**Resistance of Statin Therapy, and Methods for its Influence**

Lyudmila Georgieva Vladimirova-Kitova and Spas Ivanov Kitov

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60004

#### **1. Introduction**

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184 Hypercholesterolemia

Resistance happens when an individual has an incorrect response to the effectiveness of a drug as stated in the National Library of Medicine. It is difficult to give an accurate definition of statin resistance. Patients who fail to reach LDL-C target levels despite undergoing the best available therapy of the most highly tolerated dose of a more potent statin, are considered to be statin-resistant. Many individuals do not reach LDL-C target levels, even when compliance is taken into consideration. The reduction of LDL-C in response to statin therapy can vary from 5-70 %. This can be influenced by many factors. For instance,racial andestry, with attenuated responses in blacks compared to whites. A study comparing statin resistance patients to patients who show no resistance to statin has yet to appear.

The resistance to statins can be related to differences in drug absorption, drug transport, intrahepatic drug metabolism, drug metabolism within other organs, and drug excretion mechanisms. The same can occur due to differences in the level of the various target pathways that are unrelated to pharmacokinetics, including HMG-CoA reductase, as well as various points along the cholesterol biosynthesis and lipoprotein metabolic pathways.

#### **2. Possible causes of statin resistance**

According to Herman and Moncada the process of atherogenesis includes 28 stages. [48] Key points in this process are two - oxygenated LDL-cholesterol and endogenous nitric oxide synthase. Statin reistance may exist in both directions:

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2.1. Failed targeting LDL cholesterol**

It seems that not only genetic but environmental factors can influence the LDL-C response to statins. Studies have found that patients with hypertension have a smaller decrease than those without hypertension. Furthermore, smokers have smaller statin-induced LDL-C decrease compared with nonsmokers[47]. It also seems that inflammation might cause statin resistance. Namely, it has been shown that inflammatory cytokines, in particular IL-1b which affects sterol regulatory element binding protein cleavage-activating protein, cause statin resistance due to the disruption of LDL-R feedback regulation. Therefore, it has been suggested that in inflam‐ matory states, higher concentrations of statin may be required to achieve the appropriate LDL-C lowering [107]. Particularly interesting are observations concerning certain subpopulations of patients who might be resistant to statin treatment. Some studies have shown statins to be less effective in individuals with HIV infection. [22]. Other studies have a contraversal perspective. [55]. The role of concomitant amiodarone treatment in statin resistance was also suspected. Both amiodarone and amiodarone induced hypothyroidism influence the synthesis of LDLR, which may explain the lack of statin effect. Thyroid hormone is one of several hormones that control gene expression of the LDLR and hypothyroidism is a wellknown cause of secondary dyslipidemia characterized by elevated LDL-C levels. Similar to hypothyroidism, administration of amiodarone also increases LDL-C levels, which is the result of a decreased expression of the LDLR gene [1].

More recently, an approach was published which used metabolomics to identify markers indicative of mechanisms that contribute to differences in LDL-C response to statin. Metabolic changes were shown to be more comprehensive in responders to statin treatment than those seen in nonresponders. The baseline cholesterol ester and phospholipid metabolites correlated with LDL-C response to treatment [56]. It has also been suggested that clusters of metabolites involved in multiple pathways not directly connected with cholesterol metabolism might as well play a role in modulating the response to statin therapy - influence statin resistance [90].

Insufficient LDL-C response to statin treatment is probably the result of pseudo-resistance, which could be caused by nonadherence or nonpersistence in real life circumstances. [68].

#### **3. Lack of effect on the endothelium-dependent vasodilation after targeting LDL-C**

There is a lot of evidence that the endothelium plays a crucial role in the maintenance of vascular tone and structure. [39; 40, 41; 38; 5; 51; 9]. One of the major endothelium-derived vasoactive mediators was shown to be nitric oxide (NO). [38; 74; 51; 67]. Multifactional are the mechanism by which NO activity is reduced: reduced NO release, NO inactivation by superoxide anion, or reduced NO production by NO synthase (NOS). [91] Decrease in NOS expression by oxidized low-density lipoprotein (LDL) can cause impaired NO production [77; 91], or by the presence of asymmetric dimethylarginine (ADMA). [72; 16 ;29]

According to Herman and Moncada the basis of atherogenesis remain oxygenated LDL and eNOS. Lipid-regulating effects of statins in terms of LDL-cholesterol are undeniable, but the pleiotropic discussioncy is a particularly relevant issue of resistance of statin therapy in patients with high levels of ADMA - endogenous inhibitor of eNOS. Research of statin influence on flow-mediated vasodilation (FMD) reveals controversial results. Some studies indicate that there is an effect, whereas others document the opposite tendency [18]. There is a number of studies on simvastatin and likewise they demonstrate controversial findings. These controversies can be dismissed by studing ADMA levels.(18 ;16;6). It has been suggested that ADMA could modify the effect of statins on myocardial blood flow and on FMD% (53).

**2.1. Failed targeting LDL cholesterol**

186 Hypercholesterolemia

expression of the LDLR gene [1].

**LDL-C**

It seems that not only genetic but environmental factors can influence the LDL-C response to statins. Studies have found that patients with hypertension have a smaller decrease than those without hypertension. Furthermore, smokers have smaller statin-induced LDL-C decrease compared with nonsmokers[47]. It also seems that inflammation might cause statin resistance. Namely, it has been shown that inflammatory cytokines, in particular IL-1b which affects sterol regulatory element binding protein cleavage-activating protein, cause statin resistance due to the disruption of LDL-R feedback regulation. Therefore, it has been suggested that in inflam‐ matory states, higher concentrations of statin may be required to achieve the appropriate LDL-C lowering [107]. Particularly interesting are observations concerning certain subpopulations of patients who might be resistant to statin treatment. Some studies have shown statins to be less effective in individuals with HIV infection. [22]. Other studies have a contraversal perspective. [55]. The role of concomitant amiodarone treatment in statin resistance was also suspected. Both amiodarone and amiodarone induced hypothyroidism influence the synthesis of LDLR, which may explain the lack of statin effect. Thyroid hormone is one of several hormones that control gene expression of the LDLR and hypothyroidism is a wellknown cause of secondary dyslipidemia characterized by elevated LDL-C levels. Similar to hypothyroidism, administration of amiodarone also increases LDL-C levels, which is the result of a decreased

More recently, an approach was published which used metabolomics to identify markers indicative of mechanisms that contribute to differences in LDL-C response to statin. Metabolic changes were shown to be more comprehensive in responders to statin treatment than those seen in nonresponders. The baseline cholesterol ester and phospholipid metabolites correlated with LDL-C response to treatment [56]. It has also been suggested that clusters of metabolites involved in multiple pathways not directly connected with cholesterol metabolism might as well play a role in modulating the response to statin therapy - influence statin resistance [90]. Insufficient LDL-C response to statin treatment is probably the result of pseudo-resistance, which could be caused by nonadherence or nonpersistence in real life circumstances. [68].

**3. Lack of effect on the endothelium-dependent vasodilation after targeting**

There is a lot of evidence that the endothelium plays a crucial role in the maintenance of vascular tone and structure. [39; 40, 41; 38; 5; 51; 9]. One of the major endothelium-derived vasoactive mediators was shown to be nitric oxide (NO). [38; 74; 51; 67]. Multifactional are the mechanism by which NO activity is reduced: reduced NO release, NO inactivation by superoxide anion, or reduced NO production by NO synthase (NOS). [91] Decrease in NOS expression by oxidized low-density lipoprotein (LDL) can cause impaired NO production [77;

According to Herman and Moncada the basis of atherogenesis remain oxygenated LDL and eNOS. Lipid-regulating effects of statins in terms of LDL-cholesterol are undeniable, but the

91], or by the presence of asymmetric dimethylarginine (ADMA). [72; 16 ;29]

In our subsequent studies found in a logical sequence following facts. This facts determinate ADMA as a basic factor for statin`s resistans.

International recommendations underline the importance of diagnosis and treatment of asymptomatic individuals with high absolute cardiovascular risk [10; 37; 8], as individuals with severe hypercholesterolemia. [47; 45; 58] the levels of ADMA in patients with severe hypercholesterolemia in our study are higher than those cited in the literature in the same population patients. [13].

1. A good marker of endothelial dysfunction is considered to be ADMA, as indicated by recent publications. [16]. Subjects with cardiovascular risk – hypercholesterolemia, hyperhomocys‐ teinemia, diabetes mellitus, hypertension, smoking, erectile dysfunction having increased ADMA levels. [67; 11; 16,17]. Plasma levels of ADMA have been shown to be elevated in hypercholesterolemic rabbits [108]. The elevation of ADMA is associated with reduced activity of NOS in animal models, as well as in young asymptomatic hypercholesterolemic adults [13]. The mechanism of increased ADMA in hypercholesterolemia is not very clear - LDL cholesterol increases the expression of ADMA precursor protein and reduces the activity of the enzyme dimethyl arginine dimethyl amino hydrolase. [52; 15] Increased ADMA are associated with reduced NO synthesis and this assessed by impaired endothelium-dependent vasodilatation. Flow-mediated dilatation (FMD) - shear stress during hyperemia activates receptors on the endothelial cell surface and causes influx of intracellular calcium, which activates eNOS and NO release [54; 24; 80; 60]. The main effect that dilatation has in respons to shear stress during FMD is influenced by NO and to a smaller extent on prostaglandins and endothelial-dependent hyperpolarizing factors [78; 54; 31; 30; 73]. Ultrasound determination of flow-mediated dilatation of the brachial artery as a method has many advantages – it is non-invasive, with good reproducibility and reliable. [3; 28; 31; 36; 61]. There is convincing evidence that reduced percentage of FMD (FMD%) is a marker of coronary endothelial dysfunction [3].

Several studies have associated hypercholesterolemia with reduced FMD% and this effect can be reversed by L-arginine [34; 26; 32; 33]. However, L-arginine does not lead to the improve‐ ment of endothelial dependent vasodilatation in normocholesterolemic individuals. In this condition indicate the main role of endogenous ADMA. [11; 44] Furthermore, a recent publication demonstrated that improvement of FMD% under statin treatment depends on the ADMA levels [53; 12]. Little is know about the relationship between ADMA, and FMD%. In a small number of hypercholesterolemic patients ADMA was shown to be positively correlated with FMD% in mild hypercholesterolemia [13]. A recent paper demonstrated that low cardiovascular risk subjects have increased ADMA level. [6]. No data exist about the relation‐ ship between ADMA and FMD% in severe hypercholesterolemia patients. In our study "Relationship of asymmetric dimethylarginine with flow-mediated dilatation in subjects with newly detected severe hypercholesterolemia" was the evaluation of the relationship between ADMA and FMD, also that of ADMA and lipid parameters as well as other endothelial dysfunction in newly detected subjects with severe hypercholesterolemia. The major findings of the present study are that: (1) plasma levels of ADMA, are increased in severe hypercho‐ lesterolemia; (2) there is a significant link between ADMA and age, Apo-B, Apo-B ⁄ Apo-A<sup>1</sup> and tHcy; (3) newly detected severe hypercholesterolemia has reduced flow-mediated endothelial dependent vasodilatation, there is a correlation between plasma levels of FMD% and age, Apo-B, Apo-B ⁄ Apo-A1 and tHcy; and (4) homocystein levels has no contribution to the atherogenic risk in the patients.

