Nanostructured lipid carriers for improved oral delivery and prolonged antihyperlipidemic effect of simvastatin

Accepted Manuscript Title: Nanostructured Lipid Carriers for Improved Oral Delivery and Prolonged Antihyperlipidemic Effect of Simvastatin Authors: Heba A. Fathi, Ayat Allam, Mahmoud Elsabahy, Gihan Fetih, Mahmoud El-Badry PII: DOI: Reference:

S0927-7765(17)30819-6 https://doi.org/10.1016/j.colsurfb.2017.11.064 COLSUB 9015

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

18-9-2017 17-11-2017 27-11-2017

Please cite this article as: Heba A.Fathi, Ayat Allam, Mahmoud Elsabahy, Gihan Fetih, Mahmoud El-Badry, Nanostructured Lipid Carriers for Improved Oral Delivery and Prolonged Antihyperlipidemic Effect of Simvastatin, Colloids and Surfaces B: Biointerfaces https://doi.org/10.1016/j.colsurfb.2017.11.064 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Nanostructured Lipid Carriers for Improved Oral Delivery and Prolonged Antihyperlipidemic Effect of Simvastatin

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Heba A. Fathi1, Ayat Allam2, Mahmoud Elsabahy1, 2-4*, Gihan Fetih1,2 and Mahmoud El-Badry1,2

Assiut International Center of Nanomedicine, Al-Rajhy Liver Hospital, Assiut University, Assiut

71515, Egypt; 2Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut 71515, Egypt; 3Laboratory for Synthetic-Biologic Interactions, Department of Chemistry, Texas

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A&M University, College Station, Texas, USA; 4Misr University for Science and Technology, 6th

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of October City, Egypt Correspondence: Prof. Mahmoud Elsabahy

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Tel.: + 1 979-862-6418

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*

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Fax: + 1 979-862-3714

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E-mail: [email protected] The total number of figures and tables in this manuscript is 8 (no tables, Figure 1 (A & B), Figure 2

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(A-C), Figure 3, Figure 4 (I & II), Figure 5 (IA, IB, IIA, IIB) and Figure 6).

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Graphical abstract

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Highlights Nanoparticles showed high simvastatin entrapment efficiency.

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Nanostructured lipid carriers improved simvastatin oral delivery.

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Improved pharmacokinetic and antihyperlipidemic activity were demonstrated.

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

Abstract

The purpose of the current study is to develop nanostructured lipid carriers (NLCs) for the delivery

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of the antihyperlipidemic drug simvastatin (SIM) to increase its extremely low oral bioavailability

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(< 5%) and prolong its antihyperlipidemic effect. NLCs were prepared via emulsification-solvent

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evaporation technique followed by ultrasonication, and the effect of composition of the nanocarriers

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on the particle size, size distribution, surface charge, entrapment efficiency, drug release kinetics and physical stability was extensively studied. NLCs exhibited nanosized (< 200 nm) spherical

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morphologies with narrow size distribution and high drug entrapment efficiency (> 75%), sustained

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drug release pattern, and negative surface charge (zeta potential of -35-40 mV) that imparts sufficient electrostatic physical stability. When tested in vivo, SIM-NLCs of the optimal

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composition demonstrated improved and prolonged reduction in the total cholesterol and non-high

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density lipoprotein cholesterol levels, as compared to the drug suspension. After oral administration of a single dose of SIM-NLC, 4-fold increase in bioavailability was observed, as compared to the

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SIM suspension. Hence, NLCs might provide efficient nanodevices for the management of hyperlipidemia and promising drug delivery systems to enhance SIM oral bioavailability. Keywords: Nanostructured lipid carriers, simvastatin, hyperlipidemia, drug delivery, nanoparticles.

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1. Introduction Elevated blood cholesterol level (i.e. hypercholesterolemia) leads to development and progression of atherosclerosis, and consequently cardiovascular diseases, and it is estimated to result in 2.6

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million deaths (4.5% of total mortality) globally [1]. Successful treatment of hypercholesterolemia substantially reduces morbidity and mortality from cardiovascular diseases [2-3]. Statins have the ability to retard the accelerated atherosclerosis in hyperlipidemic individuals. Simvastatin (SIM) is one of the statins family, which is considered as the first-line treatment of hypercholesterolemia, dyslipidemia and coronary heart diseases [4]. SIM is a potent inhibitor of 3-hydroxy-3-methyl-

