the receptor molecule is adjacent to a long sequence of amino acids homologous to part of the epithelial growth factor (EGF) precursor. It should be noted that there is no homology with the portion of EGF released. Thus, although the homology may be informative about how different proteins have evolved, it does not suggest that there is a functional link between the two proteins. The same is probably true of the sequences in the receptor binding part of the molecule which have similarities with the C9 component of complement.
The next sequence of amino acids is rich in sugar, and this leads on to a hydrophobic region of the molecule, which spans the cell membrane. The final, carboxyl end of the molecule extends into the cytoplasm, and its interaction with clathrin is essential for the arrival of the receptor in the coated pit region of the cell membrane.
Synthesis of the LDL receptor is suppressed when the cell is replete in cholesterol. In this regard, LDL receptor-mediated catabolism differs from that of chylomicron remnants, for which the hepatic receptor-mediated pathway for their removal from the circulation is not downregulated by entry of cholesterol into the liver. In tissue culture, the LDL receptor is saturated when the concentration of LDL cholesterol in the medium is 2.5 mg dl–1 (0.065 mmol l–1). The concentration of LDL cholesterol in the extracellular fluid of most tissues is about one-tenth of that in the plasma. Thus, if the behaviour of cells in tissue culture can be extrapolated to whole man, a plasma LDL cholesterol of 25 mg dl–1 (0.65 mmol 1–1) should saturate the LDL receptor and provide the cholesterol requirement of most cells. In the newborn human, the serum LDL cholesterol concentration is about 30 mg dl–1 (0.8 mmol l–1). Furthermore, in humans who subsist on a diet low in fat, even as adults LDL cholesterol is generally in the range 50–80 mg dl–1 (1.3–2.1 mmol l–1). The serum LDL cholesterol concentration of much of the population of countries with a Northern European diet, however, exceeds 120 mg dl–1 (3.0 mmol 1–1), far more than is required to supply cholesterol to the tissues.
Bile-acid sequestrating agents
The bile-acid sequestrating agents or resins that are currently available are colestyramine, colestipol and colesevelam. Their mode of action is usually considered to be similar. They are anion exchange resins which bind bile acids in the intestinal lumen. They are not absorbed, so the reabsorption of bile acids, which normally occurs in the terminal ileum, is impeded and those bound are lost to the body with the bile-acid sequestrant in the faeces. The faecal bile-acid output is thus considerably increased (Hunninghake, 1999). Failure of reabsorption of bile acids is compensated by their enhanced hepatic synthesis from cholesterol, which is their precursor, via increased activity of cholesterol-7-hydroxylase, which is rate limiting for bile-acid synthesis (Myant, 1981; Schwarz et al. 1998). Therapy with bile-acid sequestrants has been shown to lower circulating LDL cholesterol by increasing hepatic catabolism via the LDL receptor-mediated pathway. The increased LDL receptor expression occurs in response to depletion of intrahepatic cholesterol as it is consumed in bile salt synthesis. Bile-acid sequestrating agents are effective in lowering serum LDL cholesterol, and even in heterozygotes for familial hypercholesterolaemia with only one fully functional LDL receptor gene, they increase receptor-mediated LDL catabolism (Hunninghake, 1999). Patients who are homozygotes for familial hypercholesterolaemia do not generally respond to bile-acid sequestrants. Colesevelam is a newer bile-acid sequestrant which causes fewer side-effects and, in combination with a statin, has been shown to decrease C-reactive protein levels more markedly than with statin alone (Bays et al. 2006), which might confer greater protection against coronary heart disease (CHD).
Although colestyramine has been in clinical use since the 1960s, it was not until the 1980s that a randomized clinical trial with cardiovascular end-points, the Lipid Research Clinics Coronary Primary Prevention Trial (LRC-CPPT), was conducted. Lowering cholesterol with cholestyramine significantly decreased coronary events (Lipid Research Clinics Program, 1984). After a mean follow-up of 7.4 years, patients assigned to cholestyramine had an average 8.5% decrease in plasma cholesterol and a 19% reduction in coronary events. In compliant patients, bile-acid sequestrants as monotherapy can decrease LDL cholesterol by substantially more than this (Betteridge et al. 1992), but their greatest current use is in combination with statin when the response to statin alone is inadequate.
