**10. New perspectives on the development of natural cholesterol-lowering products**

Cardiovascular friendly natural products are those nutritional supplements, which can provide potential health benefits in cardiovascular diseases (CVD). Experimental, epidemio‐ logical and clinical data indicates that dietary nutrients and supplements have profound cardioprotective effects in the primary as well as secondary prevention of coronary heart disease [88]. Except for the aforementioned several cholesterol-lowering natural products, the plant alkaloid berberine has recently been introduced to the filed of lipid-lowering products. Berberine has shown a moderate or comparable cholesterol- and triacylglycerol-lowering effect in humans [89]. More importantly, recent studies have demonstrated that berberine combined with plant stanols improves cholesterol-lowering efficacy through a synergistic action on cholesterol absorption and reducing plasma triacylglycerols in animals [90], [91], with the efficacies are comparable or even better cholesterol-lowering drugs, statins. The product has been patent-protected and at the preclinical stage. Clinical trials are warranted before moving this potentially highly effective natural product to the lipid-lowering market.

#### **10.1. Current status in the development of cholesterol-lowering products**

An initial approach to control the modifiable risk factors of hyperlipidemia relies on the changes of diet, lifestyle, and other factors such as smoking. When all these fail, pharmaceutical intervention is necessary. Many of the current available drugs are those either inhibit choles‐ terol synthesis, or increase synthesis of bile acids and excretion. The majority of the current research focuses on lowering the "bad" LDL cholesterol, with less attention on increasing "good" HDL cholesterol. A treatment with a combination that can lower LDL cholesterol and increase HDL cholesterol would certainly provide more benefits than single approach for the treatment of hyperlipidemia, atherosclerosis and cardiovascular disease.

#### *10.1.1. Lowering LDL cholesterol*

Health Canada has recently reconsidered the classification of food products with disease risk reduction claims or therapeutic claims in light of clarified principles for the classification of foods at the Food-Natural Health Product interface (http://www.hc-sc.gc.ca/fn-an/labeletiquet/claims-reclam/assess-evalu/sat-mono-poly-fat-gras-eng.php). Health Canada has concluded that the results of the updated literature review are consistent with the 2002 report provided by the Institute of Medicine (IOM), which forms the basis of the US and Canada dietary guidance, on the replacement of saturated fat with unsaturated fat for blood cholesterol lowering. In other words, scientific evidence exists in support of the therapeutic claim linking the replacement of saturated fat with unsaturated fat to a reduction of blood cholesterol. It is stated that the claim is relevant and generally applicable to the Canadian population as a high proportion of the Canadian population is hyperlipidemic. It is allowed now by the Health Canada's to put therapeutic claim statements such as "Replacing saturated fats with polyun‐ saturated and monounsaturated fats (from vegetable oils) helps lower/reduce cholesterol" in vegetable oils and foods containing vegetable oils when specific conditions for the food

Olive oil can reduce LDL and raise high-density lipoprotein cholesterol and decrease lipid damage due to oxidative stress. In addition, olive oil reduces inflammatory and thrombogenic

Tea catechin-especially (-)-epigallocatechin-3-gallate-inhibits the expression of soluble adhesion molecules including vascular adhesion molecule-1 and intercellular adhesion molecule-1, endothelial cell inflammatory markers, decreased oxidized LDL, and prevents the

Certainly, there are more natural products available, for instance a number of antioxidants and phenolic compounds, to lower blood cholesterol levels. However, the big challenges that the natural products have been facing in the past years lie in their relatively lower efficacies as compared with the cholesterol-lowering drugs. The apparent advantages of natural products are their better safety profiles. In order to promote the market share of cholesterol-lowering natural products in competing with the drugs, it is critical to develop new products that can offer better efficacies than the current natural products, without losing safety or introducing increased toxic or severe side effects. Considering that the majority of the current natural products fall into the same category of cholesterol absorption inhibitor, the future direction of research and development of cholesterol-lowering products, novel distinct pathways or targets should be focused. Herewith, we will provide some brief thoughts on new approaches that we believe worth to tackle into, with a hope that the new mind in the research and development direction and focus would help to discover and develop novel natural products with significantly improved cholesterol-lowering efficacy working through distinct mecha‐

nisms than the currently available natural products, without apparent side effects.

carrying the claim are met.

