Frontiers in Cardiovascular Drug Discovery: Volume 5 E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Fachliteratur
- Serie: Frontiers in Cardiovascular Drug Discovery
- Sprache: Englisch
Frontiers in Cardiovascular Drug Discovery is a book series devoted to publishing the latest advances in cardiovascular drug design and discovery. Each volume brings reviews on the biochemistry, in-silico drug design, combinatorial chemistry, high-throughput screening, drug targets, recent important patents, and structure-activity relationships of molecules used in cardiovascular therapy. The book series should prove to be of great interest to all medicinal chemists and pharmaceutical scientists involved in preclinical and clinical research in cardiology.
The fifth volume of the series covers the following topics:
-The Lipid Hypothesis: From Resins to Proprotein Convertase Subtilisin/Kexin Type-9 Inhibitors
-The Role of SGLT2i in the Prevention and Treatment of Heart Failure
-Natural Products and Semi-Synthetic Compounds as Antithrombotics: A Review of the Last Ten Years (2009-2019)
-Transient Receptor Potential Channels: Therapeutic Targets for Cardiometabolic Diseases?
-Treatment of Raynaud’s Phenomenon
-Traditional Medicine Based Cardiovascular Therapeutics
-Cardiovascular Disease: A Systems Biology Approach
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Seitenzahl: 476
Veröffentlichungsjahr: 2020
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PREFACE
According to the World Health Organization, cardiovascular diseases (CVDs) are globally the number one cause of death. Over 18 million lives are lost globally due to heart attack alone. CVDs range from benign arrhythmias to massive heart failures and from chronic hypertension to ischemic strokes. They occupy a central place in non-communicable diseases, and they are often the result of complex chronic metabolic disorders. Extensive researches are been conducted on the causes and treatments of CVDs. Changing lifestyle with high calories diets, sedentary life style, and smoking are among the key causes. Volume 5 of the book series “Frontiers in Cardiovascular Drug Discovery” covers 7 comprehensive reviews contributed by leading researchers. These reviews broadly cover various drug targets and new classes of therapies for the prevention or treatment of cardiovascular diseases.
The review by Ramachandran et al focusses on a fiercely debated topic in CVD, i.e. lipid hypothesis. Cholesterol and LDLs have since long been considered as risk factors of cardiovascular diseases. However, there is mounting evidence that challenge this dogma. The authors have carefully reviewed the scientific literature and conclude that the theory stands valid. Huynh et al present exciting new advancements of SGLT2i (Sodium-glucose cotransporters 2) inhibitors as an important new class of drugs. These inhibitors increase renal glucose excretion, and lead to natriuresis and glycosuria with subsequent reduction in blood glucose and associated CVDs in diabetic patients Platelet aggregation and thrombosis are the major causes of morbidity and mortality worldwide. Piato and Graebin have the reviewed recent literature on the development of antithrombotic agents of natural and semi-synthetic origins, with a higher level of safety. Santos et al have contributed a chapter on the significance of transient receptor potential (TRP) channels as potential drug targets against cardiometabolic diseases. Mutations in some of the TRP channels are implicated in various metabolic and cardiovascular disorders, and thus activations of TRP channels through natural products may lead to the development of a new class of drugs.
Raynaud’s phenomenon (RP), vasospasm due to cold exposure and emotional stress, is a common disorder. Lambova discuss various molecular approaches towards the treatment of RP. Traditional medicines have played an important role in the treatment of human diseases, including cardiovascular disorders. Ravindran et al have reviewed pharmacological, toxicological, and informatics studies, carried on various polyherbal formulations, in order to scientifically validate their efficacy against CVDs. The chapter by Roy and Mishra is focused on the applications of system biology approach in developing a better understanding of the molecular basis of the CVDs and its comorbidities. Special emphasis is paid to the identification of biomarkers for early diagnosis of CVD for a better management of the disease states.
We would like to express our gratitude to all the authors of above cited review articles for their excellent contributions in this dynamic and exciting field of biomedical and pharmaceutical research. The efforts of the team of Bentham Science Publishers, particularly Ms. Mariam Mehdi (Assistant Manager Publications), and Mr. Mahmood Alam (Director Publications) are deeply appreciated.
List of Contributors
The Lipid Hypothesis: From Resins to Proprotein Convertase Subtilisin/Kexin Type-9 Inhibitors
Sudarshan Ramachandran1,2,3,Mithun Bhartia4,5,Carola S. König3Abstract
The validity of the lipid hypothesis has been debated recently in both, the media and the medical press. In this chapter we review the relevant evidence to evaluate whether it is still applicable in cardiovascular prevention. After a brief description of developments leading to the lipid hypothesis we consider prospective epidemiological studies, paying particular attention to the Framingham Heart Study as it was conceived at a time when lipid lowering therapy was unavailable. We also present the predictive factors of the other commonly used cardiovascular risk scoring models. All the algorithms show cholesterol (total or low density lipoprotein – cholesterol) and high density lipoproteins to predict cardiovascular disease. Our own data from the Whickham Study where subjects were recruited in the pre-statin era also show total cholesterol to be significantly associated with coronary heart disease. We then discuss intervention randomised controlled studies using agents that lower low density lipoprotein – cholesterol (resins, statins, ezetimibe and Proprotein convertase subtilisin/kexin type 9 inhibitors) paying particular attention to studies not demonstrating reduction in cardiovascular outcomes. Apart from patients with heart failure and possibly on dialysis the lipid hypothesis appears to be true. This is reinforced by a meta-analysis carried out by the Cholesterol Treatment Trialists’ Collaboration. We do not feel that outcomes from cohort studies consisting of patients subject to multiple guideline driven treatments can be used as good quality evidence against the lipid hypothesis. We do acknowledge that more research is required rega-
rding heterogeneity and describe a non-invasive way in which atherogenesis of the individual may be measured. We would like future randomised controlled trials to incorporate study of disease mechanism(s) within the study design.