Two potentially important adverse effects of bile-acid sequestrating agents are their effect on the metabolism of other drugs and their effect on triglyceride metabolism. Bile-acid sequestrating agents bind anions non-specifically and thus may interfere with absorption of any substance which is anionic at intestinal pH. This applies to drugs such as statins, thiazide diuretics, furosemide, spironolactone, fenofibrate, tricyclic antidepressants, oral corticosteroids, some sulphonylureas, digoxin, thyroxine, vitamin K; raloxifine, lopermide and a number of non-steroidal antinflammatory drugs. These drugs should not be administered until at least 4 h have elapsed after the last dose of bile-acid sequestrant (Jacobson et al. 2007).
The effect of bile-acid sequestrants on triglyceride metabolism is not favourable. They are not suitable agents for lowering raised cholesterol associated with raised triglycerides. A slight increase in triglycerides, which is usually clinically unimportant, occurs in hypercholesterolaemic patients even when their triglycerides are initially normal. In patients where both LDL and VLDL levels are raised, the cholesterol may be lowered, but there may be an accompanying substantial rise in triglycerides. This difficulty may be overcome by adding treatment with a fibrate drug, nicotinic acid or omega-3 fatty acids. In patients with more severe hypertriglyceridaemia, bile-acid sequestrants are contraindicated. An exacerbation of hypertriglyceridaemia may precipitate acute pancreatitis. The reason for the elevation in serum triglycerides is uncertain, but may involve an increase in hepatic triglyceride synthesis, mediated by activation of phosphatidate phosphohydrolase.
In theory, cholelithiasis might be expected to be an adverse effect of the bile-acid sequestrants, because of depletion of the bile-salt pool. In hypercholesterolaemic patients prescribed these drugs, however, this does not appear to be the case, but it might be if bile-acid sequestrant were used in combination with a fibrate drug which increases biliary cholesterol saturation. Statins, in contrast, tend to decrease bile saturation (Smith et al. 2002). Steatorrhoea and fat-soluble vitamin deficiencies have been reported with the use of bile-acid sequestrants, but this is really only likely to be encountered in patients with short bowel or terminal ileal disease (which, of course, they can help symptomatically if cholorrheic enteropathy is causing diarrhoea). Folate supplements should also be given to children, pregnant women and women planning pregnancy receiving bile-acid sequestrants. Cholestyramine is prescribed for pruritus associated with pregnancy. For patients with chronic biliary obstruction which cannot be surgically relieved, for example those with primary biliary cirrhosis, bile-acid sequestrants are a means of controlling both pruritus and the rise in cholesterol, particularly where there are concerns that a statin might adversely affect liver function.
Ezetimibe
Ezetimibe is not a bile-acid sequestrant, but selectively blocks absorption of dietary and biliary cholesterol from the gut by blocking uptake of cholesterol into jejunal enterocytes (Altman et al. 2004). Ezetimibe binds to NPC1L1 located in the brush border of the proximal jejunum (Altmann et al. 2004; Garcia-Calvo et al. 2005). Niemann-Pick type C disease is an inherited disorder associated with defective cholesterol transport in the liver and elsewhere that results in excessive free cholesterol accumulation (Chang et al. 2005).
Ezetimibe is increasingly finding use in the clinic as an LDL cholesterol-lowering agent, although randomized, clinical events trials are yet to be reported. Ezetimibe has a limited LDL cholesterol-lowering effect of around 15–20%, either alone or in the presence of a statin (Mikhailidis et al. 2007). It acts by decreasing the intestinal cholesterol supply to the liver, lowering hepatic cholesterol levels and thus inducing LDL receptor expression. It is effective because it not only decreases the absorption of dietary cholesterol, but also interrupts the enterohepatic circulation of cholesterol entering the intestine in bile. Its limitation is the upregulation of hepatic cholesterol biosynthesis. The NPC1L1 haplotype is a determinant of interindividual variation of the LDL response to ezetimibe (Hegele et al. 2005). Nonetheless, the LDL decrease over and above the statin effect is critically worthwhile now that therapeutic targets for LDL cholesterol have been lowered (Grundy et al. 2004). Unlike the bile-acid sequestrants, ezetimibe has few gastrointestinal side-effects and does not interfere with absorption of fat-soluble vitamin or drugs, such as statins. Furthermore, ezetimibe does not increase serum triglycerides.