Newly detected severe hypercholesterolemia is associated with elevated ADMA, and to the proportional increase in total cholesterol. The ADMA correlates with age, Apolipoprotein-B, Apo-B ⁄ Apo-A1 and tHcy. Apo-B was found to indicate elevated ADMA in these patients. FMD % correlates most strongly with age, Apolipoprotein-B, index Apo-B ⁄ Apo-A<sup>1</sup> and tHcy. In multiple regression analysis, ADMA is the strongest predictor for FMD%. ADMA is the main modulator of FMD% - among the investigated biomarkers in newly detected severe hyper‐ cholesterolemia. Serious functional changes in the vascular wall are cause by increased level of ADMA. At the same time, ADMA is found to be a predictor of flow-modulated vasodilation of the brachial artery which also makes a probable marker for endothelial dysfunction. Therefore, measuring ADMA levels in newly detected severe hypercholesterolemia is of great importance when FMD% changes need to be clarified.

2. In the next study we investigated intima-media complex of carotid artery. The intima-media thickness (IMT) of the CCA is one of the validated measurements of subclinical atherosclerosis, as early as structural vascular abnormalities [85]. Intima-media thickening of the CCA correlates with the coronary risk factors [80] and with associated with the degree of coronary atherosclerosis. It serves as a predictor of coronary and vascular events in different patients' populations. Intima-media thickening reflects both intimal atherosclerosis and medial hypertrophy. It is used to evaluate the luminal and wall characteristics of the carotid artery. In the literature, hypercholesterolemia has an important role in early-onset IMT changes in the CCA However, there is not a lot of data about asymptomatic subjects with newly detected severe hypercholesterolemia.[72]. In the literature, data on the IMT of CCA predictors is controversial. There are a few studies of the endothelium-related biomarkers (ADMA, tHcy, soluble cell adhesion molecules), especially in asymptomatic subjects with newly detected severe hypercholesterolemia [72].

The research "Predictors of the intima-media thickness of carotid artery in asymptomatic newly detected severe hypercholesterolemic patients" age and Apo-B were established as the most important statistically significant factors related to IMT mean of CCA. This fact illustrates that they determine the slow progressive structural changes in the vascular wall. The Apo-B is a better biomarker of the total number of atherogenic particles. It might be concluded that Apo-B is a better factor for assessment of risk, as LDL cholesterol underestimates the risk in asymptomatic subjects with newly detected severe hypercholesterolemia.

In the study "Intima-Media Thickness and Flow-Mediated Vasodilation in Asymptomatic Subjects with Newly Detected Severe Hypercholesterolemia", our results show a significant correlation between IMT mean and FMD%. The correlation is still present when separating IMT on the basis of the level of thickening. This supports the idea that the two noninvasive methods complete each other. It is important with regard to building a diagnostic algorithm. These methods show early subclinical atherosclerosis but by different trigger mechanisms.

"Relationship of asymmetric dimethylarginine with flow-mediated dilatation in subjects with newly detected severe hypercholesterolemia" was the evaluation of the relationship between ADMA and FMD, also that of ADMA and lipid parameters as well as other endothelial dysfunction in newly detected subjects with severe hypercholesterolemia. The major findings of the present study are that: (1) plasma levels of ADMA, are increased in severe hypercho‐ lesterolemia; (2) there is a significant link between ADMA and age, Apo-B, Apo-B ⁄ Apo-A<sup>1</sup> and tHcy; (3) newly detected severe hypercholesterolemia has reduced flow-mediated endothelial dependent vasodilatation, there is a correlation between plasma levels of FMD% and age, Apo-B, Apo-B ⁄ Apo-A1 and tHcy; and (4) homocystein levels has no contribution to

Newly detected severe hypercholesterolemia is associated with elevated ADMA, and to the proportional increase in total cholesterol. The ADMA correlates with age, Apolipoprotein-B, Apo-B ⁄ Apo-A1 and tHcy. Apo-B was found to indicate elevated ADMA in these patients. FMD % correlates most strongly with age, Apolipoprotein-B, index Apo-B ⁄ Apo-A<sup>1</sup> and tHcy. In multiple regression analysis, ADMA is the strongest predictor for FMD%. ADMA is the main modulator of FMD% - among the investigated biomarkers in newly detected severe hyper‐ cholesterolemia. Serious functional changes in the vascular wall are cause by increased level of ADMA. At the same time, ADMA is found to be a predictor of flow-modulated vasodilation of the brachial artery which also makes a probable marker for endothelial dysfunction. Therefore, measuring ADMA levels in newly detected severe hypercholesterolemia is of great

2. In the next study we investigated intima-media complex of carotid artery. The intima-media thickness (IMT) of the CCA is one of the validated measurements of subclinical atherosclerosis, as early as structural vascular abnormalities [85]. Intima-media thickening of the CCA correlates with the coronary risk factors [80] and with associated with the degree of coronary atherosclerosis. It serves as a predictor of coronary and vascular events in different patients' populations. Intima-media thickening reflects both intimal atherosclerosis and medial hypertrophy. It is used to evaluate the luminal and wall characteristics of the carotid artery. In the literature, hypercholesterolemia has an important role in early-onset IMT changes in the CCA However, there is not a lot of data about asymptomatic subjects with newly detected severe hypercholesterolemia.[72]. In the literature, data on the IMT of CCA predictors is controversial. There are a few studies of the endothelium-related biomarkers (ADMA, tHcy, soluble cell adhesion molecules), especially in asymptomatic subjects with newly detected

The research "Predictors of the intima-media thickness of carotid artery in asymptomatic newly detected severe hypercholesterolemic patients" age and Apo-B were established as the most important statistically significant factors related to IMT mean of CCA. This fact illustrates that they determine the slow progressive structural changes in the vascular wall. The Apo-B is a better biomarker of the total number of atherogenic particles. It might be concluded that Apo-B is a better factor for assessment of risk, as LDL cholesterol underestimates the risk in

asymptomatic subjects with newly detected severe hypercholesterolemia.

the atherogenic risk in the patients.

188 Hypercholesterolemia

severe hypercholesterolemia [72].

importance when FMD% changes need to be clarified.

3. After establishing who is a predictor of FMV - ADMA, the next study proved that ADMA is the main determinant of the effect of simvastatin on FMV in severe hypercholesterolemia - "Asymmetric dimewthylarginine determines the effect of simvastatin on endotheliumdependent vasodilation in severe hypercholesterolemia" Future Medicine Clinical Lipidology 2010. With respect to their total cholesterol, LDL-cholesterol and FMD% the two groups of hypercholesterolemic patients (according to the plasma ADMA levels) differ significantly. ADMA, cell adhesion molecules or total homocysteine levels are not affected by Simvastatin in moderate dose [40 mg). Higher baseline levels of ADMA affect the ability of statins to improve endothelium-dependent vasodilation by diminishing it. Subjects from the same population, but with lower baseline levels of ADMA experience the same effect of simvastatin. Therefore, ADMA seems to be a pathophysiologycal modulator of the statin therapeutic response. The present study has been confirm by studies that there is a connection between ADMA and FMD% response to statins found by Böger et al. The different is that in our study is in the larger group of the patients.

In terms of non-randmized study "Effect of Moderate and High-Dose Simvastatin on Asym‐ metric-Homocysteine Metabolic Pathways in Patients with Newly Detected Severe Hyper‐ cholesterolemia" was demonstrated dose-dependent effect of simvastatin on the levels of ADMA.The 40 mg simvastatin has no effect on ADMA and homocysteine level in contrast to 80 mg, after target LDL-levels are reached ≤2.6 mmol/L. It is likely that statin-pleiotropic effects on ADMA-homocysteine metabolic pathways are independent of their lipid-regulating properties.

In another of our observation "Asymmetric dimethylarginine-a determinant of the effect of the high dose Simvastatin confirmed this dose-dependent effect". The two groups of patients (according to the plasma ADMA levels) differ significantly with respect to their total choles‐ terol, LDL-cholesterol and FMD%. Simvastatin in moderate dose (40 mg) does no affect ADMA, cell adhesion molecules and total homocysteine levels. The higher levels of ADMA change the ability of statins to improve the endothelium-dependent vasodilatation, by diminishing it. This shows that ADMA is a pathophysiological modulator of the statin therapeutic response. This study confirms that, for the first time, there is a correlation between ADMA levels and FMD% response to statins, found by Böger et al., but in the larger group of patients with severe hypercholesterolemia and with higher dose simvastatin. Obviously, these mechanisms require further investigation

To give a more precise answer to the question of dose-dependent manner for avoidable statin resistance subsequently conducted a randomized, placebo-controlled study "The effect of simvastatin on asymmetric dimethylarginine and flow-mediated vasodilation after optimizing the LDL level — A randomized, placebo-controlled study" The major findings of the present study are 1. Significantly higher ADMA and tHcy were seen in patients with severe hyper‐ cholesterolemia compared to the control group. 2. Administration of 40 mg simvastatin for one month results in no variation in ADMA, tHcy plasma levels and FMD%, following optimizing of the LDL. 3. Administration of 80 mg simvastatin for a month leads to a variation of ADMA and tHcy plasma levels and FMD% after optimizing the LDL. FMD%-changes can be predicted with ADMA levels and ApoB%-changes is a predictor of LDL-changes% in patients on 80 mg simvastatin (for one month) following the optimization of the LDL-C.

This study gives evidence that in experimental models and in humans (59), higher ADMA levels have a harmful effect on the coronary endothelium. On the other hand, the experimental model shows that statins have no protective effect against that harmful effect of ADMA on the endothelium. This provokes a discussion as to whether ADMA is the pathophysiological modulator of the therapeutic response of statins in hypercholesterolemia.

