![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CARDIOVASCULAR
Laboratory of Pharmacology, Medical School, Democritus University of Thrace, Alexandroupolis, Greece (A.T., V.G.M.); Onassis Cardiac Surgery Center, Department of Pathology, Athens, Greece (L.K.); ELPEN Pharmaceutical Co. Inc., Pikermi, Greece (A.P., A.C.); Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Athens, Greece (A.P.K.); and Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece (E.A.R., P.N.K.)
Received May 29, 2007; accepted September 4, 2007.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). It is unquestionably the main underlying pathology of cardiovascular disease, the leading cause of morbidity and mortality worldwide (Hennekens, 1998). There is evidence that elevated plasma levels of low-density lipoprotein (LDL)-cholesterol as well as oxidative modification of LDL are major risk factors for the development of atherosclerosis (Steinberg et al., 1989). Both primary and secondary prevention trials have shown that lipid lowering with 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors, hereafter referred to as statins, reduce morbidity and mortality from coronary heart disease (Group 4S, 1994; Shepherd et al., 1995). Apart from the lipid-lowering effect, statins have also direct antiatherosclerotic effects on the vessel wall (Bellosta et al., 1998). However, despite the results obtained with these drugs, the majority of cardiovascular events are not prevented by treatment with statins. Numerous additional factors have been associated with the initiation and progression of atherosclerosis, including monocyte/macrophage accumulation at the subendothelial space, endothelial cell dysfunction, oxidized LDL uptake by macrophages and endothelial cells leading to foam cell formation, and oxidized LDL-induced inhibition of nitric oxide production. Therefore, novel agents targeting any of these steps in atherosclerotic lesion development might prove useful in clinical practice.
The main regulatory enzyme of the cholesterol synthesis pathway, HMG-CoA reductase, is inhibited by statins. However, statins also suppress the production of mevalonate, an intermediate in cholesterol biosynthesis. Mevalonate is also a precursor of nonsterol products that are vital for diverse cellular functions. Both major known side effects of statins (hepatotoxicity and myotoxicity) have been associated with inhibition of the synthesis of the nonsterol products (Kornbrust et al., 1989; Masters et al., 1995).
The enzyme squalene synthase catalyzes the conversion of farnesyl pyrophosphate to squalene, the first committed step in the de novo cholesterol biosynthesis. Squalene synthase inhibitors are candidate hypocholesterolemic agents that decrease circulating LDL-cholesterol by an increased expression of hepatic LDL receptors in a similar manner to statins (for review, see Charlton-Menys and Durrington, 2007). They are characterized by a more specific action that leaves other nonsterol products of mevalonate metabolism unaffected. Therefore, squalene synthase inhibitors have at least one theoretical advantage compared with statins as hypocholesterolemic and antiatherosclerotic drugs because of reduced side effects. Several squalene synthase inhibitors have been reported in the literature, including ER-27586 (Hiyoshi et al., 2000), YM-53601 (Ugawa et al., 2002a,b), and TAK-475 (Amano et al., 2003; Nishimoto et al., 2003). The latter, a 4,1-benzoxazepine-3-acetic acid derivative, is an effective lipid-lowering agent in a variety of animal models. TAK-475 has become the first squalene synthase inhibitor to enter phase III clinical trials to assess its ability to reduce LDL-cholesterol alone or in combination with statins or ezetimibe (Burnett, 2006; Davidson, 2007).
Several novel 2-biphenylmorpholine derivatives have been synthesized, and preliminary results for their hypolipidemic and antioxidant properties have been reported (Chrysselis et al., 2000, 2002). Two of these compounds, EP2306 and EP2302, have been shown to possess antioxidant properties both in vitro and in vivo (Tavridou and Manolopoulos, 2004) as well as to inhibit squalene synthase activity and lipid biosynthesis in vitro (Tavridou et al., 2006). The chemical structure of EP2306 is shown in Fig. 1. To the best of our knowledge, no reports exist on the antiatherosclerotic effects of squalene synthase inhibitors in animals or humans. In the present study, we hypothesized that EP2306 can effectively modify circulating lipids and reduce atherosclerosis in the cholesterol-fed rabbit. Our main finding is that both EP2306 and simvastatin reduce atherosclerotic plaque formation in this animal model. However, only the antiatherosclerotic effect of EP2306 persists after 3 months of treatment.