Sitosterolaemia, a rare autosomal recessive lipid disorder associated with hyperabsorbtion of plant-derived sterols (as well as cholesterol) from the diet (see ‘Intestinal cholesterol absorption’), is associated with high concentrations of cholesterol and sitosterol in plasma and is characterized by tuberose and tendon xanthomas, premature cardiovascular disease and polyarthralgia (Durrington, 2007) Ezetimibe is uniquely effective in suppressing both cholesterol and sitosterol absorbtion from the gut (Salen et al. 2004), presumably acting at the NPC1L1 transporter (Garcia-Calvo et al. 2005). The bile-acid sequestrants also lower sitosterol absorption, but ezetimibe appears to more effective in improving symptoms with few, if any, side-effects.
Statins (HMG-CoA reductase inhibitors)
A major advance in therapy stemmed from the discovery of the statins (Endo et al. 1976; Endo, 1992; Steinberg, 2006), a class of fungal metabolites that inhibit HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Compactin (subsequently renamed mevastatin) was the original compound studied, but because of possible adverse effects in animals it did not undergo full clinical evaluation in man. Mevinolin, later named lovastatin, was the first analogue of compactin to receive extensive clinical use, but other related compounds have since entered clinical practice. These include simvastatin, a methylated derivative of mevinolin, and pravastatin, a hydroxylated derivative of compactin (Fig. 6). Fluvastatin was the first statin produced entirely by chemical synthesis, followed by atorvastatin and more recently rosuvastatin.
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The effect of statins on HDL metabolism is likely to be complex. Even at doses which have little effect on HDL cholesterol concentrations, the HDL particle size may be increased (Soedamah-Muthu et al. 2003). The effect of depleting the liver of cholesterol, which is after all the primary aim of statin therapy (in order to induce hepatic LDL receptors), will tend to diminish the cholesterol effluxing from the liver through ABCA1. This would otherwise enrich Apo A1 and small HDL particles to produce larger HDL particles, big enough to avoid filtration in the renal glomerulus and thus renal catabolism, as occurs in Tangier disease, in which ABCA1 is defective. There may also be an increased uptake of cholesterol from HDL circulating through hepatic sinusoids via SRB1, if the statin-induced decrease in intrahepatic cholesterol increases uptake through SRB1. Both these mechanisms would tend to lower HDL cholesterol. Operating against these would be a decrease in CETP activity in response to statins (Bhatnagar et al. 1995; Caslake et al. 2003). In part, this may be due to the decrease in the circulating VLDL, intermediate-density lipoprotein (IDL) and LDL levels, since these are acceptors for cholesteryl ester from HDL. All these mechanisms would reward further research. Probably the best documented is the statin effect on CETP activity and the least certain is the possible effect on SRB1. The combined effect of these mechanisms is generally to produce a small increase in HDL cholesterol.
The decrease in triglycerides with statins is also more variable than that in LDL cholesterol (Stein et al. 1998). It is generally smaller in terms of percentage reduction and shows less dose dependency. It is greatest in patients with raised triglycerides, although few studies have been undertaken when levels exceed 400 mg dl–1 (4.5 mmol l–1) and with statins which are more effective in lowering LDL cholesterol. The effect of statins in decreasing serum triglycerides appears to be largely, if not entirely, due to enhanced removal not only of LDL, but also of the more triglyceride-rich VLDL and IDL as a consequence of increased LDL receptor activity. They also increase postprandial triglyceride clearance, part of which is due to uptake of chylomicron remnants by the LDL receptor. Studies using turnover techniques have also revealed a decrease in VLDL, Apo B and triglyceride production associated with statin treatment which, it might be thought, would contribute to the decrease in triglyceride with statins. However, it is also the case that the effect of decreasing intrahepatic cholesterol is to induce the expression of sterol regulatory element binding proteins (SREBP; Brown & Goldstein, 1997). Whilst these induce LDL receptor expression, they also induce key enzymes in triglyceride biosynthesis.