Introduction
Atherosclerotic obstruction of arteries by plaque formation leading to cardiovascular disease (CVD) is one of the most common causes of mortality globally. Although the incidence has been decreasing [1], CVD still remains a leading cause of death in the United Kingdom [2]. Interestingly the prevalence of CVD has remained constant at about 3% [3] even though incidence has decreased, perhaps due to a fall in mortality. Thus, incidence and mortality rates and not prevalence may be the best indicators to evaluate CVD prevention measures. The Cholesterol Treatment Trialists’ (CTT) Collaboration carried out meta-analyses of randomised controlled trials (RCTs) with a minimum of 1000 participants and concluded that a 1 mmol/l reduction in low density lipoprotein (LDL) - cholesterol was associated with a reduction in myocardial infarction, revascular- isation and ischaemic stroke by just over 20% [4]. The lipid hypothesis describes this widely observed association between CVD risk and raised serum total cholesterol and LDL- cholesterol. Thus, a recent editorial in the New England Journal of Medicine describing the results of the IMPROVE-IT study, convincingly supported the hypothesis on the basis of prospective longitudinal studies showing significant decreases in CVD following use of LDL-cholesterol reducing agents, such as statins and ezetimibe [5].
However, there are publications arguing against the causative effect of cholesterol and LDL-cholesterol in the pathogenesis of atheroma and these have raised doubts regarding the benefit of lipid lowering therapy and indeed, the validity of the lipid hypothesis [6, 7]. This view contrasts with data showing statistically significant reductions in CVD using drugs that reduce LDL-cholesterol by different mechanisms. We speculate that the prevalent guideline culture in clinical medicine requires complex diseases to be simplified to aid the use of treatment pathways. Heterogeneity of populations, based on the degree of risk and mechanisms leading to risk, is often not considered [8]. After describing the history of the development of the lipid hypothesis we will consider epidemiology and interventional trials and how they fit in with the lipid hypothesis. In this chapter it is not our intention to list details of the various trials, but to discuss and place the lipid hypothesis in the context of CVD prevention.
Cholesterol
Cholesterol is found in body tissues and plasma of animals and is a ubiquitous constituent of cell membranes. It is a precursor of bile acids, vitamin D and steroid hormones such as cortisol, aldosterone, testosterone, oestrogens and progesterone. Further, it is important in the development / functioning of the nervous system, and is involved in signal transduction and sperm development. The structure of cholesterol is shown in Fig. (1) and the molecule can exist in either free or esterified (a fatty acid covalently attached to the hydroxyl group at position 3 of the ring) forms.
Fig. (1)) Structure of cholesterol with the point of esterification highlighted.Early Evolution of the Relationship between Cholesterol and Atherogenesis
We now consider major historical landmarks in the evolution of the lipid hypothesis, including the advent of evidence-based medicine via clinical trials. Controversy regarding the lipid hypothesis has ranged ever since Nikolai Anitschkow in 1913 demonstrated that rabbits when fed with purified cholesterol dissolved in sunflower oil developed vascular lesions similar to atheroma, this not being the case when the animals were fed just sunflower oil [9]. Anitschkow’s findings were not confirmed in rats or dogs, hence the observation was considered to be specific to the rabbit model and cast aside. The fact that dietary cholesterol in rats and dogs did not translate into elevated serum cholesterol, perhaps due to high conversion of cholesterol to bile acids as suggested by Anitschkow, was not considered. That atherogenesis in the rabbit model was a two-step process (ongoing feeding of cholesterol followed by elevation of blood cholesterol levels in lipoproteins) and was not recognised at the time [9]. Further, the serum cholesterol level in the rabbit was significantly higher than in humans cast doubts on the clinical relevance of Anitschkow’s work. However, continuing research confirmed the association between CVD and lipids and provided an understanding of the metabolism and transport of lipids.
The relationships between xanthomatosis, hypercholesterolemia (familial hypercholestrolaemia) and CVD were described between 1925-1938 by Francis Harbitz and Carl Müller [10]. Interestingly Müller suggested that reducing cholesterol levels may improve the prognosis [10]. John Oncley, used Cohn fractionation and electrophoresis to identify and separate the lipoproteins; their classification was based on their migration with the globulins, hence the nomenclature of alpha, prebeta and beta lipoproteins [11]. Gofman, an American scientist was convinced of the validity of Anitschkow’s experimental observations and focused on the key issue of cholesterol transport in the blood [12, 13]. This led to him to use ultracentrifugation to identify and quantify lipoproteins, the particles transporting lipids in blood and then to associate them with atherosclerosis. Further work by Fredrickson and Gordon (1958) [14] and Olson and Vester (1960) [15] resulted in some clarity of lipid transport pathways. Integrating physiology of organs such as gut, liver and adipose tissue with isotopic studies of lipoprotein metabolism led them to conclude that triglycerides were transported by chylomicrons from the gut to adipose tissue, and by very low density lipoprotein (VLDL) from the liver to adipose tissue; both processes requiring lipoprotein lipase and local uptake of free fatty acids by fat cells. Apoproteins (Apo), the protein components of lipoproteins following delipidation and fractionation of lipoproteins, was characterised by Fredrickson et al., (1967) based on size shape and amino acid composition [16]. Four families of Apo, each containing isoforms and determining metabolism of lipoproteins were identified by Jackson et al., in 1976; Apo A primarily associated with the α-lipoproteins (HDL), Apo B and Apo E with β-lipoproteins (VLDL, Intermediate Density Lipoproteins (IDL) and LDL) and chylomicrons, and Apo C with all lipoproteins other than LDL [17]. Apo B has 2 forms; Apo B 100 found in VLDL, IDL and LDL and the truncated Apo B 48 form, synthesised in the intestine following editing of mRNA, in chylomicrons [17]. Apo E has 3 isoforms (E2, E3 and E4) with E2 and E4 resulting from mutations of the E3 isoform. Both, Apo B100 (Apo B 48 is devoid of the LDL-receptor (LDLR) binding site) and Apo E are integral to lipoprotein clearance with mutations of apoproteins or receptors affecting clearance and hence, accumulation of lipoproteins. Goldstein and Brown are credited with identifying the LDLR found in coated pits of most cells, which includes a ligand binding domain for Apo B 100 and Apo E, and characterising its functional role of endocytosis of the LDL particle [18, 19]. The LDL-LDLR complex is internalised and fused with a lysosome leading to degradation of apoproteins and lipids and disorder in this process was associated with familial hypercholesterolaemia and CVD. Over a period of nearly 70 years we moved from Anitschkov’s observation to an understanding of LDL / LDLR and CVD based on a mechanistic framework devised by Goldstein and Brown. Current research is largely based on three approaches; 1. Basic science increasing our understanding of the mechanisms (e.g. lipoproteins, apoproteins and lipids) that leads to CVD, thus furthering drug development, 2. Large population based prospective studies establishing risk factors and at-risk populations, and 3. Intervention trials using therapeutic agents acting via different mechanisms resulting in the development of management guidelines. In this chapter we will mainly focus on the Framingham Heart Study and unpublished data from the Whickham study, both prospective studies, and interventional trials with LDL-cholesterol lowering agents that have led to the lipid hypothesis.
