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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 4  |  Issue : 4  |  Page : 127-131

Effect of atorvastatin nanoparticles compared to free atrovastatin on plaque properties in rabbit model of atherosclerosis


1 Department of Pharmacology, PGIMER, Chandigarh, India
2 Department of Experimental Medicine, PGIMER, Chandigarh, India

Date of Submission28-Jun-2019
Date of Decision20-Aug-2019
Date of Acceptance29-Nov-2019
Date of Web Publication31-Dec-2019

Correspondence Address:
Prof. Nusrat Shafiq
Department of Pharmacology, PGIMER, Room No. 4017, 4th Floor, Research Block B, Chandigarh - 160 012
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jncd.jncd_27_19

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  Abstract 


Background: Atherosclerosis is a systemic disease affecting the entire arterial tree involving the accumulation of inflammatory cells in the atherosclerotic plaques with larger macrophage-rich areas. Utilizing this property, we aimed to study the effect of atorvastatin nanoparticles given once in a week compared to free atorvastatin given daily on the plaque properties in a rabbit model of atherosclerosis.
Materials and Methods: Rabbits were fed with high-cholesterol diet over a period of 2 months. Thereafter, aortic wall tissues were taken for histological changes, and morphometric analysis and plasma samples were taken for the measurement of lipid profile.
Results: Our nanoformulation has been effective in improving lipid profile and decreasing plaque content in the aortic wall and showed comparable effect with that of a free drug.
Conclusion: Thus, our novelty is the development of sustained-release nanoformulation of atorvastatin having similar pharmacodynamic activity when compared to free atorvastatin.

Keywords: Atherosclerosis, atorvastatin, nanoparticles


How to cite this article:
Arora A, Bhandari RK, Pandey AK, Gani Rather II, Malhotra S, Bhatia A, Shafiq N. Effect of atorvastatin nanoparticles compared to free atrovastatin on plaque properties in rabbit model of atherosclerosis. Int J Non-Commun Dis 2019;4:127-31

How to cite this URL:
Arora A, Bhandari RK, Pandey AK, Gani Rather II, Malhotra S, Bhatia A, Shafiq N. Effect of atorvastatin nanoparticles compared to free atrovastatin on plaque properties in rabbit model of atherosclerosis. Int J Non-Commun Dis [serial online] 2019 [cited 2020 Jan 21];4:127-31. Available from: http://www.ijncd.org/text.asp?2019/4/4/127/274460




  Introduction Top


Atherosclerosis is a major risk factor for cerebrovascular and cardiovascular diseases. It is a chronic inflammatory process that involves a complex interplay between circulating cellular and blood elements and the cells of the arterial wall.[1]

Atherosclerosis results from a maladaptive inflammatory response that is initiated by the intramural retention of cholesterol-rich, apolipoprotein-B-containing lipoprotein in a susceptible area of the arterial vasculature.[2] Activation of the endothelium further results in an immune response, which is mediated by the recruitment of monocyte-derived cells into subendothelial space and these cells are further differentiated into mononuclear phagocytes to form foam cells that persist in plaque.[3] The activation products of these macrophages disturb the integrity of fibrous cap of the plaque and increase the risk of plaque rupture, and they appear with greater frequency in plaques taken from symptomatic patients.[4]

Atorvastatin, an 3-hydroxy-3-methylglutaryl (HMG) coenzyme reductase inhibitor, a well-known drug for hyperlipidemics, has a plasma concentration peak in 1–4 h and t1/2 of 20 h. It has poor oral bioavailability (14%)[5] and serious adverse effects like rhabdomyolysis on chronic administration. Due to low bioavailability, a higher dose of atorvastatin is required to achieve an optimum plasma level, which may lead to high incidence of adverse effects.