The ADMA in severe hypercholesterolemia are higher compared to those in patients in similar research protocols (13), and are similar to those in our previous research studies. Applying various laboratory methods(ELISA in the present study, high-pressure liquid chromatography in other studies) does not allow for the mean levels of ADMA to be compared directly. Using ELISA to differentiate the sample groups is less reliable than LC-MS.This is caused by the fac that the higher coefficient of variation and to the fact that the matrix dependence is likely to cloud or mimic the differences The ADMA ELISA method can be used for clinical investiga‐ tions in which groups of samples are compared and the result is the shift of the ADMA concentration in response to an intervention. The application of ELISA analysis in our study is the likely ex- planation of the higher levels of ADMA, in comparison with other studies (13). On the other hand, this is likely due to the higher levels of total cholesterol > 7.5 mmol/l and LDL-C > 4.9 mmol/l. The difference in L-arginine substitution in hypercholesterolic patients and normo-cholesterolic patients is explained by the higher levels of ADMA in hypercholes‐ terolic petients in comparison with controls with controls (11; 44).

The mechanism of an increased ADMA level in hypercholesterolemia is not clear enough. An association between ADMA and hypercholesterolemia has been previously observed [13]. Laufs et al. (1998) demonstrated that simvastatin reverse, in a dose-dependent manner, the inhibitory effect of oxidized LDL on NO production. It has been suggested that LDL- choles‐ terol increases the expression of ADMA precursor protein. This reduces the activity of the enzyme dimethylarginine dimethylaminohydrolase, which breaks down ADMA [52]. This is why, by decreasing cholesterol levels with statin therapy, ADMA plasma levels will decrease as well. The therapeutic hypothesis that the decrease of circulating ADMA levels can be achieved by lowering plasma cholesterol levels is the main idea in this publication.

In randomized, placebo-controlled research, a statistically significant reduction of ADMA plasma levels has been established following a one-month therapy with 80 mg simvastatin, yet the 40 mg simvastatin dose does not result in achieving the LDL target levels. The study showed that a 40 mg simvastatin therapy for 3 months does not produce the desired effect. Therefore, it is likely that the pleiotropic effect of the statins (respectively ADMA and tHcy) is independent from the lipid-regulation in a short-term and long-term plan. The lack of effect on 40 mg simvastatin coincides with the results presented in other studies but there is no optimizing of LDL-C level. The research in similar articles regarding the effect of 80 mg simvastatin on ADMA levels is scant. Most research works have documented a negative effect in hypercholesterolemia. However, these studies have tested a considerably smaller number of patients (64). The present study comprises 85 patients and LDL target levels have been optimized regarding the risk category. The established statistically significant therapeutic effect of 80 mg simvastatin on ADMA is comparable to the results from a recently published study — an experimental model of the effect of simvastatin on ADMA tissue levels (64). This recent experimental data shows that simvastatin regulates dimethylarginine dimethylamino‐ hydrolase transcription via the transcription factor Sterol Regulatory Element Binding Protein. The latter is activated by statins due to a reduction of plasma membrane cholesterol. These experimental models suggest that the level of asymmetric dimethylarginine will be decreased by stаtin therapy. Almost all other clinical studies (of smaller sample size and shorter duration) showed no effect of statins on ADMA (positive effect only 10 mg rosuvastatin and 80 mg fluvastatin). It is unclear whether the higher plasma levels of ADMA in human disease states correlate with a higher intracellular level. Studies testing the statin effect in vivo have reported endothelial protection without overly affecting plasma ADMA levels, however in these studies the tissue levels of ADMA have not been taken into consideration. It is likely that in the present study achieving the LDL-C target level substitutes for the LDL-cholesterol tissue levels. Similar titrations have not been carried out in any other related articles so far. The results of the present study provide further clinical evidence to the experimental model of the Ivashchenko et al., that simvastatin regulates dimethylarginine dimethylaminohydrolase transcription via the transcription factor Sterol Regulatory Element Binding Protein.

study are 1. Significantly higher ADMA and tHcy were seen in patients with severe hyper‐ cholesterolemia compared to the control group. 2. Administration of 40 mg simvastatin for one month results in no variation in ADMA, tHcy plasma levels and FMD%, following optimizing of the LDL. 3. Administration of 80 mg simvastatin for a month leads to a variation of ADMA and tHcy plasma levels and FMD% after optimizing the LDL. FMD%-changes can be predicted with ADMA levels and ApoB%-changes is a predictor of LDL-changes% in patients on 80 mg simvastatin (for one month) following the optimization of the LDL-C.

This study gives evidence that in experimental models and in humans (59), higher ADMA levels have a harmful effect on the coronary endothelium. On the other hand, the experimental model shows that statins have no protective effect against that harmful effect of ADMA on the endothelium. This provokes a discussion as to whether ADMA is the pathophysiological

The ADMA in severe hypercholesterolemia are higher compared to those in patients in similar research protocols (13), and are similar to those in our previous research studies. Applying various laboratory methods(ELISA in the present study, high-pressure liquid chromatography in other studies) does not allow for the mean levels of ADMA to be compared directly. Using ELISA to differentiate the sample groups is less reliable than LC-MS.This is caused by the fac that the higher coefficient of variation and to the fact that the matrix dependence is likely to cloud or mimic the differences The ADMA ELISA method can be used for clinical investiga‐ tions in which groups of samples are compared and the result is the shift of the ADMA concentration in response to an intervention. The application of ELISA analysis in our study is the likely ex- planation of the higher levels of ADMA, in comparison with other studies (13). On the other hand, this is likely due to the higher levels of total cholesterol > 7.5 mmol/l and LDL-C > 4.9 mmol/l. The difference in L-arginine substitution in hypercholesterolic patients and normo-cholesterolic patients is explained by the higher levels of ADMA in hypercholes‐

The mechanism of an increased ADMA level in hypercholesterolemia is not clear enough. An association between ADMA and hypercholesterolemia has been previously observed [13]. Laufs et al. (1998) demonstrated that simvastatin reverse, in a dose-dependent manner, the inhibitory effect of oxidized LDL on NO production. It has been suggested that LDL- choles‐ terol increases the expression of ADMA precursor protein. This reduces the activity of the enzyme dimethylarginine dimethylaminohydrolase, which breaks down ADMA [52]. This is why, by decreasing cholesterol levels with statin therapy, ADMA plasma levels will decrease as well. The therapeutic hypothesis that the decrease of circulating ADMA levels can be

achieved by lowering plasma cholesterol levels is the main idea in this publication.

In randomized, placebo-controlled research, a statistically significant reduction of ADMA plasma levels has been established following a one-month therapy with 80 mg simvastatin, yet the 40 mg simvastatin dose does not result in achieving the LDL target levels. The study showed that a 40 mg simvastatin therapy for 3 months does not produce the desired effect. Therefore, it is likely that the pleiotropic effect of the statins (respectively ADMA and tHcy) is independent from the lipid-regulation in a short-term and long-term plan. The lack of effect on 40 mg simvastatin coincides with the results presented in other studies but there is no

modulator of the therapeutic response of statins in hypercholesterolemia.

190 Hypercholesterolemia

terolic petients in comparison with controls with controls (11; 44).

The present study shows a statistically significant increase in FMD% in patients on 80 mg simvastatin therapy for one month in the presence of controversial results in related materials on this issue. The mechanism of this improvement is proved to be related to the enhancement of gene expression of eNOS (64). On the other hand, the FMD%-changes correlate (correlations with all biomarkers at a baseline level and the %-changes have been tested) significantly only with the baseline level of ApoB, ADMA, and tHcy. Interestingly enough,patients with ADMA levels greater than 1 μmol/l, following statin therapy, appear to have only small or no FMD% changes. A likely explanation of this finding is that in patients with ADMA greater than 1 μmol/l, competes with L-arginine as a substrate for eNOS and thus decreases the production and availability of endothelium-derived NO. For this reason, in such patients, there are no FMD% changes following statin therapy. In patients with documented small FMD% changes, the most likely explanation is the action of other mediators (endothelium-derived hyperpola‐ rizing factor or prostaglandins) that lead to vasodilation through calcium-activated potassium channels simvastatin (80 mg daily).

The high simvastatin doses should be done with caution. According to the Food and Drug Administration monitoring are also important every 3 and 6 months during the course of therapy.

In the multifactor regressive analysis only the initial ADMA levels remain predictors of an FMD%-change. For the first time, in 2007 Böger GI et al. established that ADMA determines FMD%- changes in a small hypercholesterolemic patients group (treated with a smaller simvastatin dose — 40 mg (12). Further clinical studies can be based off of this study, in order to achieve LDL-target levels and to optimize the effect of different doses statins on ADMA. Other statins are better tolerated at a high dose (atorvastatin, pravastatin, fluvastatin, lova‐ statin). There is only one study testing the effect of 80 mg fluvastatin treatment in hypercho‐ lesteromic patients with metabolic syndrome, which demonstrated decrease in plasma ADMA level at 6 weeks.

What is interesting is that the established fact that the Apo-B%-change (not the LDL%-change) is a predictor of the changes in the plasma levels of ADMA (ADMA%-change) in the linear regression model. It's very likely that this is due to the level of the smallest atherogenic and dense particles are reflected my ApoB. The fact that ApoB is a predictor of the ADMA%-change presumably is due to the higher proportion of patients with family Apo B defect (previously reported in patients with hypercholesterolemia in our previous studies.

Statins vary in their pharmacokinetics and pharmacodynamics. There is a difference in their lipid regulating and pleiotropic effect. Therefore, the data on simvastatin could not be referred to other statins. There is no other therapeutic option in cases with high ADMA levels in hypersholesterolic ptients,apart from 80 mg simvastatin. The clinical significance of our study is that high-risk patients with severe hypercholesterolemia, a family history of premature atherosclerosis and a high level of plasma ADMA, the high dose of Simvastatin is a possible therapeutic option. Substituting with L-arginine is another possible approach (11; 44; 92). These two hypotheses complete one another.

A number of factors are the cause of controversial results on the effects of statins on the endothelial-dependent vasolidation. 1. The clinical studies, testing the effect of statins on ADMA and FMD% involve only a small number of patients for a short period of time. 2. LDL levels are not optimized in accordance with the risk category of hypercholesterolemic patients (the pleiotropic effects of statins are partly connected to lipid regulating ones). 3. The im‐ provement of FMD% via increasing the activity of NO with the statin therapy is connected additionally to the effect on other inhibitors of eNOS apart from ADMA. 4. In most studies there is no testing of ADMA tissue levels.

The present study established patients with severe hypercholesterolemia have high ADMA levels in comparison with the control group. One-month treatment with 80 mg simvastatin, aimed at achieving LDL target levels of ≤ 2.6 mmol/l in high-risk contingents with severe hypercholesterolemia leads to a statistically significant reduction of ADMA and an increase of FMD% in contrast with 40 mg simvastatin therapy. The FMD%-changes correlate in a statistically significant way with the initial ApoB, ADMA and tHcy levels. The baseline ADMA levels are a predictor of FMD% changes and Apo-B%-changes is a predictor of ADMA% changes at baseline and post one-month therapy with 80 mg simvastatin. In case of optimized LDL target levels it appears that ADMA is a major modulator of FMD%-change.