Longitudinal Prospective Observational Studies and CVD Risk Algorithms
Current longitudinal studies studying complex pathologies with numerous risk factors have inherent problems due to the holistic management approach adopted. Since Scandinavian Simvastatin Survival Study (4S) [20] and West of Scotland Coronary Prevention Study (WOSCOPS) [21], statin treatment has been built into cardiovascular prevention guidelines. Thus, high risk populations would likely be on statins whose LDL-cholesterol efficacy would vary depending on the individual drug and dose. The pharmacokinetic and pharmacodynamic properties of the available statins vary. Similarly, other CVD risk factors would be treated according to national and professional organisation guidelines. This would, in our view make it very difficult to estimate the impact of individual risk factors over a long period. Thus, in this section we will primarily examine the Framingham Heart Study in depth, selected in view of it being initiated when lipid lowering therapy was not available, its length of follow-up, subsequent inclusion of children and grandchildren of the original cohort and study extension to widen the ethnicity of the original study population (https://www.nih.gov/sites/default/files/ about-nih/impact/framingham-heart-study.pdf).
The initial objectives of the study when launched in 1948 were to identify factors associated with CVD with 5,209 men and women recruited between the ages of 30 and 62 with no evidence of CVD residing in the town of Framingham in Massachusetts, USA with lifestyle details noted and physical examinations carried out (https://crimsonpublishers.com/iod/pdf/IOD.000505.pdf). Following recruit- ment, a cardiovascular focused physical examination was carried out together with updates on medical history, blood test results at 2 – 4 year intervals [22]. Importantly recruitment of children and their spouses (Offspring-Spouse Cohort) and grandchildren (Third Generation Cohort) of the original cohort was initiated in 1971 and 2002, respectively. Heterogeneity was recognised with the Framingham OMNI 1 and OMNI 2 cohorts comprising ethnic minority residents in Framingham were included in 1994 and 2003, respectively, to reflect the changing diversity [22]. In order to identify genotypes related to CVD the Framingham investigators collaborated with the Jackson Heart Study and the American Heart Association and whole genome sequencing was carried out in 4200 of the subjects [22]. The outcomes from the Framingham Heart Study also gradually evolved; the original aim was to identify factors that were associated with CVD in the study population. With the original cohort aging, outcomes expanded to include osteoporosis, cognitive decline, dementia, Alzheimer’s disease, Parkinson’s disease and atrial fibrillation [22].
Identification of factors associated with CVD was gradual. Dawber et al., in 1957 identified that the incidence of coronary heart disease (CHD) was nearly double in men compared to women [23]. Further, increased cholesterol, body weight and blood pressure were independently associated with the development of CHD in men between 45 and 62 years of age over a 4 year follow-up period [23]. Subsequently in 1961 Kannel et al., showed CHD to be related to male gender, diabetes, left ventricular hypertrophy and increased age, cholesterol and blood pressure [24]. A year later cigarette smoking was shown to be related to CHD (the analysis was carried out on combined data from the Framingham Heart Study and the Albany Cardiovascular Health Study (http://www.epi.umn.edu/cvdepi/ study-synopsis/albany-cardiovascular-health-center-study/) [25]. Following measure- ment of lipoprotein fractions by ultracentrifugation, it became apparent that CHD was associated with increased LDL-cholesterol and decreased HDL-cholesterol levels, and this led to a ratio of total cholesterol to HDL-cholesterol being incorporated subsequently in the Framingham Risk Score [26]. Subsequently serum total cholesterol was also found to be a predictor of all-cause mortality [27]. Diabetes was in 1979 established to increase risk of CHD, cerebrovascular disease, peripheral vascular disease and heart failure [28]. Interestingly in 1996 lipoprotein (a) was identified to be an independent risk factor of CVD [29].
In addition to smoking other lifestyle factors were also recognised as risk factors. Physical inactivity was inversely and independently associated with mortality due to CVD in men, but not women [30]. When investigating the effects of nutrition the Framingham investigators compared CVD risk in the Framingham Offspring Spouse cohort with that of the second National Health and Nutrition Examination Survey (NHANES 2) and recommended that national nutrition strategies should target weight reduction with recommendations including reducing of foods rich in animal and plant fats and salt together with increases in complex carbohydrates and fibre [31].