|
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). Simvastatin was obtained from ELPEN Pharmaceuticals (Pikermi, Greece). Cholesterol was purchased from Dolder AG (Basel, Switzerland). Experimental Design. Male, New Zealand White rabbits (2.5–3.5 kg), fed a standard rabbit chow, were acclimatized for 1 week before experiments started. The rabbits were housed individually in cages and given water ad libitum. Subsequently, they were fed a high-cholesterol (HC) diet consisting of standard chow supplemented with 0.5% cholesterol for 4 weeks and then were randomly divided into several treatment groups. During treatment, all animals continued to receive an HC diet. Animals were treated daily by oral gavage with the compounds suspended in 34 mM NaCl/71 mM HCl (vehicle) as indicated. Control animals received an equal volume of vehicle only (placebo). The experimental design of the study is shown in Fig. 2. In phase 1, animals were allocated to six groups and received placebo (vehicle only), several doses of EP2306 (0.2–10 mg/kg), or simvastatin (2.5 mg/kg) daily for 4 weeks. In phase 2, animals were allocated to four groups and received placebo, 2 mg/kg EP2306, 2.5 mg/kg simvastatin, or their combination daily for 4 weeks. In phase 3, animals were allocated to four groups as in phase 2, but the duration of treatment was 12 weeks. The dose of simvastatin was chosen on the basis of previous experiments designed to rule out toxicity, and it was the maximum dose tolerated by the animals without any significant increase in deaths. The weight of the animals was recorded weekly to adjust the dose of drugs. Records of food intake did not show any significant difference among animals. General symptoms were assessed daily throughout the study. At the end of the experiments, animals were killed by exsanguination by cardiac puncture while under phenobarbital anesthesia.
|
Biochemical Methods. Blood was collected after overnight fasting from the central ear artery. Serum was separated, stored at –80°C, and used within 1 week for biochemical measurements. Serum levels of total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, glutamic oxaloacetic transaminase (SGOT), and glutamate pyruvate transaminase (SGPT) were determined enzymatically on an Abbott Alcyon 300 Analyzer using commercially available reagents (Abbott Laboratories, Abbott Park, IL).
Histopathology. After euthanasia, the lungs, the kidneys, the heart, the liver, the antrum of the stomach, the gallbladder, and the spleen were removed, washed, and their weight was measured. Macroscopic examination followed, and representative sections were taken from each one of the above organs. The samples were fixed overnight in 10% buffered formalin and, after routine processing, were embedded in paraffin. Routine histochemistry was performed on sections 3 µm in thickness, including staining with hematoxylineosin, trichrome, elastic van-Gieson, and oil-red (the latter done on fixed-frozen material).
All the above organs were examined under routine light microscopy to identify the degree of lipid deposition in the parenchyma and the vessels, the degree of fibrosis, the extent and the type of the inflammation, and, if present, any other pathological changes. The pathologist who analyzed all these specimens was unaware of the compound administered to the animals.
Assessment of Atherosclerosis. Three samples (rings) from the ascending aorta were taken starting from the area above the ostia of the left main coronary artery, each one measuring approximately 1.5 to 2 mm in length. The same number of sections and all of them of similar length were taken from the descending aorta starting from the area immediately after the aortic arch. A random fragment from the carotid was also excised. All these samples were fixed in buffered 10% formalin and after routine processing, were embedded in paraffin. Tissue sections 3 µm thick were cut from paraffin-embedded blocks. The sections were dewaxed, dehydrated in graded alcohols, and stained with hematoxylin and eosin.
Serial sections were cut from all the six samples of both sides of the thoracic aorta and stained accordingly. Four parameters were analyzed, and two ratios were evaluated using an image analysis system (CAS; BD Biosciences, San Jose, CA), as follows: 1) the perimeter of the lumen (the perimeter of intima); 2) the perimeter or the length of the atherosclerotic lesions, the ratio b/a provides the percentage of the intima affected by atherosclerosis; 3) the area of the lumen; and 4) the sum of the atherosclerotic lesions occupying part of the area of the lumen, the ratio d/c provides the percentage of the luminal area affected by atherosclerosis. All the measurements from the samples of the ascending and the descending aorta were summarized, and the mean number was calculated. Apart from the usual atherosclerotic changes (e.g., fatty streaks composed of foamy macrophages filled with lipid), additional histopathological findings such as microcalcifications of the media, intramuscular degeneration, or hemorrhage were included in the histopathological analysis.