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glutaryl-coenzyme A (HMG-CoA) reductase which converts HMG-CoA into mevalonate, a

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precursor in cholesterol synthesis. The biopharmaceutical classification system classifies SIM as

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class 2 drug (i.e., low aqueous solubility and high permeability) with short plasma half-life and

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variable absorption [5]. SIM is exposed to extensive first pass metabolism by cytochrome P3A

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(CYP3A) in the intestinal guts and the liver [6]. The oral bioavailability of SIM in its intact form is

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ca. 5% due to the slow dissolution rate in the intestinal fluid and significant first-pass metabolism [7]. Various approaches have been utilized to enhance the bioavailability of SIM, such as self

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microemulsification [8], nanoemulsification [9], nanoparticles [10], solid lipid nanoparticles [11], nanocrystals [12] and nanosuspensions [13].

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Nanocarriers including biodegradable polymeric nanoparticles [14], polymeric micelles

[15], nanocrystals [16], nanosuspensions [17] and lipid colloidal nanocarriers have received

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significant consideration as delivery systems for several drugs. Lipid nanocarriers composed of natural or synthetic lipids have the advantage of controlling drug release with high biocompatibility. Solid lipid nanoparticles (SLNs) have emerged in the early nineties as an alternative carrier system to the existing traditional lipid carriers due to the feasibility of large scale production, low toxicity and availability of their excipients [18]. However, the solid lipid matrix of 4

SLNs results in the formation of a relatively perfect crystal lattice leaving a limited space for drug incorporation, which limits the loading capacity and leads to drug leakage during storage [19]. Nanostructured lipid carriers (NLCs), the second-generation of lipid nanoparticles, are composed of

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a mixture of spatially different lipid molecules, i.e., a solid lipid is blended with a liquid lipid. The addition of liquid lipid distorts the formation of perfect lipid crystals, and, thus increasing drug loading capacity, and decreasing particle size and risk of gelation and drug leakage during storage [20-21]. Additionally, NLCs promote oral absorption of encapsulated drugs via selective uptake through the lymphatic route or the payer’s patches [22]. NLCs have been previously utilized for

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enhancing the oral bioavailability of various drugs [23-24]. Moreover, SIM-NLCs have been

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reported earlier to improve SIM oral bioavailability, although pharmacodynamic profile was not

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studied [7]. Pharmacokinetic parameters are not sufficient to predict the pharmacodynamic activity

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of statins [25]. In the current study, SIM-NLCs are prepared via emulsification-solvent evaporation technique followed by ultrasonication, and the effect of composition on the physicochemical

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characteristics of the formulations has been extensively studied. In vitro release kinetics and

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stability of SIM-NLCs were also evaluated. In vivo pharmacokinetics and pharmacodynamics of

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SIM-NLCs have been investigated in normal rats and poloxamer-induced hyperlipidemic Wister rat model, respectively, to evaluate the ability of the developed nanocarriers to improve the oral

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bioavailability and antihyperlipidemic activity of the drug.

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2. Materials and methods 2.1. Materials Stearic acid was purchased from El-Nasr Chemicals Co. (Abu Zaabal, Egypt). Oleic acid was purchased from Alpha Chemicals (Cairo, Egypt). Simvastatin was purchased from El-Ryad Pharma (El-Ryad, KSA). Pluronic F-68 was provided by BASF (Luwigshafen, Germany). Lecithin was 5

purchased from Sigma-Aldrich Co. (St. Louis, MO). Other chemicals and reagents were of analytical grades. 2.2. Preparation of simvastatin-nanostructured lipid carriers (SIM-NLCs)

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SIM-NLCs were prepared via emulsification-solvent evaporation technique followed by ultrasonication as reported earlier with slight modifications [26]. Briefly, certain amount of SIM, stearic acid, oleic acid, and lecithin were dissolved in chloroform and heated at 70 °C. Aqueous phase containing the surfactant (Pluronic F-68) in 10 mL of deionized water was heated to 70 °C. The aqueous phase was then added to the lipid phase under stirring at 1200 rpm and 70 °C, and

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mixed for 5 min. The obtained pre-emulsion was sonicated by probe-type sonicator (Cole-Parmer,

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Vernon Hills, IL) for 7 min (40 W). Then, the dispersions were cooled down to the room

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temperature under agitation at 300 rpm for 1 h to obtain the aqueous NLC dispersions. Purification

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of SIM-NLCs was performed via dialysis into 250 mL of double distilled water and stirred at 300 rpm for 24 h, to remove the free drug. The dialysis water was replaced with fresh double distilled

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water after 12 h. The concentration of the drug was adjusted to ensure that the purification process

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is carried out under sink conditions (i.e. the dialysis volume exceeds the volume required to

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solubilize the total amount of the drug). Eight different formulations of different compositions were prepared (F1-F8, Supplementary Information, Table S1). The effects of oleic acid-to-stearic acid

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ratio and surfactant (Pluronic F-68) concentration on the physicochemical characteristics of the formulations were evaluated.