The initial hypothesis of the Framingham Heart Study was that CVD was multifactorial and the findings described above have shown this to be correct. CHD and CVD risk function scores have also evolved since the 1960’s [32-40]. Currently separate risk models have been derived for CVD (10 and 30 year risk), CHD, congestive heart failure, stroke and intermittent claudication in individuals without prior disease (https://www.framinghamheartstudy.org/fhs-risk-functions/ cardiovascular-disease-10-year-risk/). The factors associated with these conditions in are presented in Table 1. Interestingly, total cholesterol and HDL-cholesterol are risk factors predicting CVD (10 and 30 year models) and CHD, but not stroke, intermittent claudication and congestive heart failure (Table 1). It is worth speculating that this may be due to stroke, intermittent claudication and congestive heart failure being associated with previous CVD / CHD as both these conditions are predicted by total cholesterol and HDL-cholesterol (hence, total cholesterol and HDL-cholesterol may have lost significance when statistical models included previous CVD / CHD). It is very evident from the hazard ratios in each of the models (https://www.framing hamheartstudy.org/fhs-risk-functions/cardiovascular-disease-10-year-risk/) that all these outcomes are multifactorial with each factor being weighted differently. Heterogeneity is also hinted at as separate algorithms were derived for men and women when CVD (10 year risk), CHD and stroke were outcomes. The omission of triglycerides as a risk factor is interesting. Triglyceride levels (above 150mg/dl) along with age, gender, fasting glucose levels, HDL-cholesterol, hypertension and parental history of diabetes, predict the development of diabetes (not shown in Table 1). It could be that triglycerides levels by predicting diabetes are included in the outcomes predicted by diabetes (CVD, stroke and intermittent claudication). Receiver operated characteristic curves plotting true positive (sensitivity) against false positive rates (1- specificity) for CHD shows area under the curve of 0.79 for men and 0.83 for women [41]. The area under the receiver operated characteristic curves indicates the discriminatory ability of the models. We would consider area under the curve values of 0.9 -1.0, 0.8 - 0.9, 0.7 - 0.8 and 0.6 - 0.7 to be excellent, good, fair and poor, respectively (http://gim.unmc.edu/dxtests/roc3.htm). The values seen with the CHD predictive models also suggest that further study is required to identify new risk factors and / or to repeat the analysis for subgroups where these factors may be more strongly associated with the outcome in view of probable heterogeneity. Integrating genetic and epigenetic factors into the prediction model could potentially improve the discriminatory ability [42].
In addition to the Framingham Heart Study risk score there are numerous CVD / CHD risk calculators that are in clinical use (Table 2) [43-46]. Details of the studies which gave rise to the risk algorithms are provided in Tables 1 (Framingham Heart Study) and Table 2. Significantly all the risk algorithms presented based on observational studies include Total or LDL-cholesterol and HDL-cholesterol values. The Framingham Heart Study has many strengths including prospective recruitment at a single centre at a time when most patients were not on lipid lowering and antihypertensive treatment [47]. As all risk scores are strictly speaking only applicable to the study cohort, validation in other populations is essential. The Framingham risk algorithm has been validated in different countries with varying results. Interestingly, the scores derived appear to depend on the underlying CHD risk of the population. Whilst reasonable in non-American populations with similar CHD rates [48, 49] it appears to overestimate risk in European and Chinese populations at lower risk levels [50-54]. This was the pattern that we observed when comparing predicted and actual CHD rates in 2471 individuals during a 20 year follow-up in the Whickham Study [55] in Northeast England which has been cited in the latest NICE guidelines; CG 181 (Fig. 7 of this document) (https://www.ncbi.nlm.nih.gov/ books/ NBK248067/ pdf/ Bookshelf_NBK248067.pdf). Our results confirm that the Framingham model predicted the absolute risk of heart disease in 1700 men and women (where the Framingham risk score could be obtained) aged between 35 – 70 years without prior CVD in the United Kingdom when the annual CHD risk was above 1.5% (the observed risk falling within the 95% confidence intervals of the calculated risk using the Framingham algorithm), but underestimated the risk when the absolute risk was lower [55]. Of the 1700 participants, 529 (31.1%) developed heart disease during the 20 year follow-up. Logistic regression of the subgroup showed that CHD was significantly associated with age, male gender, smoking, diabetes, total cholesterol (also total cholesterol: HDL-cholesterol ratio using mean HDL-cholesterol values of 1.15 mmol/l and 1.4 mmol/l in men and women, respectively [56]) and systolic blood pressure, these results similar to the Framingham model (Table 3); unpublished data from [55]. Unlike in the Framingham model (Table 1) left ventricular hypertrophy was associated with CHD. Significantly, whilst triglycerides levels and diastolic blood pressure were not significantly associated with CHD, baseline HDL-cholesterol could not be considered as it was not routinely measured during the period of recruitment. The association between baseline cholesterol levels (stratified) and CHD in the total cohort is shown in Fig. (2); unpublished data from [55]. It is clear that a strong near linear association exists with no hint of CHD rates rising at the lower end or falling at the upper end of the cholesterol range. Importantly, the mean age of the stratified baseline cholesterol categories varied, cholesterol level was associated with age (linear regression analysis: c: 0.02, 95% CI: 0.02 – 0.03, p<0.001), hence it is important to be cautious with a univariate analysis. We include this figure only to demonstrate no evidence of a J or U shaped association between cholesterol and CHD, this data possibly important in the current cholesterol debate. Logistic regression analysis with age (OR: 1.04, 95% CI: 1.03 – 1.05, p<0.001) and baseline cholesterol (OR: 1.11, 95% CI: 1.02 – 1.21, p=0.015) as continuous variables included as independent variables in a single model showed both factors were independently associated with CHD. The strength of our validation was that baseline measurements were gathered between 1972 - 4 and therapeutic intervention would have been minimal with statins being unavailable for most of the follow-up period, similar to that in the Framingham Heart Study.