**9.7. Green tea products**

development of atherosclerosis and[87].

status, endothelial dysfunction, and blood pressure[86].

200 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

**9.6. Olive oil**

Statins drugs, which are currently widely prescribed, lower LDL cholesterol primarily via inhibiting cholesterol biosynthesis and a secondary effect of increase LDL receptor and LDL clearance. However this family of drugs has shown several undesirable side-effects. Niacin, can also lower LDL cholesterol but not efficiently as statin but it is only one drug proved to increase HDL cholesterol with very less side effects. Therefore, a combination of niacin and statins might be superior to these drugs alone for lowering lipid levels. The scientific evidence of long-term safety and tolerability of these combination therapies are key determinants for good compliance and cardiovascular benefits.

Proprotein convertase subtilisin kexin-9 (PCSK9) is a sterol-responsive protein that accelerates LDLR turnover in the liver. When PCSK9 transcription is reduced, there will be subsequently increases LDLR levels helping to maintain cholesterol homeostasis. The transcription of PCSK9 is increased by SREBP-2, which also regulate the transcription of many other genes. Therefore, a pharmacologically specific inhibitor of PCSK9 will have atherogenic effect by increasing LDLR levels without affecting other genes regulated by SREBP-2.

#### *10.1.2. Increasing HDL levels*

Low HDL-cholesterol (HDL-C) is a strong and independent cardiovascular risk marker. Niacin is the only drug proved to increase HDLC but associated with flushing as a side-effect. Niacininduced flushing involves both PGD2 from mast cells and serotonin from platelets. The possibility of administering or formulating niacin together with flavonoid luteolin was recently shown to inhibit niacin flush in rats. Further investigation on inhibitors on side-effect can warrant an effective medicine for CVD[92]. Aleglitazar, a dual PPARα/γ agonist, proven has beneficial effects on both lipid and glucose parameters [93]. Activation of PPARα may increase ApoA1, apolipoprotein of HDL and increased the synthesis of LPL which hydrolyzes VLDL, IDL and LDL. PPARα agonist, with good safety profile, may have a therapeutic role in modifying cardiovascular risk factors.

#### *10.1.3. CETP inhibition*

Besides established strategies to increase HDL-C, e.g. with nicotinic acid, CETP (Cholesteryl ester transfer protein)-inhibition is a promising new therapeutic option. The failure of torcetrapib, the first CETP-inhibitor, seems to be attributed to "off-target" effects. Treatment with the newer CETP-inhibitors dalcetrapib and anacetrapib has been shown to be efficacious and safe - but their usefulness in clinical practice remains to be determined in ongoing clinical trials [94].

#### *10.1.4. Inhibition of cannabinoid receptors*

G protein-coupled cannabinoid receptor, CB-1 of the endocannabinoid system, plays a crucial role in regulating feeding pattern, lipid metabolism, and energy homeostasis. CB-1 receptors are located in the central nervous system and peripheral tissues including adipocytes, pancreas, gut, liver, and muscle. Rimonabant, a selective CB-1 antagonist drug to be developed for weight loss via increasing energy expenditure actually also increases HDL-cholesterol and triglycerides.However, the clinical efficacy and safety of these new antiobesity compounds are yet to be determined. Additional cannabinoid receptor blockers have been developed and are in testing [95].

#### *10.1.5. Nuclear receptor modifiers*

Identification of nuclear receptor LXR and FXR as regulatorof genes in bile acid and cho‐ lesterol metabolism has providedpotential new targets for screening cholesterol-lowering drugsby manipulating bile acid synthesis, transport, and absorption. Therapies targeted to LXR and FXR would be ideal for drug developmentbecause nuclear receptors are acti‐ vated by natural and syntheticligands, which could be identified by high-throughput screening [96].