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2.3. Characterizations of the prepared SIM-NLCs 2.3.1. Determination of the entrapment efficiency NLCs containing SIM were separated from the free drug via dialysis. The drug was then released from the NLCs samples by addition of sufficient volume of methanol. The drug content was assessed spectrophotometrically at 238 nm using UV-visible spectrophotometer (Shimadzu 6

Seisakusho, Ltd., Kyoto, Japan). The entrapment efficiency (EE, %) was then calculated according to Equation 1: Entrapment efficiency (%)

=

amount of drug entrapped into the nanoparticles ×100 amount of drug initially added

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2.3.2. Particle size and zeta potential

(1)

Mean particle size (nm) and polydispersity index (PDI) of the prepared formulations were measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) equipped with a backscattered light detector operating at 173°. The zeta potentials (mV) were

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measured by laser Doppler anemometry using the same Malvern Zetasizer Nanoseries instrument.

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All samples were diluted ten times with double distilled water prior to measurements. All the

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measurements were performed at 25 °C in triplicates.

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2.3.3. In vitro release study

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In vitro release of SIM from the NLCs was studied under sink conditions using the dialysis method

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[5]. The dialysis bag (molecular weight cut off: 12-14 kDa) was soaked in distilled water for 12 h before use. Nanocarriers (equivalent to 1 mg of the drug) were placed into the dialysis bag and

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immersed in 100 mL of phosphate-buffered saline (PBS, pH 7.4) in a beaker. The beaker was placed in a thermostatic shaker (Gesellschaft für Labortechnik mbH, Burgwedel, Germany) at 37

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°C and 100 rpm. Five milliliter aliquots were withdrawn at pre-determined time intervals (0.5, 1, 2, 4, 6, 8 and 24 h), and replaced immediately with equal volumes of fresh PBS to maintain the same

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volume. The release of free SIM (SIM suspended in PBS) was examined as a control. The withdrawn aliquot at each time interval was analyzed for the amount of SIM spectrophotometrically at a wavelength of 238 nm. The cumulative amount of drug release was calculated as a function of time [27]. The release kinetics were analyzed by curve fitting to different kinetic models of zero order, first order, Higuchi and Korsemeyer-Peppas model [28-30]. 7

2.3.4. Stability studies Samples of the prepared formulations were stored in sealed glass vials at room temperature or at 4 °C for 30 days. Samples from each batch were withdrawn at definite time intervals and evaluated

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for physical appearance, particle size and zeta potential. In addition, further experiments were performed to study the changes of particle characteristics (size, PDI, zeta potential) for the selected optimal formulation (SIM-NLCs, F4) in media of different pHs (1.2, 6.8, 7 and 7.4). The particle characteristics were assessed immediately after mixing with the buffers (t0) and 2-h after the

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incubation (t2).

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2.3.5. The particle morphology

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Morphology of the formed particles was studied by transmission electron microscopy (TEM).

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Samples were prepared by placing a drop of SIM-NLCs onto a 400-mesh copper grid coated with a carbon film, and left for air drying. TEM images were then observed at an acceleration voltage of

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80 kV by TEM (JEM 100 CX11, Japan). The surface morphology of SIM-NLCs was visualized by

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scanning electron microscopy (SEM, JSM-5400 LV; JEOL, Japan). Samples were prepared by placing a droplet onto an aluminum specimen stub, dried overnight, and sputter-coated with gold

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prior to imaging. An acceleration voltage of 15 kV was utilized for SEM visualization.