#### **4. The impact of genetic factors on statin resistance**

The same dose of the same statin in different individuals produces different LDLC decreases. The time to reach maximum LDL-C decrease differs significantly between individuals. [81; 82] Such a wide interindividual variation as the response to statins is more and more attributed, at least partly, to the polymorphisms in genes affecting statin pharmacodynamics and pharmacokinetics. The resistance to statins has been associated with polymorphisms in the HMG-CoA-R, ABCB1, ABCG2, ABCC1, ABCC2, OATP1B1, OATP2B1, RHOA, NPC1L1, FXR, CYP7A1, ApoE, PCSK9, LDLR, LPA, CETP, and TNF-a genes. However, currently, there is still not enough evidence to advocate pharmacogenetic testing before initiating therapy with statins.

Pharmacogenetics seeks to determine the role of genetic factors in variation of statin response. However, today the origins of the notable interindividual variation in response to statins are still poorly understood. In a number of studies, genetic variability has been shown to affect statin responsiveness thus influencing statin resistance. These studies have identified numer‐ ous candidate genes (>50) and dozens of single-nucleotide polymorphisms (SNPs). It have been reported to be associated with differing aspects of statin response - pharmacokinetics and pharmacodynamics of statins being potential determinants of drug responsiveness in terms of LDL-C lowering. Although genes are supposed to be associated with statin cholesterollowering efficacy, the magnitude of variation in statin response that could be explained by these associations is still questionable. [62; 89; 35; 79; 71]

The association between SNPs in genes involved in lipid metabolism and total cholesterol and LDL-C response to statin therapy is of particular interest. The 3-hydroxyl-3- methylglutaryl coenzyme A reductase (HMG-CoA-R) gene encoding the enzyme HMG-CoA-R, which is the principal target of statins, because the foremost pharmacological action of these drugs is exactly the competitive inhibition of HMG-CoA-R. The last one might be one of the candidate genes when analyzing the SNPs as a possible cause of diminished statin responsiveness. When SNPs and the common haplotypes inferred from them were tested for association with plasma LDL-C levels and LDL-C response to statin treatment, it has been shown that HMG-CoA-R gene polymorphisms are associated with reduced plasma LDL-C levels and LDL-C response to simvastatin. [104; 42; 75; 84; 88; 49; 50]

Therefore, although it was considered that genome-wide association studies may yield a more comprehensive set of markers for predicting statin efficacy and/or resistance, this has not been proven so far and the results of these studies cannot be translated into clinical practice yet. We need future pharmacogenetic research [93].

#### **5. Conclusion**

simvastatin dose — 40 mg (12). Further clinical studies can be based off of this study, in order to achieve LDL-target levels and to optimize the effect of different doses statins on ADMA. Other statins are better tolerated at a high dose (atorvastatin, pravastatin, fluvastatin, lova‐ statin). There is only one study testing the effect of 80 mg fluvastatin treatment in hypercho‐ lesteromic patients with metabolic syndrome, which demonstrated decrease in plasma ADMA

What is interesting is that the established fact that the Apo-B%-change (not the LDL%-change) is a predictor of the changes in the plasma levels of ADMA (ADMA%-change) in the linear regression model. It's very likely that this is due to the level of the smallest atherogenic and dense particles are reflected my ApoB. The fact that ApoB is a predictor of the ADMA%-change presumably is due to the higher proportion of patients with family Apo B defect (previously

Statins vary in their pharmacokinetics and pharmacodynamics. There is a difference in their lipid regulating and pleiotropic effect. Therefore, the data on simvastatin could not be referred to other statins. There is no other therapeutic option in cases with high ADMA levels in hypersholesterolic ptients,apart from 80 mg simvastatin. The clinical significance of our study is that high-risk patients with severe hypercholesterolemia, a family history of premature atherosclerosis and a high level of plasma ADMA, the high dose of Simvastatin is a possible therapeutic option. Substituting with L-arginine is another possible approach (11; 44; 92). These

A number of factors are the cause of controversial results on the effects of statins on the endothelial-dependent vasolidation. 1. The clinical studies, testing the effect of statins on ADMA and FMD% involve only a small number of patients for a short period of time. 2. LDL levels are not optimized in accordance with the risk category of hypercholesterolemic patients (the pleiotropic effects of statins are partly connected to lipid regulating ones). 3. The im‐ provement of FMD% via increasing the activity of NO with the statin therapy is connected additionally to the effect on other inhibitors of eNOS apart from ADMA. 4. In most studies

The present study established patients with severe hypercholesterolemia have high ADMA levels in comparison with the control group. One-month treatment with 80 mg simvastatin, aimed at achieving LDL target levels of ≤ 2.6 mmol/l in high-risk contingents with severe hypercholesterolemia leads to a statistically significant reduction of ADMA and an increase of FMD% in contrast with 40 mg simvastatin therapy. The FMD%-changes correlate in a statistically significant way with the initial ApoB, ADMA and tHcy levels. The baseline ADMA levels are a predictor of FMD% changes and Apo-B%-changes is a predictor of ADMA% changes at baseline and post one-month therapy with 80 mg simvastatin. In case of optimized

The same dose of the same statin in different individuals produces different LDLC decreases. The time to reach maximum LDL-C decrease differs significantly between individuals. [81;

LDL target levels it appears that ADMA is a major modulator of FMD%-change.

**4. The impact of genetic factors on statin resistance**

reported in patients with hypercholesterolemia in our previous studies.

level at 6 weeks.

192 Hypercholesterolemia

two hypotheses complete one another.

there is no testing of ADMA tissue levels.

It is difficult to give an accurate definition of statin resistance. The patients who fail to reach LDL-C target values despite the best available therapy, mostly a highest tolerable dose of a more potent statin, are considered to be statin-resistant. Resistance to statins can be related to differences in drug absorption, transport, intrahepatic drug metabolism, drug metabolism within other organs, and drug excretion mechanisms. Possible causes of statin resistance: **1.Failed targeting LDL cholesterol** - smokers have smaller statin-induced LDL-C decrease compared with nonsmokers and the patients with hypertension have smaller decrease than those without hypertension, inflammation might cause statin resistance. The role of concom‐ itant amiodarone treatment in statin resistance was also suspected. It has also been suggested that clusters of metabolites involved in multiple pathways not directly connected with cholesterol metabolism might as well play a role in modulating the response to statin therapy and therefore influence statin resistance. **2.Lack of effect on the endothelium-dependent vasodilation after targeting LDL-C.** There is much evidence that improvement of endotheli‐ um-dependent vasodilation under statin treatment depends on the ADMA levels. At this stage of knowledge, there are two options for the management of this type of statin resistance - the use of a high dose of a statin, or the addition of L-Arginine to the statin. These two strategies are not contradictory, but complementary. 3. **The impact of genetic factors on statin resist‐ ance.** The resistance to statins has been associated with polymorphisms in the HMG-CoA-R, ABCB1, ABCG2, ABCC1, ABCC2, OATP1B1, OATP2B1, RHOA, NPC1L1, FXR, CYP7A1, ApoE, PCSK9, LDLR, LPA, CETP, and TNF-a genes. However, currently, there is still not enough evidence to advocate pharmacogenetic testing before initiating therapy with statins.

#### **Author details**

Lyudmila Georgieva Vladimirova-Kitova\* and Spas Ivanov Kitov

\*Address all correspondence to: kitov@vip.bg

Medical University, Plovdiv, Bulgaria

#### **References**


[6] Ardigo D, Stüehlinger M, Franzini L, Valtueña S, Piatti PM, Pachinger O, Reaven GM, Zavaroni I. ADMA is independently related to flow- mediated vasodilation in subjects at low cardiovascular risk. Eur J Clin Invest [2007]; 37: 263–269

those without hypertension, inflammation might cause statin resistance. The role of concom‐ itant amiodarone treatment in statin resistance was also suspected. It has also been suggested that clusters of metabolites involved in multiple pathways not directly connected with cholesterol metabolism might as well play a role in modulating the response to statin therapy and therefore influence statin resistance. **2.Lack of effect on the endothelium-dependent vasodilation after targeting LDL-C.** There is much evidence that improvement of endotheli‐ um-dependent vasodilation under statin treatment depends on the ADMA levels. At this stage of knowledge, there are two options for the management of this type of statin resistance - the use of a high dose of a statin, or the addition of L-Arginine to the statin. These two strategies are not contradictory, but complementary. 3. **The impact of genetic factors on statin resist‐ ance.** The resistance to statins has been associated with polymorphisms in the HMG-CoA-R, ABCB1, ABCG2, ABCC1, ABCC2, OATP1B1, OATP2B1, RHOA, NPC1L1, FXR, CYP7A1, ApoE, PCSK9, LDLR, LPA, CETP, and TNF-a genes. However, currently, there is still not enough evidence to advocate pharmacogenetic testing before initiating therapy with statins.

and Spas Ivanov Kitov

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Lyudmila Georgieva Vladimirova-Kitova\*

Medical University, Plovdiv, Bulgaria

\*Address all correspondence to: kitov@vip.bg

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59491

**1. Introduction**

#### **1.1. Angiotensin-Converting Enzyme Inhibitors**

#### *1.1.1. History*

In the 1950s, it was discovered that angiotensin exists as both an inactive decapeptide angio‐ tensin I and an active octapeptide angiotensin II. Human angiotensin-converting enzyme contains 277 amino-acid residues and has two homologous domains, each with a catalytic site and a region for binding Zn+2 [1, 2]. The degradation of bradykinin to inactive peptides occurs via action of ACE; ACE thus not only produces a potent vasoconstriction but also inactivates a potent vasodilator. In 1965, Ferreira [3] studied the physiological effects of snake poisoning and discovered a specific component from the venom of the pit viper, Bothrops jararaca, which inhibits degradation of the peptide bradykinin and potentiate hypotensive action of bradyki‐ nin potentiating factors (BPFs), basically amino-acid-containing peptides. Bakhle [4] reported that these same peptides had an inhibitory activity on ACE of dog lung homogenate and inhibited the enzymatic conversion of angiotensin I to angiotensin II. Brunner and Laragh [5] administered them to hypertensive patients and found them to be extremely effective in lowering blood pressure. The structural requirements for substrates of angiotensin-converting enzyme to cleave a substrate are found to be similar to those observed with carboxypeptidase A of bovine pancreas [6, 7].