Atorvastatin has been shown to reduce the macrophage content in plaque by modulating the low-density lipoprotein cholesterol level by blocking the HMG-CoA enzyme on the mevalonate cascade that leads to reduced production of isoprenoids and inhibition of the RHO/RHO kinase pathway.[4] In a clinical ATHEROMA trial, atorvastatin has shown a significant reduction in ultrasmall superparamagnetic iron oxide-enhanced magnetic resonance imaging-defined inflammation. This reveals that it has an effect on macrophage infiltration in human carotid atheroma in vivo.[6],[7]

A robust rabbit model of human atherosclerosis was developed and validated.[8] It describes a range of human-like plaques and provides a functional rather than a histological definition of vulnerable plaque which is suitable for the testing of possible dietary and pharmacological interventions.

Our group has developed “Nanopolypill” comprising commonly prescribed drugs for hypertension and coronary heart disease (HD) (ischemic HD). It has shown to have a sustained release for up to 168 h in pharmacokinetics study.[9],[10] These nanoparticulate systems have an advantage of increased oral bioavailability of drugs due to their specialized uptake mechanisms and that they bypass hepatic first-pass metabolism.[11] Meena et al. have developed oral atorvastatin nanoparticulate system and observed no/negligible myotoxicity in comparison to the marketed formulation.[12]

The aim of the present study was to evaluate the effect of previously developed atorvastatin nanoformulation on lipid profile and plaque morphology in a rabbit model of atherosclerosis.


  Materials and Methods Top


Development of atorvastatin poly(lactic-co-glycolic acid) nanoparticles

The poly (lactic-co-glycolic acid) (PLGA) nanoparticles were prepared by the multiple emulsion and solvent evaporation technique. Briefly, the drug and polymer ratio was taken as 1:1 (w/w). The drug was dissolved in water or dichloromethane (DCM) (depending on its solubility), and the polymer (PLGA) was dissolved in DCM only. To the mixture of drug-polymer in water-DCM (1:10 w/v) or DCM, the oily phase, 0.5% polyvinyl-alcohol, and the aqueous phase, were added. The oily phase and aqueous phase were added in the ratio of 1:2 (v/v). This mixture was homogenized using homogenizer for 3–4 min at a particular speed and was left on the magnetic stirrer to evaporate the DCM (organic solvent added) for 8–10 h. The solution left was centrifuged in cold centrifuge at 10,000–15,000 rpm, 4°C for 15–30 min with three subsequent washing. The pellet was resuspended in a double-distilled water and lyophilized for 24–48 h until completely dry nanoparticles were obtained. These nanoparticles were then assessed for its further characterization of particle size, entrapment efficiency (EE), drug loading (DL), zeta potential, and polydispersity index (PDI).

Pharmacodynamic study

Animal model of human atherosclerosis

New Zealand rabbits were used for the animal model of atherosclerosis. Rabbits were divided into the following groups: control high cholesterol-fed diet group, free drug-treated group, and atorvastatin nanoformulation-treated group. Atherosclerotic lesions were induced in eight rabbits by putting them on a high-cholesterol diet and 2% cholesterol (120–140 g/day) for 10 weeks/45 days.[8] For the treatment group, free atorvastatin (20mg/70kg adult) was given daily for 2 weeks; equivalent dose of nanoformulation of atorvastatin was given once in a week for 2 weeks.

Lipid profile

Blood cholesterol estimation was done by the lipid estimation kits (BioVision Inc., USA). Samples were taken from marginal ear vein at the end of 2 months of diet for the control group, and for the treatment groups, it was taken both before and after the treatment.

Histological examination and morphometric analysis

Rabbits were sacrificed after the blood samples were taken, and aortic tissues were taken and fixed in 10% formalin. Hematoxylin and eosin staining were done from these fixed aortic tissues for aortic tissue examination. Morphometric analysis of the atherosclerotic plaque for intima thickness was performed using Image J software.

Statistical analysis

Data were expressed as mean ± standard deviation, P < 0.05 was considered as statistically significant. Comparison among groups was made by ANOVA followed by post hoc t-test.