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2.3.6. Fourier transform-infrared spectroscopy Prior to characterizations by the Fourier transform-infrared (FT-IR) spectroscopy, differential

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scanning calorimetry and x-ray diffractometry, SIM-NLCs were frozen at – 80 °C overnight prior to the lyophilization process. Freeze-drying was performed using Alpha 2–4 LD plus freeze-dryer (Martin Christ GmbH, Osterode, Germany) and the operating conditions were set at a temperature of – 60 °C and a pressure of 0.011 mbar. The FT-IR spectra of the selected formulations (F4 and F6), corresponding physical mixtures and the individual solid components were recorded using FT8

IR spectrophotometer (IR-470; Shimadzu, Kyoto, Japan). Samples were mixed with potassium bromide (spectroscopic grade) and compressed into disks using hydraulic press before scanning from 4,000 to 500 cm-1.

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2.3.7. Differential scanning calorimetry The thermal behaviors of the selected formulations (F4 and F6), corresponding physical mixtures and the individual solid components were investigated by differential scanning calorimetry (DSC60; Shimadzu Corporation, Tokyo, Japan). Samples of 4–7 mg were sealed in aluminum pans and

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heated over a temperature range of 40-200 °C at a constant rate of 10 °C/min. Thermal analysis data

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were recorded using a TA 50I PC system with Shimadzu software programs. Indium standard was

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used to calibrate the DSC temperature and enthalpy scale. N2 was used as purging gas at a rate of

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50 mL/min. An empty sealed aluminum pan was used as a reference. 2.3.8. X-ray diffraction

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X-ray diffraction (XRD) patterns were studied to identify the crystal form of SIM dispersed in the

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lipid matrix. XRD patterns of the selected formulations (F4 and F6), corresponding physical

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mixtures and the individual solid components were recorded by Philips X-ray diffractometer model PW 1710 (Philips, Amsterdam, Netherlands) with CuK α radiation (λ = 1.5405 Å), at a voltage of

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40 kV and 30 mA current. Samples were scanned at room temperature from 2θ = 5° to 2θ = 50° with a step of 0.06°/min.

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2.4. In vivo studies In vivo experiments were carried out to study the pharmacodynamics and pharmacokinetics of SIMNLCs as compared to SIM suspension after oral administration in rats.

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2.4.1. Experimental animals The research protocol was reviewed and approved by the Institutional Animal Ethical Committee of the Faculty of Pharmacy, Assiut University, and it adheres to the Guide for the Care and Use of

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Laboratory Animals, 8th Edition, National Academies Press, Washington, DC. Male Wister rats (200-250 g) were obtained from the Animal House at the Assiut University Faculty of Medicine. Rats were allowed to acclimatize to the experimental conditions of temperature and humidity one week prior to the experiments. The rats were fed a standard rat pellet diet and allowed free access to water. The rats were maintained at a temperature of 25 ± 2 °C with a 12 h dark/light cycles

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2.4.2. Pharmacodynamic study of SIM-NLCs

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throughout the study.

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The anti-cholesterolemic effects of different SIM-NLCs and SIM suspension were studied using poloxamer-induced hyperlipidemic rat model [31-32]. Poloxamer is a non-ionic surfactant that

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induces hyperlipidemia via indirect stimulation of HMG-CoA reductase enzyme, which is involved

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in cholesterol biosynthesis [19-21]. Twenty rats were divided into five groups, four animals in each group, namely, normal group receiving normal saline, hyperlipidemic control group receiving

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water, standard group receiving free drug in a form of suspension, and two test groups receiving the

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selected formulations (F4 and F6). The drug suspension was prepared by suspending 10 mg of SIM in 10 mL of distilled water containing 0.5% w/v sodium carboxymethyl cellulose. The rats were

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fasted overnight prior to the study with free access to water. Hyperlipidemia was induced by a single intraperitoneal injection of 1 g/kg poloxamer F-127 solution (20% w/v). The rats were orally dosed with multiple doses of drug suspension and formulations after 12 h of the poloxamer injection for three days (dose = 5 mg/kg/day). Blood samples were withdrawn at 0, 12, 36 and 60 h.

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For further investigation of the sustained antihyperlipidemic activity of SIM-NLCs in comparison to SIM-suspension, pharmacodynamic study during the first 24 h after single oral administration of the different treatments was also examined. Eight rats were divided into two

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groups (four rats in each group) for the oral administration of a single dose of the selected SIMNLCs to one group and the drug suspension to the second group (dose = 5 mg/kg). Multiple blood samples were collected before drug administration and 2-, 6-, 12- and 24-h following drug administration. Blood samples were allowed to clot for 15-30 min at room temperature, and serum was separated by centrifugation at 3000 rpm for 10 min (Sigma Laborzentrifugen GmbH, Osterode

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am Harz, Germany). The serum samples were then frozen at – 20 °C and stored for further analysis

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[33].