Statistics. Statistical analyses were performed using the Statistical Package for Social Sciences (version 10.0; SPSS Inc., Chicago, IL). Data are expressed as mean ± S.E.M. Values of p less than 0.05 were considered statistically significant. Wilcoxon's test was used to compare paired values before and after treatment with drugs within the same treatment. The effect of different treatments on lipid levels was assessed using one-way analysis of variance (Vickers and Altman, 2001) followed by post hoc Dunnett's multiple comparison analyses or Kruskal-Wallis test followed by Mann-Whitney U test as appropriate. The effect of treatment on atherosclerosis was examined using Kruskal-Wallis test followed by Mann-Whitney U test. Bivariate association between cholesterol lowering and antiatherosclerotic effect of EP2306 was examined using Spearman's correlation coefficient. For this purpose, total cholesterol values after treatment were subtracted from values before treatment; therefore, a negative correlation indicates a positive association between cholesterol lowering and antiatherosclerotic effect.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
Four rabbits were sacrificed after 4 weeks of receiving an HC diet. Atherosclerotic lesions of significant size had been developed in the whole length of the aorta, the coronary arteries, and the carotids in all four animals. In the ascending aorta, the mean perimeter ratio was 0.32, and the mean area ratio was approximately 0.074. In one of these animals, foci of calcifications were identified in the wall of the descending aorta.
Phase 1 Experiments. The aims of phase 1 experiments were to: 1) establish and characterize in detail the hypercholesterolemic rabbit model, 2) find the dose range of EP2306 that is effective in this model, and 3) assess macro- and microscopically as well as biochemically possible adverse or toxic effects in rabbits following treatment with EP2306. A dose-response curve was performed to examine the hypolipidemic effect of EP2306 (0.2–10 mg/kg). The levels of total, LDL-, and HDL-cholesterol as well as triglycerides before and after drug treatment are shown in Table 2. Total cholesterol was significantly reduced compared with pretreatment levels by 2 mg/kg EP2306, but no other dose of the compound caused any significant changes on the levels of lipid parameters in the blood. Simvastatin (2.5 mg/kg) failed to produce any significant changes in any of the lipids measured (Table 2).
|
Quantification of morphometric analyses of cross-sections of the ascending and descending aorta is shown in Fig. 3. The ratio of the perimeter as well as the area covered by atherosclerotic lesions was assessed for increasing doses of EP2306 (0.2–10 mg/kg) or 2.5 mg/kg simvastatin, and it was compared with animals treated with placebo. A significant decrease in the ratio of the perimeter of the ascending and descending aorta covered by lesions was observed in animals treated with 2 mg/kg EP2306 (50–60% reduction) or simvastatin (
70% reduction) compared with animals treated with placebo (Fig. 3, A and B). The significant decrease in the ratio of the area covered by lesions after treatment with EP2306 was observed for several doses of EP2306 as well as for simvastatin (Fig. 3, C and D). Correlation analysis was performed to examine a possible association of the cholesterol-lowering effect of EP2306 with the decreased ratio of the perimeter or area of the ascending and descending aorta covered by lesions (Table 3). The different doses of EP2306 were examined separately or, to increase power, were combined. However, no such associations were identified as indicated by the lack of significant correlations between the reduction of total cholesterol and the ratio of the perimeter or area of the ascending or descending aorta covered by lesions.
|
|
Phase 2 Experiments. Phase 2 followed the same experimental design of phase 1. The specific aim of this set of experiments was to evaluate the effects of EP2306, at its most effective dose of 2 mg/kg (determined in phase 1), when given to the animals together with 2.5 mg/kg simvastatin. A group of animals receiving only simvastatin was also included. EP2306, simvastatin, or their combination did not significantly affect any of the lipid parameters measured, although there was a tendency of EP2306 to reduce both total (517 ± 106 before versus 413 ± 91 mg/dl after treatment) and LDL-cholesterol levels (263 ± 65 before versus 184 ± 58 mg/dl after treatment). Figure 4 shows the changes in atherosclerosis observed with the three treatments employed. There was a significant decrease of 35 ± 5% in the perimeter of the lumen covered by atherosclerotic lesions in animals treated with 2 mg/kg EP2306 compared with animals treated with placebo (p < 0.05). Simvastatin (2.5 mg/kg) caused a similar significant inhibition of atherosclerosis with EP2306. However, a further reduction of 48 ± 7% (p < 0.05) of atherosclerotic lesions was observed with the combination of EP2306 with simvastatin (Fig. 4, A and B). A less pronounced effect on atherosclerosis was observed according to the assessment of the area covered by atherosclerotic lesions but even in this case, the effect of the combination of EP2306 with simvastatin remained significant (Fig. 4, C and D). Correlation analysis was performed to examine a possible association of the total cholesterol-lowering effect of EP2306 with the decreased ratio of the perimeter or area of the ascending and descending aorta covered by lesions (Table 3), but, similarly with phase 1, no significant correlations were identified. However, when animals of phases 1 and 2 treated with 2 mg/kg EP2306 were combined, a significant (p = 0.028) association between lowering of total cholesterol and the perimeter of the ascending aorta covered by lesions was found (Table 3). It should be noted though that no such associations were seen between the other indexes of aortic atherosclerosis and total cholesterol lowering.