It is not within the scope of this chapter to analyse the use of CHD and CVD risk algorithms. As previously stated lipid levels have been significantly associated with CHD and CVD in all the major risk predictive models [38, 39, 43-46]. We have described the Framingham Heart Study in some depth as it commenced in the pre lipid lowering era and will continue to remain relevant as it is evolving with the addition of genetic data etc. We have also shown that total cholesterol levels were independently associated with CHD in a United Kingdom population also in the pre-intervention era. In our view it is very difficult to establish the role of lipids in the aetiology of CVD in the current climate. Non-lipid lowering strategies have also demonstrated significant CHD and CVD reduction. The Heart Outcomes Prevention Evaluation (HOPE) [57, 58], Captopril Prevention Project (CAPP) [59] and Appropriate Blood Pressure Control in Diabetes (ABCD) [60] trials showed antihypertensives resulting in cardiovascular benefits, often far greater than that could be expected from their effect on blood pressure in different cohorts. More recently the sodium glucose cotransporter 2 inhibitor empagliflozin reduced CVD and CVD associated mortality in patients with type 2 diabetes [61]. Observational studies have shown Phosphodiesterase type 5-inhibitors to reduce myocardial infarction [62] and all-cause mortality [63]. The above examples demonstrate the difficulty of studying in isolation, the impact of lipids and lipid lowering therapy on CVD, in view of its multifactorial aetiology. Heterogeneity of aetiology is also problematic as study results may only be applicable to the characteristics of the cohort studied [8].
Interventional Studies with Clinical Events as Outcomes
The above observational studies have clearly shown an association between lipids and CVD. In 1965, Sir Austin Bradford Hill gave the first President’s Address to the newly formed Section on Occupational Medicine discussing criteria whereby an observed association could be stated as causative [64]. The criteria consisted of strength of association, consistency, specificity, temporality, biological gradient, plausibility, coherence, experiment, and analogy. Schade et al., in 2017, after examining the association between LDL-cholesterol and CVD, concluded that LDL is the primary cause of atherosclerotic CVD [65]. This review concluded that the data studied complemented the results of RCTs in humans demonstrating that the reduction of LDL-cholesterol resulted in not only a reduction but a reversal of atherosclerosis. They rightfully acknowledged that LDL-cholesterol was not the sole factor. The progression of atherosclerosis may be accelerated by hypertension, smoking, diabetes, and obesity. They also controversially stated that without the contribution of LDL-cholesterol, clinically significant plaques would not be formed [65]. It is acknowledged that RCTs have inherent limitations and it can be often difficult to gain an understanding of a treatment in an individual patient as opposed to the study population [66]. Reporting has improved with standardisation protocols such as CONsolidated Standards Of Reporting Trials (CONSORT) [67-70]. Further, strategies have been devised for integrating the results via guidelines into clinical practice [71].
Fig. (2)) Association between CHD during 20 years of follow-up and baseline cholesterol stratified in the total Whickham cohort. Mean age is provided in the attached table to demonstrate the importance of adjusting any analysis for age in view of its association with cholesterol concentrations (linear regression analysis: c: 0.02, 95% CI: 0.02 – 0.03, p<0.001). (Unpublished data from Ramachandran S, French JM, Vanderpump MP, Croft P, Neary RH. Using the Framingham model to predict heart disease in the United Kingdom: retrospective study. BMJ 2000; 320: 676 – 677).We will now move onto RCTs evaluating the effects of various cholesterol (and LDL-cholesterol) lowering drugs have on clinical endpoints. Resins, statins and ezetimibe lower cholesterol and LDL-cholesterol via varied mechanisms.
Randomised Controlled Bile Sequestrants Trials
Bile acid sequestrants (anion exchange resins) were developed in the 1970s and by binding gut bile acids, reduced the entero-hepatic recirculation of bile acids and decreased LDL- cholesterol by 10 - 15% [72]. Most data are derived from use of cholestyramine and colestipol, but the more recently introduced colesevelam, possibly has fewer side effects [73]. The Lipid Research Clinics Coronary Primary Prevention Trial (LRC-CPPT) a double-blinded RCT studied the association between cholestyramine treatment and CHD (primary end point: composite of death due to CHD and myocardial infarction) in 3,806 asymptomatic middle-aged men with hypercholesterolemia over a mean follow-up of 7.4 years [74]. All patients followed a moderate cholesterol lowering diet. A significant reduction in the primary outcome was observed in form of a 19% relative risk reduction with cholestyramine treatment (events: 7.0%) compared to placebo (events: 8.6%). Further, the cholestyramine treatment was associated with 25%, 20%, and 21% lower rates for positive exercise stress tests, angina, and coronary bypass surgery, respectively. All-cause mortality was not significantly different in the two study arms. The LRC-CPPT showed that reducing total cholesterol and LDL-cholesterol levels significantly decreased CHD morbidity and mortality in men with elevated LDL-cholesterol levels [74]. Despite the above outcome this therapy is a minor player in lipid lowering because of low tolerability with gastrointestinal side effects and high price in the case of the better tolerated colesevalem (£0.97 - £2.91 per day: https://www.nice.org.uk/ advice/ suom22/ chapter/ evidencereview-economic-issues#cost). Further, more evidence from RCTs is required to establish benefits when bile acid sequestrants are added to statins as most statin treated patients would be expected to have lower lipid levels.