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Blood serum was analyzed for total cholesterol (TC) and high density lipoprotein

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cholesterol (HDL-C) levels. TC levels were determined by enzymatic, colorimetric assay, and HDL-C levels were measured by homogeneous enzymatic colorimetric assay on Roche/Hitachi

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cobas c 311 analyzer (Roche Ltd. Mannheim, Germany). The non-HDL-cholesterol (non-HDL-C)

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concentrations (i.e. cholesterol concentrations in the lower density lipoproteins (chylomicrons, low

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and very low density lipoproteins)) were calculated by subtracting HDL-C from the TC. Moreover, the % of initial TC level and % of initial non-HDL-C level were also calculated at each time point

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according to Equations 2 and 3:

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% of initial TC level =

Measured TC level ×100 Baseline TC level

% of initial non-HDL-C level =

(2)

Measured non-HDL-C level ×100 (3) Baseline non-HDL-C level

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2.4.3. Pharmacokinetic study Pharmacokinetic study was performed to investigate the ability of NLCs to improve SIM oral bioavailability. The rats were randomly divided into two groups (three animals per group) and

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received a single oral dose of SIM (20 mg/kg) in the form of free drug suspension for the first group and SIM-NLCs for the second group. Although this dose is higher than the clinically used dose, pharmacokinetic studies in rodents indicate that higher statin doses (as compared to human) are required to achieve similar effective concentrations [13-14]. Hence, we have selected the dose of 20 mg/kg/day, which is consistent with doses that have been used in other reported pharmacokinetic

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studies for SIM [15]. Also, a higher dose (20 mg/kg) was used in the pharmacokinetic studies, as

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compared to the pharmacodynamic studies (5 mg/kg), to allow for the detection of low

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concentrations of the drug in the plasma (on the nanogram scale). All formulations were freshly

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prepared. All rats were fasted overnight prior to the administration of the formulations and were fed

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again 4 h after treatments. Blood samples (0.5 mL) were obtained via vein puncture from the caudal

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vein at designated time points (1, 2, 4, 8, 12 and 24 h after administration) and were transferred into heparinized tubes. The plasma samples were separated by centrifugation at 3000 rpm for 10 min.

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The plasma samples were then stored frozen at – 20 °C for further analysis. The SIM concentrations in plasma were analyzed using a reversed phase-HPLC as

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described previously, with some modifications [34]. The HPLC method was performed on a Dionex Ultimat 3000 UHPLC system (Thermo Scientific, Waltham, MA) equipped with a HPG-3200 RS

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pump, a DAD-3000 RS detector, a WPS-3000TRS analytical autosampler, and a Hypersil BDS C18 analytical column (dimensions of 150 mm x 4.6 mm ID x 5 μm). The mobile phase composed of acetonitrile:deionized water (65:35 v/v) and was adjusted to pH 3.5 by phosphoric acid. The flow rate was set at 1.0 mL/min, the injection volume was 20 μL and elute was analyzed with DAD detector set at a wavelength of 239 nm. Acetonitrile (1 mL) was added to the plasma sample (200 12

µL) to precipitate the plasma proteins, and the samples were then vortexed and centrifuged at 4000 rpm for 15 min [35]. The supernatant was collected, filtered through 0.45 µm syringe filter (Millipore, Billerica, MA), and analyzed using the HPLC. A calibration curve of SIM in the plasma

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was constructed using blank plasma spiked with standard SIM solutions to obtain a concentration range of 0.01–10 µg/mL. The spiked plasma samples were then subjected to the same extraction procedure described previously.

The pharmacokinetic parameters were calculated by fitting the plasma concentration–time data to a suitable model using Win Nonlin Professional Edition software version 2.0 (Science

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Consulting, Apex, NC). The area under the plasma concentration-time curve from zero to infinity

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(AUC0-∞) was calculated using the trapezoidal rule method. The maximum concentration (Cmax),

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time to reach the maximum concentration (Tmax), elimination half-life (t½), elimination rate constant

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(Kel), mean residence time (MRT), clearance (CL) and volume of distribution (Vd) were calculated. The relative bioavailability (Fr, %) of SIM-NLCs after oral administration was calculated according

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to Equation 4:

(AUC)SIM-NLCs SIM suspension

×100

(4)

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Relative bioavailability (Fr, %) = (AUC) 2.5. Statistical analysis

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Experiments were performed in triplicates unless otherwise indicated. Statistical significance for differences between experimental groups was assessed by one-way analysis of variance or two-

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sided Student’s t-test for pairwise comparison (GraphPad Prism 6.0, GraphPad, San Diego, CA). Differences between means were considered statistically non-significant for p values > 0.05. The differences were considered as statistically significant for 0.05 > p ≥ 0.01, and highly significant for p < 0.01.