The molecule ACE is a zinc metallopeptidase and has a similar mode of action to carboxy‐ peptidase [8]. In 1970, the Bradykinin-potentiating pentapeptide BPP5a was isolated, which inhibited enzyme angiotensin and decreased blood pressure [9]. The significance of ACE in the pathogenesis of hypertension was not fully appreciated until 1977, when Ondetti [10] first isolated and then synthesized the naturally occurring non-peptide, teprotide. He proposed a hypothetical model of the active site of ACE and used it to predict and design compounds that would occupy the carboxy-terminal binding site of the enzyme captopril, a specific potent

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

inhibitor of ACE. Clinical trials showed excellent anti-hypertensive properties and these results had a major impact on the treatment of cardiovascular disease [11]. The first demon‐ stration of an orally active ACE inhibitor was made on 31 March 1975, when the succinyl group was replaced with a derivative of cysteine, increasing inhibitory potency about 2,000-fold because sulphhydryl of cysteine bound with zinc more tightly than the carboxyl of succinyl. This resulted in captopril, with a dramatic effect on renal function and on hypertension [12]. Enalapril is basically a first derivative of ACE inhibitor, which was developed to overcome the limitations of captopril. Lisinopril is a lysine analogue of enalaprilat (the active metabolite of enalapril). *In vitro* lisinopril is slightly more potent than enalaprilat. It is a non-sulphhydryl angiotensin-converting-enzyme (ACE) inhibitor active without metabolism and is absorbed in its active form.

#### *1.1.2. Chemistry*

Angiotensin enzyme inhibitors are basically ester-containing drugs that show 100-1000 times less activity than their active form; these inhibitors are synthetic in nature and can be classified on the basis of their chemical structure. They can be grouped as sulphhydral-containing (fentiapril, pivalopril, zofenopril, alacepril, etc.), dicarboxyl-containing (lisinopril, benazepril, quinapirl, perindopril, indopril, pentopril, indalapril, alazapril, moexipril, romipril, spirapril, etc.), phosphorous-containing (fosinopril) [13] and naturally occurring lactokinins and casokinins. [14]

**Table 1.** ACE Inhibitors with structure and nomenclature

In general we can say that all ACE inhibitors differ by three properties: potency, conversion from pro-drug to active form, and pharmacokinetics (i.e., ADME). They also differ in terms of tissue distribution. All ACE inhibitors have a similar antihypertensive efficacy – they effec‐ tively block the conversion of angiotensin 1 to angiotensin II – and all have similar therapeutic indications, adverse effect profiles and contraindications.

#### *1.1.3. Mechanism of action*

inhibitor of ACE. Clinical trials showed excellent anti-hypertensive properties and these results had a major impact on the treatment of cardiovascular disease [11]. The first demon‐ stration of an orally active ACE inhibitor was made on 31 March 1975, when the succinyl group was replaced with a derivative of cysteine, increasing inhibitory potency about 2,000-fold because sulphhydryl of cysteine bound with zinc more tightly than the carboxyl of succinyl. This resulted in captopril, with a dramatic effect on renal function and on hypertension [12]. Enalapril is basically a first derivative of ACE inhibitor, which was developed to overcome the limitations of captopril. Lisinopril is a lysine analogue of enalaprilat (the active metabolite of enalapril). *In vitro* lisinopril is slightly more potent than enalaprilat. It is a non-sulphhydryl angiotensin-converting-enzyme (ACE) inhibitor active without metabolism and is absorbed

Angiotensin enzyme inhibitors are basically ester-containing drugs that show 100-1000 times less activity than their active form; these inhibitors are synthetic in nature and can be classified on the basis of their chemical structure. They can be grouped as sulphhydral-containing (fentiapril, pivalopril, zofenopril, alacepril, etc.), dicarboxyl-containing (lisinopril, benazepril, quinapirl, perindopril, indopril, pentopril, indalapril, alazapril, moexipril, romipril, spirapril, etc.), phosphorous-containing (fosinopril) [13] and naturally occurring lactokinins and

**Drug Nomenclature Structure Ref**

Captopril 1-(3-mercapto-2-dmethyl-1-oxopropyl)-1-proline (S,S) [16]

In general we can say that all ACE inhibitors differ by three properties: potency, conversion from pro-drug to active form, and pharmacokinetics (i.e., ADME). They also differ in terms of

L-proline,(Z)-2-butenedioate salt [15]

dehydrate [16]

Enalapril (S)-1-[N-[1-(ethoxycarbonyl)-3-phenyl propyl]-L-alanyl]-

Lisinopril ((S)-1-[N2-(1-carboxy-3-phenylpropyl)-1-lysyl]-1-proline

**Table 1.** ACE Inhibitors with structure and nomenclature

in its active form.

204 Hypercholesterolemia

*1.1.2. Chemistry*

casokinins. [14]

These inhibitors block the converting enzyme of angiotensin, which is responsible for cleavage from angiotensin I, which is decapeptide, to angiotensin II, which is octapeptide [17, 18], and lower the BP by reducing PVR (peripheral vascular resistance). They also decrease aldosterone secretion and the resulting sodium and water retention.

#### *1.1.4. Pharmacokinetics*

The oral bioavailability of ACE inhibitors ranges from 13% to 95% [19, 20]. Most ACE inhibitors are administered as pro-drugs that remain inactive until esterified in the liver [21]. Pharma‐ kokinetic characteristics of different ACE inhibitors are given in Table 2


**Table 2.** Pharmacokinetic of ACE Inhibitors

#### *1.1.5. Therapeutic use*

ACE inhibitors are effective in patients with mild to moderately severe hypertension, normal or low plasma renin activity, collagen vascular disease and cardiovascular disease [22, 23]. They are also used in the prevention and treatment of myocardial infarction [24, 25] and in the management of cardiac arrhythmias [26]. They can decrease the progression of atherosclerosis, microalbuminuria and diabetic retinopathy, and produce a beneficial effect in patients with Bartter's syndrome [27].

#### *1.1.6. Adverse effects*

ACE inhibitors have a relatively low incidence of side effects and are well tolerated; however, dry cough is common, appearing in 10-30% of patients. This appears to be related to the elevation in bradykinin [28-30]. Hypotension is seen especially in patients with heart failure [31], angiooedema (life-threatening airway swelling and obstruction; 0.1-0.2% of patients) and hyperkalaemia. ACE inhibitors are contraindicated in pregnancy, in the first trimester associated with a risk of major congenital malformations, particularly affecting the cardiovas‐ cular and central nervous systems [32]. The most common (≥1% of patients) adverse effects include hypotension, fatigue, dizziness, headache, nausea and other gastrointestinal distur‐ bances, dry cough, hyperkalaemia and renal impairment. Rash and taste disturbances are more prevalent with captopril and are attributed to its sulphhydryl moiety; eosinophilia has also been reported. Most of the adverse effects are reversible on withdrawing therapy [33]. Treatment with ACE inhibitor has been associated with the development of anaphylactoid reaction [34].

#### *1.1.7. Drug interactions*

Hypotensive effect of ACE inhibitors decreased when given in combination with non-steroidal anti-inflammatory drugs [35], but this effect was enhanced with calcium-channel blockers and beta-blockers [36]. Granulocytopaenia occurs after combined therapy of ACE inhibitors and interferones [37]. ACE inhibitors interact with different drugs, like NSAIDs [38]. Cytokines antagonize the hypotensive effect of ACE inhibitors [39]; severe hypokalaemia occurs with potassium-depleting diuretics [40] and potassium-sparing diuretics produce hyperkalaemia [41, 42]. ACE inhibitors were shown to increase potassium levels in the body [43]. Alphablockers enhance the hypotensive effect of ACE inhibitors [44]. Iron supplementation success‐ fully decreases cough induced by ACE inhibitors [45] and can interfere with the absorption of ACE inhibitors [46]. Hypoglycaemic effect is enhanced with anti-diabetics and insulin [47, 48]. Combination of azathioprine and ACE inhibitors is associated with anaemia [49]. The risk of bone marrow depression is increased in patients taking concomitant therapy of ACE inhibitors and immunosuppressive agents.

#### **1.2. HMG-CoA reductase inhibitors (statins)**

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are the most effective among all hypolipidaemic agents [50]. These lipid-lowering drugs are increas‐ ingly used for primary and secondary hindrance of cardiovascular disease [51]; they have only been recognized for treatment of hyperlipidaemia. In clinical studies, statins are highly effective in enhancing HDL levels while reducing total cholesterol, LDL cholesterol, apolipo‐ protein B and triglyceride levels.

The normal treatment regimen for these drugs involves daily exposure over a period of many years [52, 53]. They have also been examined in combination with cures of multiple sclerosis, osteoporosis, Alzheimer's disease and dropping the superfluous increased occurrence in CHD in women on HRT treatment [54]. They have anti-thrombogenic, anti-inflammatory and anticoagulant properties [55, 56]. These therapeutic properties are independent of lipid lowering [57], and the benefits of statins appear to be independent of baseline cholesterol [58]. They can be classified into subclasses: the naturally or fungi-derived first generation, and the synthetic second generation. The first generation includes simvastatin, lovastatin and pravas‐ tatin, and the second atorvastatin and rosuvastatin. They can be further divided into the lipophilic group (simvastatin, lovastatin and atorvastatin) and the OH hydrophilic group (pravastatin and rosuvastatin) [59].


**Table 3.** Statins with structure and chemical name

#### **2. Experiment**

#### **2.1. Materials**

include hypotension, fatigue, dizziness, headache, nausea and other gastrointestinal distur‐ bances, dry cough, hyperkalaemia and renal impairment. Rash and taste disturbances are more prevalent with captopril and are attributed to its sulphhydryl moiety; eosinophilia has also been reported. Most of the adverse effects are reversible on withdrawing therapy [33]. Treatment with ACE inhibitor has been associated with the development of anaphylactoid

Hypotensive effect of ACE inhibitors decreased when given in combination with non-steroidal anti-inflammatory drugs [35], but this effect was enhanced with calcium-channel blockers and beta-blockers [36]. Granulocytopaenia occurs after combined therapy of ACE inhibitors and interferones [37]. ACE inhibitors interact with different drugs, like NSAIDs [38]. Cytokines antagonize the hypotensive effect of ACE inhibitors [39]; severe hypokalaemia occurs with potassium-depleting diuretics [40] and potassium-sparing diuretics produce hyperkalaemia [41, 42]. ACE inhibitors were shown to increase potassium levels in the body [43]. Alphablockers enhance the hypotensive effect of ACE inhibitors [44]. Iron supplementation success‐ fully decreases cough induced by ACE inhibitors [45] and can interfere with the absorption of ACE inhibitors [46]. Hypoglycaemic effect is enhanced with anti-diabetics and insulin [47, 48]. Combination of azathioprine and ACE inhibitors is associated with anaemia [49]. The risk of bone marrow depression is increased in patients taking concomitant therapy of ACE

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are the most effective among all hypolipidaemic agents [50]. These lipid-lowering drugs are increas‐ ingly used for primary and secondary hindrance of cardiovascular disease [51]; they have only been recognized for treatment of hyperlipidaemia. In clinical studies, statins are highly effective in enhancing HDL levels while reducing total cholesterol, LDL cholesterol, apolipo‐

The normal treatment regimen for these drugs involves daily exposure over a period of many years [52, 53]. They have also been examined in combination with cures of multiple sclerosis, osteoporosis, Alzheimer's disease and dropping the superfluous increased occurrence in CHD in women on HRT treatment [54]. They have anti-thrombogenic, anti-inflammatory and anticoagulant properties [55, 56]. These therapeutic properties are independent of lipid lowering [57], and the benefits of statins appear to be independent of baseline cholesterol [58]. They can be classified into subclasses: the naturally or fungi-derived first generation, and the synthetic second generation. The first generation includes simvastatin, lovastatin and pravas‐ tatin, and the second atorvastatin and rosuvastatin. They can be further divided into the lipophilic group (simvastatin, lovastatin and atorvastatin) and the OH hydrophilic group

reaction [34].