The increased SREBP expression associated with statin treatment has another major adverse effect: it increases the synthesis of HMG-CoA reductase, which thus opposes the competitive inhibition of the statin on cholesterol biosynthesis. This is the main pharmacological limitation imposed on the degree of LDL lowering which can be achieved with statin treatment. Inhibition of HMG-CoA reductase with drugs which are non-competitive, such as apomine (Roitelman et al. 2004), could theoretically improve on the LDL lowering effect of statins. So also could the introduction of drugs which inhibit the cholesterol biosynthetic pathway downstream of mevalonate, such as the squalene synthase inhibitors (Charlton-Menys & Durrington, 2007; see squalene synthase inhibitors), which are currently undergoing active clinical evaluation.
Pleiotropic effects of statins
There has been much speculation that statins may have effects which are unexplained by the changes in serum lipoprotein levels they produce. The concept is given plausibility because mevalonic acid produced by HMG-CoA reductase is not only rate limiting for cholesterol biosynthesis, but also for geranylgeranyl pyrophosphate and farnesyl pyrophosphate, which are known to be important metabolic regulators. Prenylation of proteins (post-translational modification of proteins by farnesyl pyrophosphate or geranylgeranyl pyrophosphate) regulates the subcellular location of G-proteins influencing many signalling cascades within the cell (Edwards & Ericsson, 1999). Oxysterols and farnesyl pyrophosphate derived from the cholesterol biosynthetic pathway after mevalonate also affect the activity of nuclear orphan receptors, such as liver X receptors and farnesoid X-activated receptors (Edwards et al. 2002).
The case for the pleiotropic effects of statins on atherosclerosis is advanced on the basis that the variation in reduction in CHD risk in statin trials is only partly explicable on the basis of changes in LDL cholesterol, HDL cholesterol and triglycerides. Effects on other atherogenic risk markers, particularly C-reactive protein, platelet aggregation or even homocysteine, have been suggested as the basis for this, and experiments with statins in tissue culture have also shown potential antiatherogenic direct actions of statins on the cells involved in atherogenesis.
However, before invoking pleiotropy to explain some of the effect of statins in preventing CHD and stroke, it should be realized that the methods of measuring LDL cholesterol used in large clinical trials may underestimate the beneficial effect of statins on lipoproteins. The LDL cholesterol measurement embraces the whole of the LDL spectrum from 1.006 to 1.063 g ml–1. Much of the cholesterol-rich LDL in the middle of this range many not be particularly atherogenic and, were we to have more direct measurement of the statin effect on the intermediate-density lipoprotein in the range 1.006–1.019 g ml–1 and the dense, cholesterol-depleted LDL in the 1.044–1.063 g ml–1 range (LDL III), we should have a better explanation for their effect in reducing CHD risk. The effect of statins in lowering C-reactive protein (CRP), which may be a direct effect on hepatic production of CRP, could, however, be a true pleiotropic effect, since it appears to be unrelated to LDL lowering. The question about CRP is whether its association with atherosclerosis is causal. A trial of rosuvastatin in people identified as at high CVD risk by raised CRP levels is currently underway (Mora & Ridker, 2006). The putative direct effect of statins on the cells of the arterial wall and other extrahepatic tissue must also at this stage be viewed with caution, because probably < 1% of the statin dose is distributed outside the liver. In contrast, it must be admitted that the statin effect in decreasing stroke risk (Law et al. 2003; Baigent et al. 2005) would not be anticipated from epidemiological studies, which often show very little relationship between serum cholesterol and stroke risk (Kurth et al. 2007). Therefore, either epidemiology is misleading in this respect and LDL really is important in cerebral atherosclerosis or perhaps CRP or some other factor modified by statins is critical for stroke.