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3. Results and discussion 3.1. Preparation and characterizations of the SIM-NLCs SIM-NLCs were prepared via emulsification-solvent evaporation technique followed by

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ultrasonication. The effects of oleic acid-to-stearic acid ratio and Pluronic F-68 concentration were evaluated for further optimization of the formulations (Supplementary Information, Table S1). Since oleic acid concentration affects the viscosity inside NLCs, different formulations were prepared using constant Pluronic F-68 concentration (1.5 % w/v) and various oleic acid-to-stearic acid ratios (15, 30, 50, 70 and 80 wt% of oleic acid/total lipid), and coded as F1, F2, F3, F4 and F5,

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respectively. On the other hand, the surfactant at the aqueous/organic interface governs the

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effectiveness of the emulsification process and stabilization of the oil nanodroplets during the

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solvent evaporation process. Therefore, Pluronic F-68 was used at different concentrations of 1.5, 1,

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3 and 5 % w/v (coded as F4, F6, F7 and F8, respectively) while maintaining the oleic acid-to-stearic acid ratio constant. Evaluation of the prepared NLCs was carried out by measuring the particle size,

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PDI, zeta potential and entrapment efficiency.

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Table S2 (Supplementary Information) shows the particle size, PDI and zeta potential of

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the freshly prepared NLCs at different oleic acid and surfactant concentrations. All formulations were in the nanosized range, and demonstrated a highly significant decrease (p < 0.001) in both the

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particle size and PDI upon increasing oleic acid concentration from 15% to 70%. The decrease in size is due to the difference in viscosity between the liquid lipid (oleic acid) and solid lipid (stearic

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acid) [19,36]. The high oleic acid content reduced the viscosity inside the NLCs, and, thus reducing the interfacial tension and afford the formation of smaller particles with homogeneous size distribution and smooth surface [36-37]. On the contrary, there was an increase in particle size upon increasing the concentration of solid lipid (stearic acid) due to the increased viscosity and interfacial tension, which is consistent with previously reported data [37-38]. The small particle size 14

and high negative zeta potential values of NLCs indicate the stability of the formed colloidal dispersion. No change in the zeta potential values was observed upon increasing the oleic acid content of nanoparticles, which is consistent with previously reported data for clobetasol-loaded

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NLCs [36]. Regarding the effect of surfactant concentration, highly significant reduction (p < 0.001) in particle size and increase in the zeta potential values (p < 0.001) were observed upon increasing the surfactant concentration from 1 to 5% w/v (Supplementary Information, Table S2). The reduction of particle size might be attributed to the decreased interfacial tension between the lipid

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matrix and the aqueous phase upon increasing the surfactant concentration. The nonionic nature of

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the surfactant (Pluronic F-68) provides steric stabilization, and, thus preventing aggregation of the

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particles, and consequently preventing the coalescence of the droplets and preserving the physical

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stability of the lipid nanoparticles. Since Pluronic F-68 is nonionic, the increase of zeta potential values that is observed upon increasing the surfactant concentration can be attributed to the coating

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layers of the surfactant that shield the negative surface charge of stearic acid [39-40]. It was

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observed that there was no significant change in the PDI of NLCs upon increasing surfactant

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concentration from 1 to 3% (p > 0.05). On the other hand, further increase of surfactant concentration to 5% led to a highly significant increase (p < 0.01) of PDI of NLCs from 0.18 to

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0.30.