206 Hypercholesterolemia

*1.1.7. Drug interactions*

inhibitors and immunosuppressive agents.

protein B and triglyceride levels.

(pravastatin and rosuvastatin) [59].

**1.2. HMG-CoA reductase inhibitors (statins)**

Raw materials used were of pharmaceutical purity and were obtained from different phar‐ maceutical companies (Table 4). Tablets were purchased from a local pharmacy; each product was labelled with an expiry date not earlier than two years from the time of these studies.


#### **Table 4.** Drugs, brands and manufacturers

#### *2.1.1. Reagents*

Analytical-grade solutions were used for the performance of the experiment. Methanol and acetonitrile were of HPLC grade and other reagents included HCl, sodium hydroxide (NaOH), sodium chloride (NaCl), disodium hydrogen orthophosphate, potassium dihydrogen ortho‐ phosphate, ammonium chloride, NH3 solution (10%), phosphoric acid (8%) (Merck Germany). Organic solvents used were methanol, ethanol, ethyl acetate, chloroform, acetronitrile, triethylamine and DMSO (Merck HPLC Grade Germany).

#### *2.1.2. Equipment*

A UV-visible spectrophotometer (Shimadzu Model 1601, Japan) with 10-mm path length was connected to a computer with UVPC version 3.9 software. A Stedec CSW-300 was used for deionization of water. Dissolution was accomplished using BP 2009 standards. Chromato‐ graphic studies were carried out by using two Shimadzu HPLC systems, one equipped with an LC-10 AT VP pump (SPD-10 A VP), and the second with an LC-20AT UV/VIS detector utilizing Hypersil, ODS, C18 (150×4.6 mm, 5 micron) and a Purospher® STAR RP-18 column. Chromatographic data peaks were analysed using Shimadzu Japan CBM-102, class GC 10 software.

Infrared studies were performed using Shimadzu FTIR Prestige-21. Spectral analysis was performed using Shimadzu software. The proton H1 -NMR spectra were calculated on a Bruker (AMX 500 MHz) spectrometer using TMS as an internal standard. Melting points were recorded using Gallen kamp melting-point apparatus Minnesota Mining And Manufacturing Company.

#### **2.2. Methods**

#### *2.2.1. Preparation of simulated gastric juice and buffers*

0.1 N HCl was prepared by using 9 mL HCl (11 N) in a volumetric flask; the volume was made up with de-ionized water. Chloride buffer at pH 4 was prepared by dissolving 3.725 g of KCl (potassium chloride) in deionized water and 0.1N HCl was used for pH adjustment. For preparation of PO4 (phosphate buffer pH 7.4) 0.6 gm of potassium dihydrogen orthophosphate was used, plus 6.4 g of disodium hydrogen orthophosphate and 5.85 g of NaCl (sodium chloride), and the pH was adjusted. Preparation of NH3 ammonia buffer at pH 9 was done using 4.98 g of NH4Cl ammonium chloride and pH-adjusted with 10% ammonia.

#### *2.2.2. Construction of the calibration curve of drugs*

**Class Drugs Brands Potency**

triethylamine and DMSO (Merck HPLC Grade Germany).

performed using Shimadzu software. The proton H1

*2.2.1. Preparation of simulated gastric juice and buffers*

ACE inhibitors

208 Hypercholesterolemia

*2.1.1. Reagents*

*2.1.2. Equipment*

software.

Company.

**2.2. Methods**

**Table 4.** Drugs, brands and manufacturers

Statins

**(mg)**

Captopril Capoten 25 Bristol Meyers (Pvt.) Ltd. Lisinopril Lisinopril 5 Atco Laboratories Ltd.

Rosuvastatin X-plended 20 Pharm Evo (Pvt.) Ltd. Atorvastatin Atopitar 10 Atco Pharma (Pvt.) Ltd. Pravastatin Pravachol 20 Bristol Meyers (Pvt.) Ltd. Simvastatin Atcol 10 Geofman Pharma (Pvt.) Ltd.

Enalapril Renitec 10 MSD

Analytical-grade solutions were used for the performance of the experiment. Methanol and acetonitrile were of HPLC grade and other reagents included HCl, sodium hydroxide (NaOH), sodium chloride (NaCl), disodium hydrogen orthophosphate, potassium dihydrogen ortho‐ phosphate, ammonium chloride, NH3 solution (10%), phosphoric acid (8%) (Merck Germany). Organic solvents used were methanol, ethanol, ethyl acetate, chloroform, acetronitrile,

A UV-visible spectrophotometer (Shimadzu Model 1601, Japan) with 10-mm path length was connected to a computer with UVPC version 3.9 software. A Stedec CSW-300 was used for deionization of water. Dissolution was accomplished using BP 2009 standards. Chromato‐ graphic studies were carried out by using two Shimadzu HPLC systems, one equipped with an LC-10 AT VP pump (SPD-10 A VP), and the second with an LC-20AT UV/VIS detector utilizing Hypersil, ODS, C18 (150×4.6 mm, 5 micron) and a Purospher® STAR RP-18 column. Chromatographic data peaks were analysed using Shimadzu Japan CBM-102, class GC 10

Infrared studies were performed using Shimadzu FTIR Prestige-21. Spectral analysis was

(AMX 500 MHz) spectrometer using TMS as an internal standard. Melting points were recorded using Gallen kamp melting-point apparatus Minnesota Mining And Manufacturing

0.1 N HCl was prepared by using 9 mL HCl (11 N) in a volumetric flask; the volume was made up with de-ionized water. Chloride buffer at pH 4 was prepared by dissolving 3.725 g of KCl (potassium chloride) in deionized water and 0.1N HCl was used for pH adjustment. For

**Pharmaceutical industry**


The above standard solutions of all drugs were scanned in the region 200-700 nm against the reagent blank, and absorbance maxima were recorded as shown in Table 5. Calibration curves were constructed between concentration and absorbance. Epsilon values and linear coeffi‐ cients were calculated in each case at all the above-described pH values. Beer Lambert's law was obeyed at all concentrations and pHs.


**Table 5.** Please Add Caption

*2.2.3. Monitoring of drug interactions of enalapril, captopril and lisinopril by high-performance liquid chromatography*

HPLC methods for simultaneous determination of enalapril, captopril and lisinopril with statins in raw materials, pharmaceutical dosage forms or in human serum were developed and validated according to ICH guidelines. These methods were then applied to drug-drug, drugmetals and drug-antacid interaction studies.

#### *2.2.4. Chromatographic conditions*

Isocratic elution was performed at ambient temperature with two different types of column. Hypersil, ODS, C18 (150×4.6 mm, 5 micron) and Purospher® STAR RP-18, for assay of enalapril, captopril and lisinopril and simultaneous determination of these drugs with interacting drugs, respectively. The mobile phase, flow rate, wavelength and UV detection were varied as shown in Table 6. A sample volume of 20 μL was injected in triplicate onto the HPLC column and the elute was monitored at different wavelengths.

#### *2.2.5. Preparation of standard solutions*

Stock reference standard solutions of all drugs were prepared daily by dissolving appropriate amounts of each drug in mobile phase to yield final concentration of 300 μgmL-1. For the calibration standards, calibrators of each drug were prepared by making serial dilutions from stock solutions. All solutions were filtered through 0.45 μm filter and degassed using sonicator.

#### *2.2.6. Preparation of pharmaceutical dosage from samples*

Pharmaceutical formulations of the respective brands commercially available in Pakistan were evaluated. In each case, groups of 20 tablets were individually weighed and finely ground in a mortar. The portion of the powder equivalent to the amount of drug was transferred into a volumetric flask and completely dissolved in mobile phase, and then diluted with this solvent up to the mark. After filtration using a 0.45 micrometre μm filter this was then injected.

#### *2.2.7. Preparation of standard plasma solutions*

Samples of blood used were collected then centrifuged at 3000 rpm for at least ten minutes, Supernatant solution was stored at –20°C. The solution serum was deprotinated by using (ACN) acetonitrile, and this solution was spiked daily with working solutions for required concentrations of ACE inhibitors and interacting drugs (statins). 10 μL of sample was injected and chromatographed under the above conditions.


**Table 6.** Chromatographic conditions of HPLC methods

#### *2.2.8. Method development and optimization*

HPLC methods were developed and optimized for certain parameters before method valida‐ tion. The optimization of the analytical procedure was carried out by varying the mobile-phase composition, flow rate, pH of the mobile phase, diluent of solutions and wavelength of analytes in order to achieve symmetrical peaks with good resolution at reasonable retention time.

#### *2.2.9. Method validation*

All validation parameters were established according to the guidelines given by ICH: system suitability, linearity, selectivity of drugs, specificity, (concentration-detector response rela‐ tionship), accuracy or precision and sensitivity with systems, i.e., D and Q (detection and quantification) limit.

#### Specificity and linearity

*2.2.5. Preparation of standard solutions*

210 Hypercholesterolemia

*2.2.6. Preparation of pharmaceutical dosage from samples*

*2.2.7. Preparation of standard plasma solutions*

and chromatographed under the above conditions.

**Table 6.** Chromatographic conditions of HPLC methods

*2.2.8. Method development and optimization*

*2.2.9. Method validation*

Stock reference standard solutions of all drugs were prepared daily by dissolving appropriate amounts of each drug in mobile phase to yield final concentration of 300 μgmL-1. For the calibration standards, calibrators of each drug were prepared by making serial dilutions from stock solutions. All solutions were filtered through 0.45 μm filter and degassed using sonicator.

Pharmaceutical formulations of the respective brands commercially available in Pakistan were evaluated. In each case, groups of 20 tablets were individually weighed and finely ground in a mortar. The portion of the powder equivalent to the amount of drug was transferred into a volumetric flask and completely dissolved in mobile phase, and then diluted with this solvent up to the mark. After filtration using a 0.45 micrometre μm filter this was then injected.