#### *10.1.6. Acyl-coenzyme A: Cholesterol acyltransferas (ACAT) inhibitors*

a pharmacologically specific inhibitor of PCSK9 will have atherogenic effect by increasing

Low HDL-cholesterol (HDL-C) is a strong and independent cardiovascular risk marker. Niacin is the only drug proved to increase HDLC but associated with flushing as a side-effect. Niacininduced flushing involves both PGD2 from mast cells and serotonin from platelets. The possibility of administering or formulating niacin together with flavonoid luteolin was recently shown to inhibit niacin flush in rats. Further investigation on inhibitors on side-effect can warrant an effective medicine for CVD[92]. Aleglitazar, a dual PPARα/γ agonist, proven has beneficial effects on both lipid and glucose parameters [93]. Activation of PPARα may increase ApoA1, apolipoprotein of HDL and increased the synthesis of LPL which hydrolyzes VLDL, IDL and LDL. PPARα agonist, with good safety profile, may have a therapeutic role in

Besides established strategies to increase HDL-C, e.g. with nicotinic acid, CETP (Cholesteryl ester transfer protein)-inhibition is a promising new therapeutic option. The failure of torcetrapib, the first CETP-inhibitor, seems to be attributed to "off-target" effects. Treatment with the newer CETP-inhibitors dalcetrapib and anacetrapib has been shown to be efficacious and safe - but their usefulness in clinical practice remains to be determined in ongoing clinical

G protein-coupled cannabinoid receptor, CB-1 of the endocannabinoid system, plays a crucial role in regulating feeding pattern, lipid metabolism, and energy homeostasis. CB-1 receptors are located in the central nervous system and peripheral tissues including adipocytes, pancreas, gut, liver, and muscle. Rimonabant, a selective CB-1 antagonist drug to be developed for weight loss via increasing energy expenditure actually also increases HDL-cholesterol and triglycerides.However, the clinical efficacy and safety of these new antiobesity compounds are yet to be determined. Additional cannabinoid receptor blockers have been developed and are

Identification of nuclear receptor LXR and FXR as regulatorof genes in bile acid and cho‐ lesterol metabolism has providedpotential new targets for screening cholesterol-lowering drugsby manipulating bile acid synthesis, transport, and absorption. Therapies targeted to LXR and FXR would be ideal for drug developmentbecause nuclear receptors are acti‐ vated by natural and syntheticligands, which could be identified by high-throughput

LDLR levels without affecting other genes regulated by SREBP-2.

202 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

*10.1.2. Increasing HDL levels*

modifying cardiovascular risk factors.

*10.1.4. Inhibition of cannabinoid receptors*

*10.1.3. CETP inhibition*

trials [94].

in testing [95].

screening [96].

*10.1.5. Nuclear receptor modifiers*

ACAT 1 and ACAT2 play an important role in cellular cholesterol esterification and thus modulate intestinal cholesterol absorption and hepatic lipoprotein secretion. ACAT2 is proposed to play a central role in cholesterol absorption from the intestine. ACAT1, on the other hand is widely distributed in tissues, and plays a pivotal role in cholesterol metabolism in macrophages and steroidogenic tissues. It is anticipated that inhibitors of ACAT2 activity could be developed that would decrease cholesterol absorption, whereas specific ACAT1 inhibitors could be used to reduce foam cell formation and prevent atherosclerosis. However, the results of studies have indicated that ACAT1 inhibition is not a good strategy and, in fact, could have detrimental consequence [97]. In contrast, mice lacking ACAT2 exhibited attractive metabolic findings. These include a restricted capacity to absorb cholesterol and protection against diet-induced hypercholesterolemia and gallstone formation [98, 99]. Further, ACAT2 inhibitor reduces cholesterol esters in apolipoprotein B-containing lipoproteins and protects from atherosclerosis in murine models of the disease [100]. A fundamental question remains: would ACAT2 specific inhibition in humans, either pharmacologically or with anti-sense oligonucleotides, prevent or reduce atherosclerosis? "The sole test of the validity of an idea is experiment." Until a potent and specific inhibitor of ACAT2 is tested in humans, the hypothesis remains uncertain [101].

