Elsevier

Journal of Controlled Release

Volume 180, 28 April 2014, Pages 117-124
Journal of Controlled Release

Comparative pharmacokinetics and tissue distribution analysis of systemically administered 17-β-estradiol and its metabolites in vivo delivered using a cationic nanoemulsion or a peptide-modified nanoemulsion system for targeting atherosclerosis

https://doi.org/10.1016/j.jconrel.2014.02.009Get rights and content

Abstract

The primary objective of this study was to compare the biodistribution and pharmacokinetic profile of 17-β-estradiol (17-βE) on systemic delivery using either the cationic or the CREKA-peptide-modified (Cysteine–Arginine–Glutamic-acid–Lysine–Alanine) omega-3-fatty acid oil containing nanoemulsion system in vivo in the wild type C57BL/6 mice. Higher blood concentrations of 17-βE, higher accumulation in the tissues of interest – heart and aorta, and higher accumulation within the other tissues – liver and kidney was observed on delivering 17-βE using the CREKA-peptide-modified nanoemulsion system (AUClast in plasma — 263.89 ± 21.81 min*%/injected dose/ml) as compared to the cationic nanoemulsion (AUClast in plasma — 20.2 ± 1.86 min*%/injected dose/ml) and solution form (AUClast in plasma — 44.9 ± 1.24 min*%/injected dose/ml) respectively. Both, the cationic nanoemulsion and the CREKA-peptide-modified nanoemulsion showed a higher relative targeting efficiency of 4.57 and 4.86 respectively for 17-βE than the relative targeting efficiency of 1.78 observed with the solution form. In conclusion, since the maximum exposure (highest AUClast for plasma and tissues) for 17-βE was observed with the CREKA-peptide-modified nanoemulsion system, the study shows that CREKA-peptide-modified nanoemulsion system was the most suitable vehicle for systemic delivery of 17-βE in the wild type C57BL/6 mice.

Introduction

Occlusive vascular disorders like atherosclerosis are the primary cause of deaths associated with cardiovascular diseases [1]. Severely occluded vessels and critical stenosis leads to acute coronary syndromes and myocardial infarction, however sudden deaths due to these events are mainly associated with the highly rupture prone-vulnerable plaque that shows only 30% vessel stenosis [2]. Systemic therapy with statins as well as local approaches aimed at focal plaque pacification have shown limited success in targeting and management of vulnerable plaque [2]. In recent years, the field of cardiovascular research has seen some promising research involving the application of nanotechnology for diagnosis as well as treatment of atherosclerosis [3]. The cardioprotective benefits of 17-βE have been reported in several pre-clinical models [4], [5], [6]. While observational studies have reported about 50% reduced incidences of coronary artery disease in postmenopausal women receiving hormone therapy, the results from clinical trials evaluating hormone replacement therapy have always shown large discrepancies and limited success in the treatment of the disease [7], [8], [9]. Current research is now focused on designing techniques for improved delivery of this hormone [10]. In our previous study, we have demonstrated the successful encapsulation of 17-β-estradiol (17-βE) in an omega-3-fatty acid containing flaxseed oil-based cationic nanoemulsion system and the therapeutically beneficial responses of this formulation on vascular cells in vitro [11]. In addition to this formulation system, we have developed a slightly modified 17-βE loaded CREKA-peptide-modified nanoemulsion system (Cysteine-Arginine-Glutamic-acid-Lysine-Alanine). The peptide CREKA shows selective targeting of fibrin clots that are observed in atherosclerotic lesions [12], [13]. In the current study, we have compared and contrasted the pharmacokinetic profile and biodistribution of 17-βE when administered using the solution system, cationic nanoemulsion system and the CREKA-peptide modified nanoemulsion system to identify the most suitable formulation for systemic delivery of 17-βE in vivo in the C57BL/6 mice for treatment of atherosclerosis.