Squalene synthase inhibitors
Squalene synthase inhibitors decrease circulating LDL cholesterol by the induction of hepatic LDL receptors in a similar manner to statins. They have fewer secondary effects mediated by a decrease in non-cholesterol products of mevalonate metabolism distal to HMG-CoA reductase, but have the potential to increase intermediates proximal to squalene. It might be expected that there would be important differences between cholesterol-independent effects of squalene synthase inhibitors and those of HMG-CoA reductase, because squalene synthase, which acts downstream of mevalonate, is the first committed step of hepatic cholesterol biosynthesis at the final branch point of the cholesterol biosynthetic pathway (Fig. 4; Gibbons et al. 1982). Inhibition of squalene synthase may avoid the risk of myotoxicity, because intermediates formed before squalene, responsible for example, for prenylation of proteins, are not depleted (Flint et al. 1997). This, however, requires more confirmation in vivo. The squalene synthase inhibitor TAK-45 (Nishimoto et al. 2003) decreases circulating LDL levels and lacks the hepatotoxicity found with atorvastatin in cynomolgus monkeys. More recently, two new potent squalene synthase inhibitors (EP2306 and EP2302) have been described (Tavridou et al. 2006). The squalene synthase inhibitor EP2302 inhibited cholesterol synthesis in a dose-dependent manner with a similar potency to that of simvastatin.
3-Hydroxy-3-methyl-glutaryl-CoA reductase is the site of physiological regulation of cholesterol biosynthesis, making it unlikely that accumulation of metabolites earlier in the pathway (Fig. 3) would be toxic, but this is not necessarily true of squalene synthase inhibition. Triparanolol, another inhibitor of cholesterol biosynthesis downstream of mevalonate, was found to cause cataract formation (Laughlin & Carey, 1962; Steinberg, 2006). More recently, an association between lanosterol synthase (Fig. 4) mutations and cholesterol deficiency resulting in cataract formation in a rat model was described (Mori et al. 2006). However, in these rats the cholesterol deficiency was confined largely to the lens. In contrast, hepatic and serum levels of cholesterol were not decreased. Since vertebrate eye lenses are not vascularized, de novo cholesterol synthesis in the lens is necessary for normal proliferation of epithelial cells in the lens. This suggests that the gene or isoform of the gene regulating lens cholesterol synthesis differs from the gene regulating hepatic cholesterol synthesis. Statins have not been found to cause cataract formation in clinical trials. There is, however, evidence that in much higher doses than used in the clinic they can cause cataract formation in rats, and this would appear to be due to a failure of upregulation of lens HMG CoA reductase (Cenedella et al. 2005). Decreased ubiquinone levels may also be a factor. Selective inhibition of another enzyme, oxidosqualene cyclase, with U18666A has also more recently been reported to induce cataract formation in rats. However, this may not be wholly due to inhibition of cholesterol synthesis, because U18666A has been shown to have a direct toxic effect on lens epithelial cells (Cenedella et al. 2004), perhaps by increasing formation of oxysterols. These findings suggest that squalene synthase inhibitors may prove to have less muscle toxicity than statins and not to have the toxic effect on the lens of the eye of oxidosqualene cyclase inhibitors or triparanol. However, it remains for more detailed clinical assessments to be undertaken. Squalene synthase inhibitors might lack some of the favourable pleiotropism of statins and could have other side-effects related to accumulation of metabolites upstream of squalene synthase.
Microsomal triglyceride transfer protein (MTP) inhibitors and Apo B mRNA antisense oligonucleotides
An alternative approach to lowering LDL-C is to limit hepatic assembly of VLDL, the precursor of LDL. One strategy is to develop inhibitors of MTP. Whilst this may effectively lower LDL cholesterol, it also causes hepatic triglyceride accumulation (Cuchel et al. 2007). Another approach is to use Apo B mRNA antisense oligonucelotides (Crooke et al. 2005). These hold the promise of preventing VLDL formation without causing hepatic steatosis (Akdim et al. 2007). It is known that in the disorder hypobetalipoproteinaemia, hepatic steatosis is a likely complication only when the Apo B truncation occurs after less than 27% of the Apo B message has been translated and that truncation after 48% of the Apo B sequence has been translated leaves chylomicron formation unaffected whilst still decreasing LDL (Schonfeld et al. 2005). Such truncations are possible with antisense oligonucleotides (Khoo et al. 2007). Interestingly, many species, including the rat, which have substantially lower circulating LDL levels than man, physiologically produce both Apo B48 and Apo B100 from the liver.