Randomised Controlled Statin Trials
Cholesterol is synthesised via a multistep pathway from acytyl-CoA taking place in the cytosol and endoplasmic reticulum [75]. From the endoplasmic reticulum it is transported to the plasma membrane and other organelles such as mitochondria, and lipid droplets. Fig. (3) shows many of the enzymes involved in the synthetic pathway with HMG-CoA reductase (targeted by statins) catalysing the rate-limiting step converting HMG-CoA to mavelonic acid [76]. This was demonstrated by the Japanese biochemist Akira Endo in the 1970s using citrinin produced by the fungus Pythium ultimum to inhibit cholesterol synthesis in rats [77]. Since then we have had lovastatin (1987), simvastatin, a semi synthetic derivative of lovastatin (1987), pravastatin (1991), fluvastatin (1994), atorvastatin (1997), cerivastatin (1998: withdrawn in 2001), rosuvastatin (2003) and pitavastatin (2003) [78].
The expression of genes encoding HMG-CoA reductase is regulated by the sterol response element binding protein (SREBP), a membrane protein residing in the endoplasmic reticulum, binding to upstream sterol response elements sequences and activating transcription. SREBP contains two transmembrane domains and two cytosol facing DNA-binding domains and is held in the endoplasmic reticulum via interactions with other proteins containing a sterol-sensing domain. Binding of this domain and cholesterol determines regulation of HMG-CoA reductase; following association with cholesterol the conformation allows other parts of the protein to interact with SREBP. However, when cholesterol levels decrease, interaction between the sterol-sensing domain and cholesterol is lost leading to a change in the structure resulting in disassociation of the protein from SREBP. Free SREBP is transported to the Golgi where DNA-binding domains are released into the cytosol following the actions of proteases. In the nucleus, the DNA-binding domains bind sterol response elements and activate genes transcription promoting cholesterol synthesis and uptake [74].
Fig. (3)) Pathway leading to cholesterol synthesis.Since 4S in 1994 there have been numerous RCTs conducted to study the effect of reducing LDL-cholesterol on clinical outcomes. We picked the RCTs including >1000 individuals from Pubmed (Fig. 4, abbreviations of the trials are provided in the footnote of the figure) to discuss further [5, 20, 21, 78-109]. Whilst many of the trial cohorts comprised only individuals with established CVD (secondary prevention) some trials had individuals with no previous CVD (primary prevention). The proportion of individuals without previous CVD includes WOSCOPS: 92% [21], AFCAPS/TEXCAPS: >99% [81], HPS: 15% [85], PROSPER: 56% [88], ALLHAT-LLT: 78% [89], ASCOT-LLT: 86% [90], ALERT: 81% [91], CARDS: 96% [92], 4D: 27% [97], MEGA: 99% [99], JUPITER: 100% [103] and AURORA: 60% [104]. Fig. (4) shows the RCTs that did not show benefit regarding the clinical CVD outcomes (trials with red backgrounds). We consider these to see if cohort properties (e.g. underlying risk, type of dyslipidaemia etc) or scale of LDL-cholesterol reduction could contribute to them not achieving significant CVD reduction. RCTs showing statins reducing CVD outcomes are assumed to adhere to the lipid hypothesis and will not be discussed.
The post CABG trial included 1351 individuals who had undergone coronary artery bypass grafting with LDL- cholesterol between 3.4 – 4.5 mmol/l and triglycerides < 3.4 mmol/l randomised to either intensive (to achieve LDL-cholesterol of 1.6 – 2.5 mmol/l) or standard (> 2.5 mmol/l) lipid lowering therapy (lovastatin ± cholestyramine if required) and warfarin or placebo over a mean 4.3 years and an extended follow-up of 3 further years [80]. The progression of atherosclerosis was significantly reduced in the intensive lipid lowering arm. Though the clinical CVD end-points were lower they did not achieve statistical significance in the intensive lipid lowering arm at the end of the follow-up [80]. However, at the end of the extended follow-up both end points were significantly lower in the intensive lipid lowering arm [110]. Thus, though the study did not meet strict statistical criteria, it did suggest lowering LDL-cholesterol was beneficial. Interestingly, warfarin did not demonstrate any benefit.