|
|
Figure 6 shows the changes in atherosclerosis observed after 3 months of treatment with the different treatments employed. There was a significant decrease by more than 50% of atherosclerotic lesions in the ascending (Fig. 6, A and C) and descending aorta (Fig. 6, B and D) in animals treated with EP2306 as assessed both by the perimeter and the area covered by atherosclerotic lesions (p < 0.05). However, simvastatin alone or in combination with EP2306 did not result in any significant reduction of atherosclerosis. Figure 7 shows representative images of stained atherosclerotic lesions after 3 months of treatment with EP2306, simvastatin, or their combination compared with animals treated with placebo. There was an extensive accumulation of foamy macrophages forming a thick layer protruding into the vascular lumen in animals treated with placebo or the combination of EP2306 with simvastatin, whereas treatment with EP2306 or simvastatin resulted in thinner layers of foamy macrophages.
|
|
Effect of EP2306 or Simvastatin on Liver Transaminases. In phase 1, liver transaminases were measured in a subset of animals before and after 4 weeks treatment with different doses of EP2306 (0.2, 0.5, 2, and 10 mg/kg) or simvastatin (2.5 mg/kg) (Fig. 8). A small but significant decrease of SGPT levels was observed after treatment with EP2306 (46.2 ± 4.1 before versus 37.2 ± 2.9 U/l after treatment, p = 0.045). Simvastatin increased SGPT levels approximately 2-fold (40.6 ± 3.6 before versus 89.7 ± 39.2 U/l after treatment), but this difference did not reach significance. Treatment with EP2306 did not affect significantly SGOT levels, whereas there was a significant 3-fold increase of SGOT levels after treatment with simvastatin (30.0 ± 2.3 before versus 90.4 ± 18.9 U/l after treatment, p = 0.021).
|
Toxicity of EP2306 and Simvastatin on Various Organs. In all animals of the three phases of experiments, sections from various organs were also analyzed to detect any histopathological changes caused by EP2306, simvastatin, or their combination. The main findings per organ were the following.
Heart. Many atheromatic changes were observed mainly in intracardiac arteries with the form of subintimal accumulation of lipids. These features were present in the animals treated with placebo and to a lesser extent in the other groups. No evidence of myocarditis, necrosis, fibrosis, or thrombosis was seen.
Liver. Multifocal deposition of lipids in the form of micro- and macrovesicular fatty changes were observed. In addition, there was evidence of mild fibrosis in the periportal areas but no evidence of necrosis.
Lung. There was no evidence of edema, peribronchial inflammation, or vasculitis in any of the animals.
Kidney. No features of interstitial nephritis, glomerular changes, vascular lesions, necrosis, or thrombosis were observed.
Spleen. Numerous foamy macrophages were present in most of the animals fed with high-cholesterol diet.
Stomach. No evidence of interstitial inflammation, ulceration, necrosis, metaplasia, or atrophy was observed.
Gallbladder. No features of ulceration, inflammation, or necrosis were noted.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). A significantly greater potency for inhibition of the enzyme has been reported for other squalene synthase inhibitors. For example, YM-53601 and TAK-475 inhibit human liver squalene synthase with IC50 values of 79 and 78 nM, respectively. However, it is not possible to directly compare the lipid-lowering properties of EP2306 with these compounds since different animal models were used for this purpose.
Despite the existence of several studies showing a lipid-lowering effect of squalene synthase inhibitors, so far, no data exist on the effect of any such compound on atherosclerosis in vivo. The experimental model of diet-induced atherosclerosis in rabbits has been extensively used to study the role of different pathways in atherogenesis as well as to evaluate the ability of pharmacological treatment to modulate atherosclerosis mainly because the atherosclerotic lesions in this model resemble those occurring in humans (Sugano et al., 1996; Yang et al., 1998). High cholesterol levels are easily achieved in rabbits fed an HC diet. However, the cholesterol-lowering effect of statins is a phenomenon restricted to higher animal species, including primates and man, because in lower animals, enzyme inhibition is followed by a dramatic rise of tissue enzyme levels blunting the plasma cholesterol-lowering response (Endo, 1992). Therefore, it is not unexpected that inconsistent results have been obtained regarding the cholesterol-lowering effect of statins in this animal model (Alfon et al., 1999; Hernandez-Presa et al., 2003). Moreover, the cholesterol-fed rabbit is not a good model for studying the mechanisms affecting triglycerides and HDL-cholesterol levels, as shown in previous reports where HC diet resulted only in modest changes in these lipids (Hernandez-Presa et al., 2003; Nachtigal et al., 2005). Our results confirm these reports because no significant changes were observed following 1 month of HC diet in the levels of HDL-cholesterol, whereas a significant but modest increase was observed in triglyceride levels.