#### **10.2. Future perspectives of cholesterol-lowering products and their potential impact on CVD**

#### *10.2.1. Identification of new molecular targets*

Target identification is an essential first step in drug development. Although cholesterol metabolism has been studied for decades and relatively well understood, the specific molec‐ ular targets that can be used for drug discovery and development are far from well-known. It is believe that with the development and use of modern molecular technology and techniques in nutritional and physiological research, more targets will be indentified and used for future drug development.

#### *10.2.2. Application of gene silencing technology*

Apart from the identification of new targets, several new approaches have been introduced or shown great potential for the development of new cholesterol-lowering products. Of them is gene-silencing technology. One technology that has generated a lot of excitement is RNA interference (RNAi) [102]. For example, clinical trials have been launched using RNA inter‐ ference approaches to reduce PCSK9 expression or specific antibodies targeting and inhibiting PCSK9 interaction with the LDL receptor. They constitute very promising approaches to reducing cholesterol levels and coronary heart disease. Understanding of PCSK9 and its potential as a therapeutic target through which to reduce LDL cholesterol for prevention and treatment of coronary heart disease has been of great interest recently [103]. The administration of chemically modified small interference RNAs (siRNAs) results in silencing of a target mRNA, such as apolipoprotein B (apoB) mRNA in liver and jejunum and decreasing plasma levels of apoB protein. ApoB is a molecule involved in the metabolism of cholesterol; the concentrations of this protein in human blood samples correlate with those of cholesterol. Higher levels of both compounds are associated with an increased risk of coronary heart disease. Intravenous injections of the siRNA-cholesterol conjugates in mice resulted in a lowering of the levels of blood cholesterol comparable to that in mice in which the apoB gene had been deleted. These results demonstrate that siRNA can be delivered systemically to target the liver and suggest that RNAi has the potential to become a new therapeutic for the treatment of metabolic diseases [102].

#### *10.2.3. Development of target-specific inhibitors or enhancers*

For those molecular targets that have several different isoforms, the biggest challenge in the drug development is the specificity. A good example is ACAT inhibitors. To date, it has been demonstrated that there are two sioforms, ACAT1 and ACAT2. ACAT1 is ex‐ pressed universally while ACAT2 is expressed mainly in the liver and small intestine. Both isoforms can esterify cholesterol and other sterols. However, ACAT1 primarily works on the esterification of plant sterols and other sterol products, while ACAT2 main‐ ly converts cholesterol to its esters. Due to their different distribution and activities in the body, a universal inhibitor of both enzymes has been shown to be detrimental and recent humans trials are disappointing [104, 105]. The study of Bell et al. in 2006 breathes life back into the idea of ACAT2-specific inhibition [106]. In atherosclerosis-prone mice, ACAT2 was specifically ihibhied in the liver with antisense oligonucleotides. Biweekly intraperitoneal injections, which reduced ACAT2 expression by a remarkable 80%, de‐ creased diet-induced hypercholesterolemia and sharply reduced cholesterol ester deposi‐ tion in the aorta. The specific inhibition can be achieved from the antisense gene technology or small molecules that inhibit only ACAT2. There is no mature product in this category and lots more work needs to be done.

It has been demonstrated by numerous studies that inhibition of cholesterol absorption is associated with much less side-effects than other approaches. It remains challenge but highly promising that a potent inhibitor of cholesterol absorption would sufficiently low‐ er blood cholesterol or keep blood cholesterol levels within the recommended healthy range. To achieve this goal, other approaches have to be take in addition to the specific inhibitor of cholesterol absorption, such as a combination of two or more products, in particular the combinations that work synergistically through one or multiple pathways of cholesterol homeostasis. Recent studies on the combination of the plant alkaloid ber‐ berine and plant sterols/stanols have shown an excellent example of this approach. The researchers have demonstrated consistently that plant sterols or stanols and berberine have a moderate effect to lower blood cholesterol levels in different animal models. However, when plant sterols/stanols were combined with berberine, a significant syner‐ gism was produced. They remarkably improved cholesterol-lowering efficacy by synerg‐ istically inhibiting cholesterol absorption [90,91].