Fig. (4)) Time line of RCTs with LDL-cholesterol reducing agents (statins, ezetimibe and PCSK9 inhibitors) and CVD reduction since the 4S in 1994. The RCTs significantly achieving and not achieving reduction in CVD outcomes are coloured green and red respectively. Statin RCTs are denoted in black print whilst ezetimibe RCTs (SHARP, IMPROVE-IT) are in red and PCSK9 inhibitor RCTs (FOURIER, ODYSSEY OUTCOMES) are in blue print. 4S: Scandinavian Simvastatin Survival Study [18] 4D: Die Deutsche Diabetes Dialyze Studie [95] A to Z: Aggrastat-to-Zocor Trial [93] AFCAPS/TEXCAPS: Air Force/Texas Coronary Atherosclerosis Prevention Study [79] ALERT: Assessment of Lescol in Renal Transplantation [89] ALLHAT-LLT: Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial Lipid-Lowering Trial [87] ALLIANCE: Aggressive Lipid-Lowering Initiation Abates New Cardiac Events [91] ASCOT: Anglo-Scandinavian Cardiac Outcomes Trial [88] AURORA: A Study to Evaluate the Use of Rosuvastatin in Subjects on Regular Hemodialysis: An Assessment of Survival and Cardiovascular Events [102] CARDS: Collaborative Atorvastatin Diabetes Study [90] CARE: The Cholesterol and Recurrent Events Trial [77] CORONA: The Controlled Rosuvastatin Multinational Trial in Heart Failure [99] FOURIER: Further Cardiovascular Outcomes Research with PCSK9 Inhibition in Subjects with Elevated Risk [106] GISSI HF: The Gruppo Italiano per lo Studio della Sopravvivenza nell’Insufficienza cardiac [100] GISSI Prevenzione: The Gruppo Italiano per lo Studio della Sopravvivenza nell’Infarto Miocardico [81] GREACE: The Greek Atorvastatin and Coronary Heart-disease Evaluation Study [85] HOPE-3: Heart Outcomes Prevention Evaluation – 3 [105] HPS: Heart Protection Study [83] IDEAL: Incremental Decrease in End Points Through Aggressive Lipid Lowering study [96] IMPROVE-IT: Improved Reduction of Outcomes: Vytorin Efficacy International Trial [5] JUPITER: Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin [101] LIPID: The Long-Term Intervention with Pravastatin in Ischemic Disease [80] LIPS: The Lescol Intervention Prevention Study [84] MEGA: Management of Elevated Cholesterol in the Primary Prevention Group of Adult Japanese study [97] MIRACL: The Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering study [82] ODYSSEY OUTCOME: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Study to Evaluate the Effect of Alirocumab on the Occurrence of Cardiovascular Events in Patients Who Have Recently Experienced an Acute Coronary Syndrome [107] Post CABG: coronary artery bypass graft [78] PROSPER: The Prospective Study of Pravastatin in the Elderly at Risk [86] PROVE IT: The Pravastatin or Atorvastatin Evaluation and Infection Therapy study [92] SEARCH: Study of the Effectiveness of Additional Reductions in Cholesterol and Homocysteine [103] SHARP: Study of Heart and Renal Protection [104] SPARCL: The Stroke Prevention by Aggressive Reduction in Cholesterol Levels study [98] TNT: Treating to New Targets study [94] WOSCOPS: The West of Scotland Coronary Prevention Study [19]The ALLHAT-LLT investigated a mixed primary (LDL-cholesterol: 3.1 – 4.9 mmol/l) and secondary prevention (LDL-cholesterol: 2.6 – 3.3 mmol/l) multiethnic (38% black, 23% Hispanic) cohort comprising 10,355 individuals aged >55 years and compared the effects of pravastatin 40mg vs usual care (in this group 32% and 29% of individuals with and without CHD were commenced on lipid lowering therapy) over a mean 4.8 years [89]. All-cause mortality was similar in the groups (pravastatin: 14.9%, usual care: 15.3%) and though CHD was lower in the pravastatin subjects (pravastatin: 9.3%, usual care: 10.4%) risk reduction did not achieve significance (RR: 0.91, 95% CI: 0.79 – 1.04, p=0.16). This non-significant outcome may be due to the modest LDL-cholesterol difference (16.7%) between the 2 arms.
The ALERT trial compared fluvastatin and placebo treatments in 2102 renal transplant recipients with total cholesterol values between 4.0 – 9.0 mmol/l [91]. Although major adverse cardiac events were lower in the fluvastatin arm compared to the placebo arm statistical significance was not achieved (RR: 0.83, 95% CI: 0.64 – 1.06, p=0.14). However, it is important to note that fewer cardiac deaths and non-fatal myocardial infarctions, a secondary outcome, were seen in the fluvastatin arm (RR: 0.65, 95% CI: 0.48 – 0.88, p=0.005).
The A to Z trial compared patients commenced on simvastatin 40mg, increased after 1 month to 80mg arm (LDL-cholesterol after 8 months was 1.63 mmo/l) with those on placebo who were converted after 4 months to simvastatin 20mg (LDL-cholesterol after 8 months was 1.99 mg) in 4497 individuals [95]. The primary outcome was a composite of cardiovascular death, non-fatal myocardial infarction hospitalisation with acute coronary syndrome and stroke over 6 – 24 months and this was not achieved; simvastatin event rate: 14.4%, placebo / simvastatin event rate: 16.7% (HR: 0.89, 95% CI: 0.76 – 1.04, p=0.14). Primary outcome events after the initial 4 months of follow-up, showed a significant reduction in the simvastatin arm (HR: 0.75, 95% CI: 0.60 – 0.95, p=0.02). In our view, this study with a relatively short follow-up and results that do not reach statistical significance does not disprove the lipid theory.
The SEARCH study included 12,064 patients aged 18 – 80 years with a history of myocardial infarction were randomised to either simvastatin 20mg or 80mg for a mean follow-up of 6.7 years [105]. The primary endpoint consisted of major vascular events; coronary death, non-fatal myocardial infarction, stroke, or arterial revascularisation. The LDL-cholesterol difference between the groups was a modest 0.35mmol/l. This probably accounted for the primary outcome not being significantly different between the simvastatin 80mg (24.5%) and simvastatin 20mg (25.7%) groups; RR: 0·94, 95% CI: 0·88 – 1·01, p=0·10.