In preclinical studies, several squalene synthase inhibitors have been shown to possess lipid-lowering properties (Hiyoshi et al., 2000; Ugawa et al., 2002; Amano et al., 2003; Nishimoto et al., 2003). So far, EP2306 has been shown to be an effective inhibitor of cholesterol and triglyceride biosynthesis in vitro (Tavridou et al., 2006). Moreover, it decreased total cholesterol, LDL-cholesterol, and triglycerides in plasma of Triton WR-1339-induced hyperlipidemic rats (Chrysselis et al., 2000). However, in the present study, we did not observe consistent reductions in total and LDL-cholesterol levels after treatment with EP2306 in the cholesterol-fed rabbit. One shortcoming of this model is that, to establish atherosclerotic lesions in these animals, serum cholesterol must reach much higher levels than those occurring in clinical situations. Further studies are required to evaluate the lipid-lowering effect of EP2306 in this animal model, performed in milder hypercholesterolemic conditions that would be more relevant to the clinical situation.
We found significant reductions of atherosclerotic lesions in animals treated with EP2306 compared with control animals. The weak and inconsistent effect of the compound on lipids and the lack of association between lipid-lowering and most atheromatic indexes assessed suggest that it is unlikely the antiatherosclerotic effects of EP2306 in HC rabbits to be solely mediated by the cholesterol-lowering properties of this drug. It appears that there are other mechanisms preventing atherosclerosis development in response to EP2306 in these animals. Interestingly, there is clinical evidence that inhibition of LDL-oxidation can inhibit atherosclerosis independently of lowering plasma cholesterol levels. Among statins, fluvastatin and simvastatin possess significant antioxidant properties (Franzoni et al., 2003). The antiatherosclerotic effects of fluvastatin have been extensively studied in the cholesterol-fed rabbit and have been mainly attributed to its antioxidant properties. Baetta et al. (2002) have shown that fluvastatin reduces tissue factor expression and macrophage accumulation in carotid lesions of cholesterol-fed rabbits in the absence of lipid lowering. Furthermore, fluvastatin, at a dose insufficient to reduce plasma cholesterol levels in cholesterol-fed rabbits, decreased atheromatous plaque formation by preserving endothelial function (Rikitake et al., 2001; Mitani et al., 2003). We have already shown that EP2306 inhibits dose dependently the in vitro oxidation of LDL induced by copper ions as well as 12-lipoxygenase activity (Tavridou and Manolopoulos, 2004). In addition, in hyperlipidemic rabbits treated with EP2306 for 4 weeks, there was a decrease in thiobarbituric acid-reactive substance levels and a significant increase in total peroxyl radical-trapping potential levels compared with control animals (Tavridou and Manolopoulos, 2004). Unfortunately, in the present study, no such markers of oxidative status were assessed to examine a possible association with the antiatherosclerotic effect of EP2306. However, these antioxidant properties of EP2306 could contribute to its antiatherosclerotic effect by several different manners. For example, decreased LDL-oxidation may suppress the expression of adhesion molecules by endothelial cells and the expression of tissue factor, colony-stimulating factor, and monocyte-chemoattractant protein-1 in macrophages, molecules that modulate the atherosclerotic process. All these findings suggest that inhibition of LDL-oxidation might contribute directly and/or indirectly to the antiatherosclerotic effects of EP2306 and the same appears to be true for simvastatin.
Although both EP2306 and simvastatin monotherapy as well as their combination significantly reduced atherosclerosis after 1 month of treatment, the results of the long-term study (3 months of treatment) showed a differential effect on atherosclerosis by these two compounds. EP2306 sustained its antiatherosclerotic effect for this longer period, whereas there was a reversal of the effect of simvastatin either administered as monotherapy or in combination with EP2306. A possible explanation of this finding could be differential toxicity exerted by the compounds studied in this long-term study, but because we did not find any differences in organ toxicity among treatment groups, this is not very likely. Interestingly, our finding with simvastatin is in agreement with earlier reports questioning the sustained efficacy of statins. Long-term administration of statins was shown to be associated with the escape phenomenon, in which plasma cholesterol levels of human subjects are initially reduced but return to pretreatment levels (Pazzucconi et al., 1995). These investigators classified patients treated with statins (including simvastatin) in three groups: 1) responders, 2) nonresponders, and 3) response losers. In routine clinical practice, this is often masked either by increasing drug dosage or by adding drugs with different modes of action. The mechanism by which HMG-CoA reductase inhibitors induce the escape phenomenon has been a subject of considerable research and is believed to involve their causing inhibition of not only cholesterol but also mevalonate derivatives such as farnesol. For example, Ugawa et al. (2002b) described an animal model of the escape phenomenon observed in humans and showed that the squalene synthase inhibitor YM-53601 consistently decreased plasma non-HDL-cholesterol in this model, whereas pravastatin lost its hypolipidemic effect between 17 and 27 days of treatment.