3.2. Entrapment efficiency

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The effect of concentrations of oleic acid and Pluronic F-68 on the EE of the drug in the prepared NLCs was also studied (Supplementary Information, Table S2). Increasing the concentration of oleic acid from 15 to 80 wt% resulted in a highly significant increase (p < 0.001) in the EE of the drug into the NLCs from 40.44 ± 2.7 to 78.40 ± 2.9%, respectively. The resulted improve in EE upon increasing oleic acid content is in agreement with a previous study, which reported that 15

incorporation of liquid lipids into solid lipids led to a massive crystal order disturbance that left enough space to accommodate drug molecules, thus leading to improved drug entrapment efficiency [36]. On the other hand, no significant change (p > 0.05) in EE was detected upon

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increasing surfactant concentration from 1 to 3 %. Moreover, further increase of surfactant concentration to 5 % led to a highly significant decrease (p < 0.01) in EE from 75.77 ± 4.9 to 54.58 ± 3.7%, which might be attributed to the hydrophobic nature of the drug that tends to diffuse out from the oil nanodroplets and solubilize in the micelles formed in the aqueous phase upon increasing the surfactant concentration [41]. Drug loading did not increase the size of NLCs, which

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is consistent with a previous report [42].

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3.3. In vitro drug release

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The release profiles of SIM from the lipid nanocarriers of various compositions were studied over 24 h (Figure 1). The drug release patterns demonstrated initial burst release followed by sustained

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release at a constant rate over 24 h. The initial rapid release may be attributed to the fast release of

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drug entrapped near the surface of the nanoparticles. The sustained release of drug after the initial

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burst release could be related to the lipophilic nature of SIM that is entrapped deeply in the core matrix of the NLCs. SIM in the core of the nanoparticles has a longer diffusion path to reach the

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surface, as compared to the drug entrapped near the surface [43]. Faster drug release patterns were observed upon increasing the concentrations of oleic acid (Figure 1A) and the surfactant

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concentration (Figure 1B). This enhanced drug release from the NLCs might be attributed to the significant decrease in particle size, and thus increasing the specific surface area and, subsequently, the release rate of the drug [36]. Table S3 (Supplementary Information) summarizes the release kinetics and correlation coefficients (R2) calculated for the tested formulations. The in vitro release data indicated that the release of SIM from NLC formulations followed first order kinetics except 16

for F1 (15 wt% OA) and F2 (30 wt% OA), which were fitted to a diffusion–controlled mechanism (Higuchi model). Korsmeyer-Peppas was utilized to provide further information on drug release mechanisms, whether it is Fickian (diffusion), non-Fickian (anomalous), or erosion-mediated (zero

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order) release. The values of the diffusional release exponents for all formulations were < 0.5 except for F6 (1% w/v Pluronic F-68), which indicates that Fickian mechanism is the dominant mechanism that controls the drug release from the nanoparticles. 3.4. Stability studies

The stability of the various NLC formulations was monitored at 4 ºC and at room temperature for

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30 days to assess their long-term stability (Supplementary Information, Table S4). The visual

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observation of all the prepared NLCs did not indicate any sign of gelation, creaming, color change

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or particle aggregation either at 4 ºC or at room temperature. After 4 weeks, it could be noticed that

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there were no significant differences (p > 0.05) in either particle size or zeta potential values for all the formulations except for F5, F7 and F8, which had a significant increase (p < 0.001) in the mean

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particle size after 30 days of storage. Among all NLC formulations, formulations F4 and F6 were

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selected for further investigations due to their small particle sizes, higher entrapment efficiencies,

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slower in vitro cumulative drug release, higher stability upon storage, and their lower surfactant contents. Moreover, neither F4 nor F6 exhibited any significant change (p > 0.05) in the EE after 30

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days of storage either at 4 ºC (F4: 70.64 ± 7.6%, F6: 66.30 ± 8.2%) or at room temperature (F4:

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68.62 ± 6.61%, F6: 51.30 ± 15%). The effect of pH on the particle size, PDI and zeta potential was also studied

(Supplementary information, Tables S5 and S6). At low pH, there was no significant change (p > 0.05) in size and PDI of the NLCs in comparison to the original sample (NLCs in water), which may be attributed to the steric stabilization effect of the nonionic surfactant (Pluronic F-68) [12]. At pH 7.4, there was a slight increase in particle size, which is consistent with earlier studies [12-13]. 17

The zeta potential of the SIM-NLCs reached 1.27 ± 1.67 mV when the pH was decreased to 1.2, which might be attributed to protonation of the anionic groups of lecithin and oleic acid, thereby reducing the net negative charge [14].