The 4D study included 1255 subjects with type 2 diabetes receiving haemodialysis randomised to atorvastatin 20mg or placebo [97]. The primary end point was a composite of cardiovascular death, nonfatal myocardial infarction, and stroke. Primary outcome event rate in the total cohort after a median follow-up of 4 years was high at 37% (atorvastatin arm: 37%, placebo arm: 38%, RR: 0.92, 95% CI: 0.77 – 1.10, p=0.37). It is also worth stating that fatal strokes were more common in the atorvastatin arm (4.0%) than the placebo arm (2.0%); RR: 2.03, 95% CI: 1.05 – 3.93, p= 0.04), this result not driven by haemorrhagic stroke as was seen in the SPARCL study [100]. When all cardiac events such as death from CVD, non-fatal myocardial infarction, and interventions were combined (secondary outcome), the atorvastatin arm showed benefit (33%) compared to placebo (39%); HR: 0.82, 95% CI: 0.68 – 0.99, p=0.03. The AURORA study included 2776 patients between 50 – 80 years of age receiving haemodialysis randomised to either rosuvastatin 10mg or placebo during a median follow-up of 3.8 years [104]. The primary end point was a composite of death from cardiovascular causes, nonfatal myocardial infarction and nonfatal stroke. No significant difference was observed in the primary end-point with between the rosuvastatin group (9.2 events / 100 patient years) and the placebo group (9.5 events / 100 patient years); HR: 0.96; 95% CI: 0.84 - 1.11; p=0.59. We speculate that the reason for the primary outcome not being reached could be related to cohort characteristics (e.g. renal failure, dialysis etc). However, this speculation is not supported by the SHARP study which included 9270 individuals with renal impairment showing the primary outcome of the first major cardiovascular event was significantly lower in the simvastatin + ezetimibe group (11.3%) compared to the simvastatin + placebo group (13.4%) during a median follow-up of 4.9 years; RR: 0·83, 95% CI: 0·74–0·94, p=0·0021 [106]. Importantly, 27% of patients in both arms were receiving haemodialysis. Interestingly patients on dialysis were not treated as a subgroup. However, it must be emphasised that the LDL-cholesterol difference between the groups was a result of ezetimibe and not statin therapy.
The CORONA study included 5011 patients > 60 years of age with heart failure (New York Heart Association class 2 – 4) who were randomised to receive either rosuvastatin 10mg or placebo over a median follow-up of 32.8 months [101]. The primary outcome was a composite of death from cardiovascular causes, nonfatal myocardial infarction and nonfatal stroke. No significant difference (HR: 0.92, 95% CI: 0.83 – 1.02, p=0.12) in primary outcome was evident between the rosuvastatin (11.4%) and placebo (12.3%) arms. The GISSI-HF trial was carried out in 4574 patients > 18 years of age with heart failure (New York Heart Association class 2 – 4) over a median follow-up of 3.9 years [102]. Primary endpoints consisted of time to death, and time to death or admission to hospital for cardiovascular reasons. No benefit was observed with rosuvastatin therapy regarding either end-point. In the rosuvastatin group 29% patients died from any cause and in the placebo group 28% (HR: 1·00, 95% CI: 0·90 – 1·12, p=0·94). The second end-point of death or hospital admission for cardiovascular reasons was seen in 57% and 56% of patients rosuvastatin and placebo, respectively (HR: 1·01, 99% CI: 0·91 – 1·11, p=0·90). The above 2 trials (CORONA and GISSI-HF) raise the question whether rosuvastatin (it is important to further study whether this phenomenon is a class effect or not) treatment is of any benefit in patients with heart failure, although it must be emphasised that in neither study did it result in any harm. Once again we speculate whether lipid lowering therapy is of benefit in patients with heart failure.
Fig. (4) shows most RCTs led to significant reduction in CVD, the primary end point in virtually all the studies. It must be emphasised that none of the RCTs showed statins being associated with any increase in CVD. We have also briefly described the studies where the primary outcome was not met. Perhaps the benefit of lipid lowering therapy in patients with heart failure or haemodialysis needs to be reconsidered, although for the latter group the results of the SHARP study are perhaps reassuring [106].
We did not consider the ASPEN (Atorvastatin Study for Prevention of coronary heart disease Endpoints in Non-insulin dependent diabetes mellitus) as it was originally designed as a secondary cardiovascular prevention trial in patients with prior myocardial infarction or interventions for CVD [111]. However, in view of changing treatment guidelines for patients with CHD, recruitment was compromised which led to a change in protocol after 2 years leading to inclusion of individuals without CVD. Further, all patients with CVD developed either before or during the trial were commenced on active therapy. In view of this we did not include ASPEN in Fig. (4).
The CTT Collaboration carried out a meta-analysis of 26 trials (5 trials (39,612 patients) comparing more vs less intensive statin therapy and 21 trials (129,526 patients) comparing statins vs placebo) [4]. All trials in this meta-analysis, apart from ASPEN are included in Fig. (4). The results suggested a 1mmol/l decrease in LDL-cholesterol was associated with a significant (RR: 0.78, 95% CI: 0.76 – 0.80, p<0.0001) 22% reduction in major vascular events. Even the negative studies described above showed event reduction close to what would be predicted from the CTT collaboration meta-analysis. All-cause mortality was reduced by 10% (RR: 0.90, 95% CI: 0.87 – 0.93, p <0.0001), mainly driven by lower CHD deaths. No significant differences were observed in cancer rates or deaths due to non-vascular causes.
Randomised Controlled Ezetimibe Trials
The source of cholesterol in the intestinal lumen is mainly dietary (300 – 500mg/day) and biliary (800 – 1200mg/day). Absorption (about 50-60% of intestinal content, although saturation of absorption takes place at higher cholesterol content) takes place principally in the duodenum and proximal jejunum, the process facilitated by bile salts [112]. Cholesterol and phytosterols are taken up both from the intestinal lumen of the enterocyte (the uptake of cholesterol being greater than phytosterols) by what appears to be a common unidirectional sterol transporter involving a Neiman–Pick C1 Like 1 (NPC1L1) protein in the jejunal brush border membrane [112, 113]. Reverse transport of the sterols into the intestinal lumen is mediated by the ATP-binding cassette (ABC) hemitransporters (ABCG5 and ABCG8) function. Ezetimibe, or 1-(4-fluorophenyl)-(3R)-[3-{4-fluorophenyl}-{3S}-hydroxyprophyl]-(4S)-(4-hydroxyphenyl)-(2-azetidinone), inhibits intestinal cholesterol absorption by selectively blocking the NPC1L1 protein [113].
The large RCTs with clinical outcomes assessing the association between ezetimibe and CVD are included in Fig. (4); SHARP [106] and IMPROVE-IT [5