Hepatotoxicity remains one of the concerns of statin use, and frequent follow-up of liver markers is recommended. To exclude such an adverse effect with EP2306 treatment, we examined the effect of EP2306 on serum liver transaminases. In comparison with simvastatin, EP2306 showed a more favorable effect both on SGOT and SGPT enzymes, indicating a lack of hepatotoxicity. Moreover, there was no evidence of toxicity of EP2306 on any other vital organs including the heart, liver, lungs, kidney, spleen, stomach, and gallbladder. Overall, the safety profile of EP2306 on this animal model was good, and this is encouraging given the safety concerns raised for many of the therapeutic agents aiming at the cardiovascular system.
Hitherto, strategies for prevention of cardiovascular disease mainly focus on improving dyslipidemia with the use of statins. Frequently, monotherapy with statins or any other agent is insufficient. Meta-analyses have shown that even with the most aggressive treatment with statins, the reduction of the risk for major coronary events is only 30% (LaRosa et al., 1999). Clearly, there is a need for combining drugs with different or complementary mechanisms of action to achieve the proposed lower LDL-cholesterol therapeutic targets. Recently, the effects of ezetimibe, an inhibitor of dietary cholesterol absorption, have been described (Sudhop et al., 2002; Garcia-Calvo et al., 2005), and the agent is currently widely used as an add-on hypolipidemic agent to statins (Gagne et al., 2002; Davidson et al., 2004). Squalene synthase inhibitors might prove to be an additional useful tool in the context of combination therapy of hyperlipidemia.
In conclusion, we have shown that EP2306 reduces the development of atherosclerotic lesions in an animal model of diet-induced atherosclerosis. This is the first report showing an inhibitory effect of a squalene synthase inhibitor on atherosclerosis in vivo. Although more research is needed to further elucidate its mechanisms of action and its pharmacological profile in humans, such a molecule that confers vascular protection indicates potential as an additional therapeutic approach for coronary artery disease and other atherosclerosis-related disorders.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: LDL, low-density lipoprotein; HMG-CoA, 3-hydroxy-3-methyl glutaryl coenzyme A; EP2306, 2-(4-biphenyl)-4-methyl-octahydro-1,4-benzoxazin-2-ol, hydrobromide; EP2302, 2-(4-biphenyl)-2-(3-nitrooxypropoxy)-4-methylmorpholine, hydrobromide; HC, high-cholesterol; HDL, high-density cholesterol; SGOT, (serum) glutamic oxaloacetic transaminase; SGPT, (serum) glutamate pyruvate transaminase; TAK-475, 2-(1-(2-((3R,5S)-1-(3-acetoxy-2,2-dimethylpropyl)-7-chloro-5-(2,3-dimethoxyphenyl)-2-oxo-1,2,3,5-tetrahydrobenzo[e][1,4]oxazepin-3-yl)acetyl)piperidin-4-yl)acetic acid; ER-27856, 5-{N-[2-butenyl-3-(2-methoxyphenyl)]-N-methylamino}-1,1-pentylidenebis(phosphonic acid)-tripivaloyloxymethyl ester; YM-53601, (E)-2-[2-fluoro-2-(quinuclidin-3-ylidene)ethoxy]-9H-carbazole monohydrochloride.
Address correspondence to: Vangelis Manolopoulos, Laboratory of Pharmacology, Medical School, Democritus University of Thrace, Dragana Campus, 68100 Alexandroupolis, Greece. E-mail: manolopoulos{at}med.duth.gr
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Alfon J, Guasch JF, Berrozpe M, and Badimon L (1999) Nitric oxide synthase II (NOS II) gene expression correlates with atherosclerotic intimal thickening: preventive effects of HMG-CoA reductase inhibitors. Atherosclerosis 145: 325–331.[CrossRef][Medline]
Amano Y, Nishimoto T, Tozawa R, Ishikawa E, Imura Y, and Sugiyama Y (2003) Lipid-lowering effects of TAK-475, a squalene synthase inhibitor, in animal models of familial hypercholesterolemia. Eur J Pharmacol 466: 155–161.[CrossRef][Medline]
Baetta R, Camera M, Comparato C, Altana C, Ezekowitz MD, and Tremoli E (2002) Fluvastatin reduces tissue factor expression and macrophage accumulation in carotid lesions of cholesterol-fed rabbits in the absence of lipid lowering. Arterioscler Thromb Vasc Biol 22: 692–698.