Samples of blood used were collected then centrifuged at 3000 rpm for at least ten minutes, Supernatant solution was stored at –20°C. The solution serum was deprotinated by using (ACN) acetonitrile, and this solution was spiked daily with working solutions for required concentrations of ACE inhibitors and interacting drugs (statins). 10 μL of sample was injected

Enalapril assay 70 - 30 3.5 1 215 Enalapril + statins 60 40 3 1.8 230 Captopril 50 - 50 2.9 1 220 Captopril + statins - 60 40 2.9 1.5 230 Lisinopril 80 2.5 17.5 3 1 225 Lisinopril + statins - 60 40 3 1 225

HPLC methods were developed and optimized for certain parameters before method valida‐ tion. The optimization of the analytical procedure was carried out by varying the mobile-phase composition, flow rate, pH of the mobile phase, diluent of solutions and wavelength of analytes in order to achieve symmetrical peaks with good resolution at reasonable retention time.

All validation parameters were established according to the guidelines given by ICH: system suitability, linearity, selectivity of drugs, specificity, (concentration-detector response rela‐

**Drug Mobile phase pH Flow rate Detection**

MeOH ACN H2O mLmin-1 nm

The drugs were spiked with pharmaceutical formulations containing different excepients. The linearity of the proposed method was checked at different levels of concentration with different groups. Correlation coefficient was linear; intercept and slope values were also calculated.

#### Suitability of system

The system suitability of the method was evaluated by analysing five replicate analyses of the drug at a specific concentration for repeatability, (peaks) symmetry factor, theoretical plates for columns, resolution of peaks between interacting drugs, and relative retention of drugs.

#### Accuracy and precision

Accuracy was calculated at three different levels of concentration (80±20%) by spiking a known amount of the drug. Three or four injections of each drug were injected into the system and the percentage recovery was calculated.

For precision, six replicates of each level were injected into the system on two different nonconsecutive days in each case, and the %RSD was calculated.

Limit of detection and quantification

The detection limit (LOD) of the method was calculated by the formula LOD = 3.3 SD/slope. The quantitation limit (LOQ) – the lowest level of analyte that is accurately measured – was set at ten times the noise level (LOQ = 10ơ/S, where ơ is the standard deviation of the lowest standard concentration and S is the slope of the standard curve).

#### Robustness

Robustness was established by changing the concentration of mobile phase (water, methanol and acetonitrile), wave length, flow rate and pH. At least five repeated solutions were used with small variations of different parameters. Parameters that were changed mainly had a small deviation: ± 0.2% flow rate/pH, and ±5% for wave length.

#### Ruggedness

Ruggedness was determined in different labs. Lab 1 was the (RIPS) Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Karachi University, and the other at the same university in the Department of Chemistry. Two different instruments (LC 10/LC 20) and two different columns (Purospher STAR C18/Hypersil ODS) were used.

#### *2.2.10. Interaction studies by HPLC*

Enalapril solution was mixed with a solution of the interacting drug (statins), which gave a final concentration of 100 μgmL-1 for each constituent). These solutions were kept in a water bath at 37 °C for three hours. An aliquot of 5 mL was withdrawn at 30-minute intervals; after making appropriate dilutions it was filtered through 0.45 μ filter paper and three replicates were injected into the HPLC system. The concentration of each drug was determined and the percentage recovery was calculated; the same procedure was applied for captopril and lisinopril.

#### **2.3. Synthesis of ACE inhibitors and interacting-drugs complexes**

Complexes of enalapril, captopril and lisinopril with all interacting drugs were synthesized. Equimolar solutions of enalapril and interacting drugs were prepared in methanol. An equivolume solution of enalapril was mixed with each drug individually and the respective pH was adjusted either by 1-2 drops of ammonia or 0.1 N HCl. These mixtures were refluxed for three hours then filtered and left for crystallization at room temperature. Melting points and physical characteristics of these complexes were noted. Solubility of all these complexes was checked in different solvents: water, methanol, ethanol, chloroform and DMSO. A similar procedure was adopted for captopril and lisinoril.

#### *2.3.1. Spectroscopic studies of complexes*

#### *2.3.1.1. Infrared studies*

ACE inhibitors and their complexes were characterized by using a FT-IR spectrophotometer in the region 400-4000 cm-1. The infrared spectra were recorded using a potassium bromide disc. ATR (attenuated total reflection) or smart performer accessory was used for the sample (minimum amount).

#### *2.3.1.2. Proton NMR analysis*

Proton 1 H NMR analysis was performed using a Bruker instrument in deuterated H2O, chloroform and methanol using (TMS) tetramethyl silane as an IS (internal standard).

#### **3. Results and discussion**

#### **3.1. Method development/validation by HPLC**

Simple, cheap and very precise, HPLC was used for the determination of ACE inhibitors (captopril, enalapril and lisinopril) in the presence of different statins: ROS (rosuvastatin), ATR (atorvastatin) and SMV(simvastatin) in active ingredients as well as in formulations. It was developed according to guidelines ICH. All inhibitors with statins separated out in less than 10 mins without interference from any ingredients. The recovery of drugs was within the desired range (99-102%). These methods were validated according to ICH and the criteria for acceptance (accuracy/linearity/precision/specificity) and for system suitability were met. The methods can easily be used for quantitative analysis of ACE inhibitors and statins as single drugs or in formulations.

#### **3.2. Interaction of ACE inhibitors with statins**

Hyperlipidaemia and hypertension correlate with each other. They can effect coronary heart disease (CHD), because cardiovascular disease (CVD) is closely related to different factors, such as hypertension (HT) or high cholesterol levels. Factors include family history, age, sex, and diabetes [60-66]. Co-administration of antihypertensive, lipid-lowering and antidiabetic drugs is used in the treatment [67-72]. The most commonly used combinations of diuretic (chlorthalidone, hydrochloroth-iazide, etc.) and an angiotensin II receptor antagonist to control hypertension, as well as with a statin (fluvastatin, simvastatin, etc.) to reduce the cholesterol [73]. Co-administration of an antihypertensive agent with statin is an effective therapeutic option for treatment of multiple cardiovascular risk factors, and especially for high blood pressure (BP) and LDL-C [74-78]. In addition, statins may improve the vasodilatation capacity of large arteries and may thus contribute to BP-lowering in patients treated with both an anti-hypertensive and a statin [79]. Hypercholesterolaemia is often accompanied by hypertension, an associated risk factor for coronary-artery disease (CAD) [80-82]. ACE inhibitors are effective for the management of hypertension, supraventricular arrhythmias and angina pectoris. Other antihypertensive drugs such as propranolol [83] and atenolol [84] also interact with HMG-CoA reductase inhibitor. In the light of the above results, ACE inhibitors may interact and effect a change in each other's availabilities. Methods were developed by HPLC for both ACE inhibitors and statins before starting interaction studies [85-88]. *In vitro"* interactions of ACE inhibitors with statins (atorvastatin, rosuvastatin, pravastatin and simvastatin) were studied in stimulated body environments utilizing the HPLC technique.

#### *3.2.1. Interaction of enalapril with statins using HPLC*

percentage recovery was calculated; the same procedure was applied for captopril and

Complexes of enalapril, captopril and lisinopril with all interacting drugs were synthesized. Equimolar solutions of enalapril and interacting drugs were prepared in methanol. An equivolume solution of enalapril was mixed with each drug individually and the respective pH was adjusted either by 1-2 drops of ammonia or 0.1 N HCl. These mixtures were refluxed for three hours then filtered and left for crystallization at room temperature. Melting points and physical characteristics of these complexes were noted. Solubility of all these complexes was checked in different solvents: water, methanol, ethanol, chloroform and DMSO. A similar

ACE inhibitors and their complexes were characterized by using a FT-IR spectrophotometer in the region 400-4000 cm-1. The infrared spectra were recorded using a potassium bromide disc. ATR (attenuated total reflection) or smart performer accessory was used for the sample

chloroform and methanol using (TMS) tetramethyl silane as an IS (internal standard).

Simple, cheap and very precise, HPLC was used for the determination of ACE inhibitors (captopril, enalapril and lisinopril) in the presence of different statins: ROS (rosuvastatin), ATR (atorvastatin) and SMV(simvastatin) in active ingredients as well as in formulations. It was developed according to guidelines ICH. All inhibitors with statins separated out in less than 10 mins without interference from any ingredients. The recovery of drugs was within the desired range (99-102%). These methods were validated according to ICH and the criteria for acceptance (accuracy/linearity/precision/specificity) and for system suitability were met. The methods can easily be used for quantitative analysis of ACE inhibitors and statins as single

Hyperlipidaemia and hypertension correlate with each other. They can effect coronary heart disease (CHD), because cardiovascular disease (CVD) is closely related to different factors,

H NMR analysis was performed using a Bruker instrument in deuterated H2O,

**2.3. Synthesis of ACE inhibitors and interacting-drugs complexes**

procedure was adopted for captopril and lisinoril.

*2.3.1. Spectroscopic studies of complexes*

*2.3.1.1. Infrared studies*

(minimum amount).

Proton 1

*2.3.1.2. Proton NMR analysis*

**3. Results and discussion**

drugs or in formulations.

**3.1. Method development/validation by HPLC**

**3.2. Interaction of ACE inhibitors with statins**

lisinopril.

212 Hypercholesterolemia

*In vitro* interactions of enalapril in the presence of statins drugs (rosuvastatin, atorvastatin and simvastatin) were observed in 1:1 ratio buffers of pH 4 and 7.4 at 37°C. Simultaneous deter‐ mination of both interacting drugs was also developed, as described above. The results are summarized in Table 7 and Figures 1-3. There was no significant increase or decrease in the concentration of enalapril and interacting drugs at pH 4 and pH 7.4. When enalapril interacted with rosuvastatin, atorvastatin and simvastatin, concentration remained at nearly 99-103% at pH 4 and 99-107% at pH 7.4. Collectively, *in vitro* interaction of enalapril with rosuvastatin, atorvastatin and simvastatin using HPLC at pH 4 and pH 7.4 did not show any significant interactions.


**Table 7.** Percentage availability of enalapril and statins at pH 4 and 7.4 using HPLC

**Figure 1.** Percentage availability of enalapril and statins at pH 4

**Figure 2.** Percentage availability of enalapril and statins at pH 7.4

#### *3.2.2. Interaction of captopril with statins using HPLC*

*In vitro* interactions of captopril in the presence of statins drugs (rosuvastatin, atorvastatin and simvastatin) were observed in 1:1 ratio solutions at 37°C. Simultaneous determination of both interacting drugs was also developed, as described above. Interaction results (Table 8 and Figure 3) show that availability of all drugs was 100% at zero minutes; after that, availability of atorvastatin and simvastatin increased in ascending order, but the percentage availability of captopril decreased in the presence of atorvastatin and simvastatin and remained the same in the presence of rosuvastatin. The availability of atorvastatin and simvastatin was 173% and 115% after 180 min, respectively. Retardation effect was observed at availability of captopril of 97.8%, and 58.2% was available at the end of experiment. Rosuvastatin showed no effect on

captopril and availability of rosuvastatin and captopril at the end of experiment was 100% was 99.12%, respectively. end of experiment. Rosuvastatin showed no effect on captopril and availability of rosuvastatin and captopril at


**Table 8.** Percentage availability of captopril and statins using HPLC Table 8. Percentage availability of captopril and statins using HPLC

**3.2.3. Interaction of lisinopril with statins using HPLC**

significant results.

the end of experiment was 100% was 99.12%, respectively.