17-βE exerts atheroprotection through local and other pleiotropic mechanisms and circulating plasma levels of 17-βE correlate inversely with the size, development, number and distribution of atherosclerotic lesions in mice [6], [14]. 17-βE is a lipophilic molecule and is mainly eliminated through urinary/fecal excretion by conversion into less active, hydrophilic metabolites [15]. In humans, intravenously administered 17-βE is rapidly metabolized into estrones and other conjugates, with a plasma half-life of 27.45 ± 5.65 min and a low volume of distribution (0.082 ± 0.015 l/kg) [16]. The major pathways involved in the metabolism of 17-βE include mainly oxidative transformation which involves hydroxylation at 2- and 16-positions and the conjugative metabolism that includes sulfonation, glucuronidation and O-methylation [15]. Estrogen metabolism mainly occurs in the liver, catalyzed by enzymes of the cytochrome P450 family and some isoforms of the estrogen metabolizing enzymes are also found in extrahepatic tissues [15], [17]. Hydroxylation at the 16-position leads to the formation of estriol and estriol conjugates predominate the metabolic profile of 17-βE in mice, as compared to the estrone conjugates observed in the metabolic profile of humans [18], [19]. In addition, whereas urinary excretion is prevalent in women (63% of intravenously administered 17-βE mainly excreted through the urine), 17-βE is mainly excreted through the feces in mice [18], [20]. Estradiol sulfate has been reported as one of the major metabolites observed in the plasma of mice on intravenous administration [18]. Thus, in our study we have measured the levels of 17-βE (parent compound), Estradiol sulfate (ESS) and Estriol (EST), observed in the plasma and other tissues on intravenous administration of 17-βE using either solution, cationic nanoemulsion or the CREKA-peptide-modified nanoemulsion system. The pharmacokinetics and biodistribution studies were performed using tritiated estradiol and the radioactive fractions of the parent compound and two metabolites –ESS and EST were first separated using high-performance liquid chromatography (HPLC) and then measured by liquid scintillation counting.

Section snippets

Chemicals and reagents

17-β-Estradiol, 17-β-estradiol-3-sulfate (ESS) and (17β)-estra 1,3,5(10)-triene-3,17-diol EST were purchased from Sigma-Aldrich (St Louis, MO). Extra-virgin, ω-3-PUFA-containing flaxseed oil was kindly provided by Jedwards International (Quincy, MA). Egg phosphatidylcholine (Lipoid® E80) and 1,2-dioleolyl-3-trimethylammonium-propane (DOTAP) was kindly provided by Lipoid GmbH (Ludwigshafen, Germany), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(polyethylene glycol)-2000]

Biodistribution analysis of 17-βE and its metabolites ESS and EST in plasma

As reported in our previous study, the 17-βE loaded cationic nanoemulsion had a size of ~ 140 nm and a charge of ~ 30 mV,[11] while the 17-βE loaded CREKA-peptide modified nanoemulsion system had a size of ~ 175 nm and charge of ~  53 mV (Table 1). On intravenous administration, at all time points, an increase in levels of 17-βE in plasma was observed with the CREKA-peptide-modified nanoemulsion system relative to the solution and the cationic nanoemulsion system (DOTAP) (Fig. 1). Plasma 17-βE

Conclusions

The pharmacokinetics parameters, biodistribution studies and calculated targeting efficiency ratios clearly demonstrated the increased exposure of 17-βE in the plasma, heart and aorta and even in the liver and kidney when 17-βE was delivered using the CREKA-peptide modified nanoemulsion formulation as compared to the solution and cationic (DOTAP) nanoemulsion system. The CREKA-peptide modified nanoemulsion system showed greater exposure of 17-βE to plasma and target organs, had a high targeting

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

This study was performed in the radioactive core facility at Northeastern University in Dr. Samuel Gatley's laboratory and the authors wish to thank him for his help with the study design and experimental setup. The authors would also like to thank Kinjal Sankhe for assisting in the beginning of this study.

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