Bellosta S, Bernini F, Ferri N, Quarato P, Canavesi M, Arnaboldi L, Fumagalli R, Paoletti R, and Corsini A (1998) Direct vascular effects of HMG-CoA reductase inhibitors. Atherosclerosis 137 (Suppl): S101–S109.[CrossRef][Medline]
Burnett JR (2006) Drug evaluation: TAK-475: an oral inhibitor of squalene synthase for hyperlipidemia. Curr Opin Investig Drugs 7: 850–856.[Medline]
Charlton-Menys V, and Durrington PN (2007) Squalene synthase inhibitors: clinical pharmacology and cholesterol-lowering potential. Drugs 67: 11–16.[CrossRef][Medline]
Chrysselis MC, Rekka EA, and Kourounakis PN (2000) Hypocholesterolemic and hypolipidemic activity of some novel morpholine derivatives with antioxidant activity. J Med Chem 43: 609–612.[CrossRef][Medline]
Chrysselis MC, Rekka EA, Siskou IC, and Kourounakis PN (2002) Nitric oxide releasing morpholine derivatives as hypolipidemic and antioxidant agents. J Med Chem 45: 5406–5409.[CrossRef][Medline]
Davidson MH (2007) Squalene synthase inhibition: a novel target for the management of dyslipidemia. Cur Atheroscler Rep 9: 78–80.[CrossRef]
Davidson MH, Ballantyne CM, Kerzner B, Melani L, Sager PT, Lipka L, Strony J, Suresh R, and Veltri E (2004) Efficacy and safety of ezetimibe coadministered with statins: randomised, placebo-controlled, blinded experience in 2382 patients with primary hypercholesterolemia. Int J Clin Pract 58: 746–755.[CrossRef][Medline]
Endo A (1992) The discovery and development of HMG-CoA reductase inhibitors. J Lipid Res 33: 1569–1582.[Medline]
Franzoni F, Quinones-Galvan A, Regoli F, Ferrannini E, and Galetta F (2003) A comparative study of the in vitro antioxidant activity of statins. Int J Cardiol 90: 317–321.[CrossRef][Medline]
Gagne C, Bays HE, Weiss SR, Mata P, Quinto K, Melino M, Cho M, Musliner TA, and Gumbiner B (2002) Efficacy and safety of ezetimibe added to ongoing statin therapy for treatment of patients with primary hypercholesterolemia. Am J Cardiol 90: 1084–1091.[CrossRef][Medline]
Garcia-Calvo M, Lisnock J, Bull HG, Hawes BE, Burnett DA, Braun MP, Crona JH, Davis HR Jr, Dean DC, Detmers PA, et al. (2005) The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1). Proc Natl Acad SciUSA 102: 8132–8137.
Group 4S (1994) Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 344: 1383–1389.[CrossRef][Medline]
Hennekens CH (1998) Increasing burden of cardiovascular disease: current knowledge and future directions for research on risk factors. Circulation 97: 1095–1102.
Hernandez-Presa MA, Ortego M, Tunon J, Martin-Ventura JL, Mas S, Blanco-Colio LM, Aparicio C, Ortega L, Gomez-Gerique J, Vivanco F, et al. (2003) Simvastatin reduces NF-kappaB activity in peripheral mononuclear and in plaque cells of rabbit atheroma more markedly than lipid lowering diet. Cardiovasc Res 57: 168–177.[CrossRef][Medline]
Hiyoshi H, Yanagimachi M, Ito M, Ohtsuka I, Yoshida I, Saeki T, and Tanaka H (2000) Effect of ER-27856, a novel squalene synthase inhibitor, on plasma cholesterol in rhesus monkeys: comparison with 3-hydroxy-3-methylglutaryl-coa reductase inhibitors. J Lipid Res 41: 1136–1144.
Kornbrust DJ, MacDonald JS, Peter CP, Duchai DM, Stubbs RJ, Germershausen JI, and Alberts AW (1989) Toxicity of the HMG-coenzyme A reductase inhibitor, lovastatin, to rabbits. J Pharmacol Exp Ther 248: 498–505.
LaRosa JC, He J, Vupputuri S (1999) Effect of statins on risk of coronary disease: a meta-analysis of randomized controlled trials. J Am Med Assoc 282: 2340–2346.