  Results Top


Characterization of nanoparticles

PLGA-based atorvastatin nanoparticles prepared by the single emulsion and solvent evaporation method showed EE % of 62.09% and DL of 29.06%. The average particle size of nanoparticles was observed to be 798 nm, with a PDI of 0.033 and negative zeta potential of −10.1 [Table 1].
Table 1: Characterization parameters of atorvastatin loaded nanoparticles

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Pharmacodynamic study

Human atherosclerosis model establishment

After 2 months of high-fat diet (HFD) to rabbits; we have been closer to develop atherosclerosis in their aorta. Histological examination of the cross-section of the rabbit aorta demonstrates lipid-laden macrophages in atherosclerotic plaque visible in the lumen [Figure 1]. Lipid profile examination showed significantly increased cholesterol levels in HFD group as compared to normal-fed rabbits [Figure 2]. All other lipid parameters were also seen to increase [Table 2].
Figure 1: Cross section of the rabbit aorta with lipid-laden macrophages visible in the atherosclerotic plaque

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Figure 2: Lipid profile of high cholesterol-fed diet rabbit as compared to normal-fed rabbits

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Table 2: Pharmacodynamic parameters after intervention

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Effect of atorvastatin poly(lactic-co-glycolic acid) nanoparticles

For both the treatment groups, free atorvastatin was given daily for 2 weeks; atorvastatin nanoformulation was given once a week for 2 weeks. It was observed that the effects on both groups were comparable to each other. As lipid profiles of both the groups after the treatment have shown that daily administration of atorvastatin is comparable with a once a week administration of PLGA-AT nanoparticles (PLGA Atorvastatin nanoparticles) [Figure 3], [Figure 4] and [Table 2]. These results were consistent with the histological examination of the rabbit aorta, as no plaque could be clearly seen in the intima for free drug [Figure 5] as well as for the nanoformulation group. Intact lumen was seen with some area of cholesterol plaques [Figure 6]. Morphometric analysis of intima thickness of stained aortic tissues of control, free, and nanoformulation group (P < 0.01) showed a significant difference in the intima thickness between the groups [Figure 7].
Figure 3: Lipid profile of untreated high cholesterol-fed diet rabbit as compared to free atorvastatin-treated high cholesterol-fed diet rabbits

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Figure 4: Lipid profile of untreated high cholesterol-fed diet rabbit as compared to atrovastatin nanoparticles-treated high cholesterol-fed diet rabbits

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Figure 5: The three layers of the aorta wall can be clearly noticed, as no plaque in the intima

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Figure 6: Intact lumen with some area of cholesterol plaque

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Figure 7: Morphometric analysis of the aortic intima thickness

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  Discussion Top


The atherosclerotic plaque is one of the primary focuses of all complications related to a precipitating cardiovascular event. The immune cells including the macrophages, mast cells, and T-cells, majorly populate the plaque, along with lipids such as esterified cholesterol and cholesterol crystals.[13] The macrophages are largely in an activated state, producing cytokines, proteases, pro-thrombotic molecules, and vasoactive substances, all of which can affect plaque inflammation and vascular function.[9] When the macrophage incorporates lipoproteins, it transforms into a lipid-rich foam cell, which is a hallmark feature of atherosclerosis and leads to lesion expansion.[14] Cytokine release from macrophages augments the inflammatory response and increases the lesion size. Cytotoxic substances, including peroxynitrite and tumor necrosis factor-α, released by the macrophage results in cell death of lesion-resident endothelial and smooth muscle cells and thereby disrupting the vessel structure.

Following our demonstration of sustained action of atorvastatin nanoformulation for a week,[9] we have in the current study demonstrated a significant correlation with the pharmacodynamics response. As compared to the daily administration of free atorvastatin, weekly administration of nanoformulation of atorvastatin was found to produce similar results in lipid profile, intima thickness, and plaque morphology.

PLGA-based nanoparticles are extensively taken up by no-phagocytic eukaryotic cells, macrophages, and dendritic cells.[15] The current study did not explore the uptake of PLGA nanoparticles by the plaque components, we cannot say whether this factor contributed to the reduction in the plaque morphology or not, but it would be interesting to see.