**Figure 1.** Percentage availability of enalapril and statins at pH 4

214 Hypercholesterolemia

**Figure 2.** Percentage availability of enalapril and statins at pH 7.4

*3.2.2. Interaction of captopril with statins using HPLC*

*In vitro* interactions of captopril in the presence of statins drugs (rosuvastatin, atorvastatin and simvastatin) were observed in 1:1 ratio solutions at 37°C. Simultaneous determination of both interacting drugs was also developed, as described above. Interaction results (Table 8 and Figure 3) show that availability of all drugs was 100% at zero minutes; after that, availability of atorvastatin and simvastatin increased in ascending order, but the percentage availability of captopril decreased in the presence of atorvastatin and simvastatin and remained the same in the presence of rosuvastatin. The availability of atorvastatin and simvastatin was 173% and 115% after 180 min, respectively. Retardation effect was observed at availability of captopril of 97.8%, and 58.2% was available at the end of experiment. Rosuvastatin showed no effect on

Figure 3. Chromatogram showing change in AUC of drugs. CAP+ATR, CAP+ROS and CAP+SIM (pink before and black after interaction). **Figure 3.** Chromatogram showing change in AUC of drugs. CAP+ATR, CAP+ROS and CAP+SIM (pink before and black after interaction).

*In vitro* interactions of lisinopril in the presence of statin drugs (pravastatin, rosuvastatin and atorvastatin) were carried out in solution of 1:1 ratio at 37°C. Simultaneous determination of these interacting drugs was also developed as described above. Results of these interactions (Table 9 and Figure 4) show that when lisinopril interacted with pravastatin, rosuvastatin and atorvastatin its concentration remained in the range 100.3- 102%, 99.9-101.4% and 99.36-102% at 37oC. Pravastatin, rosuvastatin and atorvastatin amplified the availability of lisinopril, unaffected at 37oC using HPLC. Availability of pravastatin and rosuvastatin after interaction almost stayed unchanged at 37oC. Interactions of lisinopril in the presence of atorvastatin showed that availability of atorvastatin was enhanced, while that of lisinopril after interaction was unchanged. Collectively, *in vitro* interaction of lisinopril with rosuvastatin, atorvastatin and simvastatin using HPLC at 37oC did not show any

#### *3.2.3. Interaction of lisinopril with statins using HPLC*

*In vitro* interactions of lisinopril in the presence of statin drugs (pravastatin, rosuvastatin and atorvastatin) were carried out in solution of 1:1 ratio at 37°C. Simultaneous determination of these interacting drugs was also developed as described above. Results of these interactions (Table 9 and Figure 4) show that when lisinopril interacted with pravastatin, rosuvastatin and atorvastatin its concentration remained in the range 100.3- 102%, 99.9-101.4% and 99.36-102% at 37°C. Pravastatin, rosuvastatin and atorvastatin amplified the availability of lisinopril, unaffected at 37 °C using HPLC. Availability of pravastatin and rosuvastatin after interaction almost stayed unchanged at 37 °C. Interactions of lisinopril in the presence of atorvastatin showed that availability of atorvastatin was enhanced, while that of lisinopril after interaction was unchanged. Collectively, *in vitro* interaction of lisinopril with rosuvastatin, atorvastatin and simvastatin using HPLC at 37 °C did not show any significant results.


**Table 9.** Percentage availability of lisinopril and statins using HPLC 150 102.0 100.3 102.3 100.4 102.4 106.2 180 102.3 101.3 101.4 100.5 102.4 106.02

**Interaction studies suggest that with enalapril and lisinopril not affected by statins but captopril have change availability**

1 Bernstein KE, Martin BM, Edwards AS and Bernstein EA (1989) Mouse Angiotensin ‐converting enzyme is a

2 Soubrier F, Alhene‐Gelas F, Hubert C, Allegrini J, John M, Tregear G and Corvol P (1988) Two putative active centres in human angiotensin I‐converting enzyme revealed by molecular cloning, Proc. Natl. Acad. Sci., **85**, 9386‐9390. 3 Ferreira SH (1965) A bradykinin‐potentiating factor present in the venom of Bothrops jararaca, Brit. J.

4 Bakhle YS (1968) Conversion of angiotensin I to angiotensin II by cell free extracts of dog lung, Nature., **220**,

5 Brunner HR, Laragh JH, Sealey JE, Gavras I and Vukovich RA (1974) An angiotensin converting enzyme inhibitor to identify and treat vasoconstrictor and volume factors in hypertensive patients, New. Eng. J. Med., **291**,

15

**Fig 3.4** *% Availability of lisinopril and statins* **Figure 4.** Percentage availability of lisinopril and statins

**of drugs but invivo studies requires for prove this relationship.**

protein composed of two homologous domains, J. Biol. Chem., **264**, 11945‐11951.

**Conclusion:**

**References**

919‐21.

817‐821.

Pharmacol., **24**, 163‐169.

#### **4. Conclusion**

*3.2.3. Interaction of lisinopril with statins using HPLC*

216 Hypercholesterolemia

**Table 9.** Percentage availability of lisinopril and statins using HPLC

0 50 100 150 200 **T i me( mi n)**

**of drugs but invivo studies requires for prove this relationship.**

**Figure 4.** Percentage availability of lisinopril and statins

protein composed of two homologous domains, J. Biol. Chem., **264**, 11945‐11951.

99 99.5 100 100.5 101 101.5 102 102.5

**Conclusion:**

**References**

919‐21.

817‐821.

Pharmacol., **24**, 163‐169.

*In vitro* interactions of lisinopril in the presence of statin drugs (pravastatin, rosuvastatin and atorvastatin) were carried out in solution of 1:1 ratio at 37°C. Simultaneous determination of these interacting drugs was also developed as described above. Results of these interactions (Table 9 and Figure 4) show that when lisinopril interacted with pravastatin, rosuvastatin and atorvastatin its concentration remained in the range 100.3- 102%, 99.9-101.4% and 99.36-102% at 37°C. Pravastatin, rosuvastatin and atorvastatin amplified the availability of lisinopril, unaffected at 37 °C using HPLC. Availability of pravastatin and rosuvastatin after interaction almost stayed unchanged at 37 °C. Interactions of lisinopril in the presence of atorvastatin showed that availability of atorvastatin was enhanced, while that of lisinopril after interaction was unchanged. Collectively, *in vitro* interaction of lisinopril with rosuvastatin, atorvastatin

Time min LIS PRA LIS ROS LIS ATOR 100.3 99.98 99.98 100.3 99.36 97.34 100.6 99.63 99.36 100.2 101.5 100.2 100.3 99.36 101 100.6 101.3 105.2 100.6 100.3 101 100.2 102.5 106.3 100.9 100.3 102.3 100.9 101.1 106.20 102.0 100.3 102.3 100.4 102.4 106.2 102.3 101.3 101.4 100.5 102.4 106.02

 100.6 99.63 99.36 100.2 101.5 100.2 100.3 99.36 101 100.6 101.3 105.2 100.6 100.3 101 100.2 102.5 106.3 100.9 100.3 102.3 100.9 101.1 106.20 102.0 100.3 102.3 100.4 102.4 106.2 102.3 101.3 101.4 100.5 102.4 106.02

> li si nopr i l Pr avastati n

0 50 100 150 200 **T i me( mi n)**

**Fig 3.4** *% Availability of lisinopril and statins*

**Interaction studies suggest that with enalapril and lisinopril not affected by statins but captopril have change availability**

1 Bernstein KE, Martin BM, Edwards AS and Bernstein EA (1989) Mouse Angiotensin ‐converting enzyme is a

2 Soubrier F, Alhene‐Gelas F, Hubert C, Allegrini J, John M, Tregear G and Corvol P (1988) Two putative active centres in human angiotensin I‐converting enzyme revealed by molecular cloning, Proc. Natl. Acad. Sci., **85**, 9386‐9390. 3 Ferreira SH (1965) A bradykinin‐potentiating factor present in the venom of Bothrops jararaca, Brit. J.

4 Bakhle YS (1968) Conversion of angiotensin I to angiotensin II by cell free extracts of dog lung, Nature., **220**,

5 Brunner HR, Laragh JH, Sealey JE, Gavras I and Vukovich RA (1974) An angiotensin converting enzyme inhibitor to identify and treat vasoconstrictor and volume factors in hypertensive patients, New. Eng. J. Med., **291**,

99 99.5 100 100.5 101 101.5 102 102.5

> 0 50 100 150 200 **Time( min)**

> > l i si nopr i l Ator vastati n

lisinopril Rosuvast at in

and simvastatin using HPLC at 37 °C did not show any significant results.

Interaction studies suggest that enalapril and lisinopril are not affected by statins but captopril changes the availability of drugs. *In vivo* studies are required to prove this relationship.

#### **Author details**

Safila Naveed\*

Address all correspondence to: safila117@gmail.com

Faculty of pharmacy, Jinnah University for Women Karachi, Pakistan

#### **References**

15


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222 Hypercholesterolemia


## *Edited by Sekar Ashok Kumar*

This book is aimed to accentuate the importance of hypercholesterolemia, since targeting and treating the hypercholesterolemia is increasingly well known as an essential strategy in the prevention of atherosclerosis-induced cardiovascular disease. It is important to look at hypercholesterolemia as it is proved to be crucial as well as the early stage of atherogenesis and can also be managed with appropriate treatment. This book describes the basics of hypercholesterolemia and its causes and various experimental animal models to understand and study the pathophysiology of hypercholesterolemia as well as to present practice-based clinical approaches to treat hypercholesterolemia. Further, the book describes various treatment strategies of hypercholesterolemia in detail, especially the appropriate use of statin. It is well known that the use of statin is an ideal as well as a potent therapy to lower cholesterol level and also has various beneficial pharmacological effects to prevent cardiovascular diseases. However, there exists less awareness about the use of statin. Hence, it is important to understand the appropriate use of statin in terms of doses for different stages of hypercholesterolemia, side effects, resistance of its use, and also interaction of statin with other drugs, which are well described in this book. In short, the major aim of this compendium is to present to the readers comprehensive, updated, and current research perspectives on hypercholesterolemia.

Hypercholesterolemia

Hypercholesterolemia

*Edited by Sekar Ashok Kumar*

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