Libby P (2002) Inflammation in atherosclerosis. Nature 420: 868–874.[CrossRef][Medline]
Masters BA, Palmoski MJ, Flint OP, Gregg RE, Wang-Iverson D, and Durham SK (1995) In vitro myotoxicity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, pravastatin, lovastatin, and simvastatin, using neonatal rat skeletal myocytes. Toxicol Appl Pharmacol 131: 163–174.[CrossRef][Medline]
Mitani H, Egashira K, Ohashi N, Yoshikawa M, Niwa S, Nonomura K, Nakashima A, and Kimura M (2003) Preservation of endothelial function by the HMG-CoA reductase inhibitor fluvastatin through its lipid-lowering independent antioxidant properties in atherosclerotic rabbits. Pharmacology 68: 121–130.[CrossRef][Medline]
Nachtigal P, Kopecky M, Solichova D, Zdansky P, and Semecky V (2005) The changes in the endothelial expression of cell adhesion molecules and iNOS in the vessel wall after the short-term administration of simvastatin in rabbit model of atherosclerosis. J Pharm Pharmacol 57: 197–203.[CrossRef][Medline]
Nishimoto T, Amano Y, Tozawa R, Ishikawa E, Imura Y, Yukimasa H, and Sugiyama Y (2003) Lipid-lowering properties of TAK-475, a squalene synthase inhibitor, in vivo and in vitro. Br J Pharmacol 139: 911–918.[CrossRef][Medline]
Pazzucconi F, Dorigotti F, Gianfranceschi G, Campagnoli G, Sirtori M, Franceschini G, and Sirtori CR (1995) Therapy with HMG CoA reductase inhibitors: characteristics of the long-term permanence of hypocholesterolemic activity. Atherosclerosis 117: 189–198.[CrossRef][Medline]
Rikitake Y, Kawashima S, Takeshita S, Yamashita T, Azumi H, Yasuhara M, Nishi H, Inoue N, and Yokoyama M (2001) Anti-oxidative properties of fluvastatin, an HMG-CoA reductase inhibitor, contribute to prevention of atherosclerosis in cholesterol-fed rabbits. Atherosclerosis 154: 87–96.[CrossRef][Medline]
Shepherd J, Cobbe SM, Ford I, Isles CG, Lorimer AR, MacFarlane PW, McKillop JH, and Packard CJ (1995) Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia: West of Scotland Coronary Prevention Study Group. N Engl J Med 333: 1301–1307.
Steinberg D, Parthasarathy S, Carew TE, Khoo JC, and Witztum JL (1989) Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 320: 915–924.[Medline]
Sudhop T, Lutjohann D, Kodal A, Igel M, Tribble DL, Shah S, Perevozskaya I, and von Bergmann K (2002) Inhibition of intestinal cholesterol absorption by ezetimibe in humans. Circulation 106: 1943–1948.
Sugano M, Makino N, and Yanaga T (1996) The effects of renin-angiotensin system inhibition on aortic cholesterol content in cholesterol-fed rabbits. Atherosclerosis 127: 123–129.[CrossRef][Medline]
Tavridou A, Kaklamanis L, Megaritis G, Kourounakis AP, Papalois A, Roukounas D, Rekka EA, Kourounakis PN, Charalambous A, and Manolopoulos VG (2006) Pharmacological characterization in vitro of EP2306 and EP2302, potent inhibitors of squalene synthase and lipid biosynthesis. Eur J Pharmacol 535: 34–42.[CrossRef][Medline]
Tavridou A and Manolopoulos VG (2004) Antioxidant properties of two novel 2-biphenylmorpholine compounds (EP2306 and EP2302) in vitro and in vivo. Eur J Pharmacol 505: 213–221.[CrossRef][Medline]
Ugawa T, Kakuta H, Moritani H, and Inagaki O (2002a) Effect of YM-53601, a novel squalene synthase inhibitor, on the clearance rate of plasma LDL and VLDL in hamsters. Br J Pharmacol 137: 561–569.[CrossRef][Medline]
Ugawa T, Kakuta H, Moritani H, and Shikama H (2002b) Experimental model of escape phenomenon in hamsters and the effectiveness of YM-53601 in the model. Br J Pharmacol 135: 1572–1578.[CrossRef][Medline]
Vickers AJ, and Altman DG (2001) Statistics notes: analysing controlled trials with baseline and follow up measurements. Br Med J 323: 1123–1124.
Yang BC, Phillips MI, Mohuczy D, Meng H, Shen L, Mehta P, and Mehta JL (1998) Increased angiotensin II type 1 receptor expression in hypercholesterolemic atherosclerosis in rabbits. Arterioscler Thromb Vasc Biol 18: 1433–1439.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