  Conclusion Top


Our results have shown that once-weekly dosing of atorvastatin encapsulated inside the PLGA nanoparticles can improve the lipid profile and similar to that produced by daily dosing of free atorvastatin. Some limitations of our proof of concept studies are that we have not seen the uptake of nanoparticles by the macrophages. Another one is that we have not observed the fate of PLGA nanoparticles inside the body.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Degnan AJ, Patterson AJ, Tang TY, Howarth SP, Gillard JH. Evaluation of ultrasmall superparamagnetic iron oxide-enhanced MRI of carotid atherosclerosis to assess risk of cerebrovascular and cardiovascular events: Follow-up of the ATHEROMA trial. Cerebrovasc Dis 2012;34:169-73.  Back to cited text no. 1
    
2.
Moore KJ, Tabas I. Macrophages in the pathogenesis of atherosclerosis. Cell 2011;145:341-55.  Back to cited text no. 2
    
3.
Moore KJ, Sheedy FJ, Fisher EA. Macrophages in atherosclerosis: A dynamic balance. Nat Rev Immunol 2013;13:709-21.  Back to cited text no. 3
    
4.
Puato M, Faggin E, Rattazzi M, Zambon A, Cipollone F, Grego F, et al. Atorvastatin reduces macrophage accumulation in atherosclerotic plaques: A comparison of a nonstatin-based regimen in patients undergoing carotid endarterectomy. Stroke 2010;41:1163-8.  Back to cited text no. 4
    
5.
Lennernäs H. Clinical pharmacokinetics of atorvastatin. Clin Pharmacokinet 2003;42:1141-60.  Back to cited text no. 5
    
6.
Tang TY, Howarth SP, Miller SR, Graves MJ, Patterson AJ, U-King-Im JM, et al. The ATHEROMA (atorvastatin therapy: Effects on reduction of macrophage activity) study. Evaluation using ultrasmall superparamagnetic iron oxide-enhanced magnetic resonance imaging in carotid disease. J Am Coll Cardiol 2009;53:2039-50.  Back to cited text no. 6
    
7.
Reilly SD, Litovsky SH, Steinkampf MP, Caulfield JB. Statins improve human coronary atherosclerotic plaque morphology. Tex Heart Inst J 2008;35:99-103.  Back to cited text no. 7
    
8.
Phinikaridou A, Hallock KJ, Qiao Y, Hamilton JA. A robust rabbit model of human atherosclerosis and atherothrombosis. J Lipid Res 2009;50:787-97.  Back to cited text no. 8
    
9.
Arora A, Shafiq N, Jain S, Khuller GK, Sharma S, Malhotra S. An development of sustained release “Nanopolypill” of ischemic heart disease drugs – An experimental study. CNANO 2014;10:816-26.  Back to cited text no. 9
    
10.
Arora A, Shafiq N, Jain S, Khuller GK, Sharma S, Malhotra S. Development of sustained release “NanoFDC (Fixed Dose Combination)” for hypertension – An Experimental Study. PLoS One 2015;10:e0128208.  Back to cited text no. 10
    
11.
Kumar G, Sharma S, Shafiq N, Khuller GK, Malhotra S. Optimization, in vitro-in vivo evaluation, and short-term tolerability of novel levofloxacin-loaded PLGA nanoparticle formulation. J Pharm Sci 2012;101:2165-76.  Back to cited text no. 11
    
12.
Meena AK, Ratnam DV, Chandraiah G, Ankola DD, Rao PR, Kumar MN. Oral nanoparticulate atorvastatin calcium is more efficient and safe in comparison to lipicure in treating hyperlipidemia. Lipids 2008;43:231-41.  Back to cited text no. 12
    
13.
Hansson GK, Libby P. The immune response in atherosclerosis: A double-edged sword. Nat Rev Immunol 2006;6:508-19.  Back to cited text no. 13
    
14.
Dickhout JG, Basseri S, Austin RC. Macrophage function and its impact on atherosclerotic lesion composition, progression, and stability: The good, the bad, and the ugly. Arterioscler Thromb Vasc Biol 2008;28:1413-5.  Back to cited text no. 14
    
15.
Lutsiak ME, Robinson DR, Coester C, Kwon GS, Samuel J. Analysis of poly(D, L-lactic-co-glycolic acid) nanosphere uptake by human dendritic cells and macrophages in vitro. Pharm Res 2002;19:1480-7.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2]



 

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