Inhibitors of CETP
Cholesteryl ester transfer protein allows the transfer of cholesteryl ester from HDL to VLDL and LDL. When its activity is high, HDL levels are generally low. Although the movement of cholesteryl ester from HDL to VLDL and LDL can complete the process of reverse cholesterol transport when LDL is removed from the circulation by the liver, the process also potentially adversely contributes to the pool of circulating atherogenic VLDL and LDL. The activity of CETP is not generally determined by its concentration except in extreme circumstances, such as genetic CETP deficiency (Boekholdt et al. 2004). Often more important as a determinant of CETP activity is the concentration of serum triglycerides. For physicochemical reasons, triglyceride-rich VLDL appears to be a highly efficient acceptor of cholesteryl ester from CETP. This is probably the major reason that drugs such as fibrates and statins decrease CETP activity, through their action in lowering triglycerides. It has been difficult for the scientific community to decide whether CETP is a good thing or a bad thing. By removing cholesteryl ester from larger HDL, it creates a smaller HDL particle which can accommodate more cholesterol and may be a better acceptor of cholesterol from tissues. Also, as has already been stated, the transfer of cholesteryl ester to Apo B-containing lipoproteins can, if they are then cleared by the liver, be viewed as an important component of reverse cholesterol transport. This may be why probucol, which raises CETP activity (and lowers HDL levels), anecdotally sometimes caused rapid resolution of xanthomata (Durrington, 2007). Many animal species, such as the rat and mouse, lack CETP. Such creatures tend to be resistant to atherosclerosis and to have hardly any LDL-like lipoproteins. In these species, HDL (in an Apo E-rich form) is employed to transport cholesterol to the tissues. Expressing CETP in some animal models that naturally lack it makes them susceptible to atherosclerosis, whereas in others it may protect them against atherosclerosis (Barter & Kastelein, 2006). Furthermore, there has been dispute about whether human CETP deficiency, most frequently reported in Japanese men and associated with very high HDL levels, does reduce CHD risk (Barter & Kastelein, 2006). Perhaps a complete absence of CETP in species such as man, with high levels of Apo B-containing lipoproteins, compromises reverse cholesterol transport. The abnormally large HDL in genetic CETP deficiency is replete in cholesterol and a poor acceptor of tissue cholesterol. In contrast, a partial decrease in CETP activity might help to decrease LDL formation and perhaps be beneficial if the raised levels of HDL assist in preventing atherosclerosis (Link et al. 2007) by retaining their direct anti-inflammatory and antioxidative properties (Durrington et al. 2001; Schaefer & Asztalos, 2006; Ansell et al. 2007).
Recently, two types of CETP inhibitors, exemplified by JTT-705 and torcetrapib, have undergone development to the point of human testing. In humans, at a dose of 600 mg daily, JTT-705 raised HDL cholesterol by 28% compared with placebo in patients also receiving pravastatin 40 mg daily (Kuivenhoven et al. 2005). Torcetrapib at a dose of 120 mg daily or twice daily raised HDL by more than 50% over and above any effect of atorvastatin 20 mg daily (Brousseau et al. 2004). The increase in HDL with both drugs was due to large HDL particles and was accompanied by a decrease in Apo A1 catabolism. It has been strongly argued that increases in circulating large HDL particles with CETP inhibition are not as extreme as in genetic CETP deficiency and that the larger particles produced by CETP inhibition are likely to decrease the risk of atherosclerosis (Schaefer & Asztalos, 2006). A reduction in the smaller LDL particles also occurs (Brousseau et al. 2004), which might be expected to have a favourable effect. The early discontinuation of a randomized clinical trial of torcetrapib because of its unfavourable effect on the incidence of cardiovascular disease was thus unanticipated (Tall et al. 2007). Torcetrapib may have some unfavourable effect not related to its action in inhibiting CETP, but nonetheless the concept that CETP is proatherogenic remains to be proven.
Conclusion
Although coronary mortality is falling in the USA and Europe owing to improved pharmacotherapy, including statins, and surgical interventions, morbidity continues to increase. Furthermore, the favourable trend in mortality may be reversed because of the increasing incidence of obesity in young people. Globally, coronary disease rates are set to rise dramatically as the more populace parts of Asia become increasingly wealthy and adopt a coronary-prone lifestyle. There will thus be a continuing impetus for on-going research into cholesterol metabolism and the development of drugs to modify it favourably. Currently, statins are the most successful example of the link between basic scientific advances and drug development.
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