SC RI PT

3.5. Morphology of the SIM-NLCs Figure 2A shows the transmission electron micrograph of SIM-NLCs. The lipid nanocarriers appeared as spherical or elliptical vesicles with smooth surfaces. There were several dark spots on the spherical structures of the NLCs, which might be attributed to the liquid lipids sticking on the

U

surface of the lipid nanoparticles [44-45]. The average particle size was ca. 169.4 ± 33.9 nm (n =

N

20), which is in agreement with the DLS results (Figure 2C). Scanning electron micrograph clearly

A

demonstrates the spherical shape of the prepared NLCs with characteristic smooth surface (Figure

M

2B).

3.6. Fourier transform-infrared spectroscopy

D

The FT-IR spectra of the pure drug, the selected formulations (F4 and F6), physical mixtures, and

TE

the individual solid components, were recorded (Figure 3). The spectrum of pure SIM drug showed characteristic peaks at 3012 cm-1 (C=CH), 1698.9 cm-1 (aromatic C=O), 3551.4 cm-1 (aliphatic O-

EP

H) and 1466.7 cm-1 (benzene ring). This pattern is in agreement with previously recorded spectrum

CC

for the same drug [46]. The characteristic peaks of the drug were also observed in the FT-IR spectrum of the physical mixture of pure drug/Pluronic F-68 due to the absence of predominant

A

chemical interactions between the drug and the surfactant. However, in the spectrum of SIM-NLCs, the characteristic peaks of the drug disappeared, shifted or replaced by the peak of stearic acid, and thus indicating the drug entrapment in the lipid matrix [47].

18

3.7. Differential scanning calorimetry DSC thermograms of the pure drug, the selected formulations (F4 and F6), physical mixtures, and solid components, were recorded (Figure 4I). The thermogram of the pure SIM showed a sharp

SC RI PT

single melting peak at ca. 138.5 °C, which corresponds to the melting of the crystalline drug [7,11,34]. In the physical mixture of SIM and stearic acid, the characteristic peak of the drug disappeared with slight increase in the onset temperature of the stearic acid (62.2 °C). The disappearance of the drug characteristic peak can be ascribed to solubilization of the drug within the lipid matrix [47-48]. DSC thermograms of F4 and F6 showed a decrease in the onset of melting

U

peak of the used lipid and disappearance of the characteristic melting peak of the drug, which may

N

be attributed to drug solubilization within the lipid matrix in amorphous and/or molecularly

A

dispersed form [49]. On the other hand, the decrease in the melting temperature of the used lipid

M

might be due to the interactions between the solid lipid and the liquid lipid and the surfactant. Also, it may be attributed to the small particle size of the prepared NLCs, as a result of the kelvin effect,

D

which is described by the Thomson equation. According to this equation, small isolated particles

TE

(SIM-NLCs) would melt at a lower temperature than the bulk material (stearic acid) [38].

EP

3.8. X-ray diffraction

X-ray diffractograms of the pure drug, F4, F6, physical mixtures, and the individual solid

CC

components are presented in Figure 4II. The crystalline nature of pure SIM was demonstrated upon detection of the diffraction peaks in the XDR pattern. These characteristic diffraction peaks of the

A

pure drug were also detected on the XDR pattern of the physical mixtures, however, they disappeared in the diffractogram of SIM-NLCs, which clearly confirm that SIM did not crystallize in the lipid matrix. The crystalline nature of the pure lipid and surfactant was also confirmed by the diffraction peaks observed in the XDR. However the disappearance of the characteristic peaks of the surfactant on diffractogram of SIM-NLCs indicates that the crystallinity of Pluronic F-68 is lost 19

when it is adsorbed on the stearic acid nanoparticles, due to the change in the surfactant conformation [47].

SC RI PT

3.9. Pharmacodynamic study of SIM-NLCs Hypercholesterolemia is usually managed by lowering the serum concentration of TC and low density lipoprotein cholesterol (LDL-C) or non-HDL-C. Non-HDL-C contains more atherogenic cholesterol than LDL-C, and is considered as a more accurate measurement of the total amount of atherogenic particles in the circulation [50]. Moreover, recent investigations indicate that non-HDL-

N

U

C ≥ LDL-C is a predictor of coronary artery diseases [51]. SIM is a potent lipid-lowering drug that

A

suppresses the formation of bad cholesterol in the liver through inhibiting the HMG-CoA reductase

M

enzyme, thus resulting in substantial decrease in mortality from cardiovascular diseases. For statins, the pharmacokinetic parameters are not sufficient to predict the pharmacodynamic activity [52].

D

This issue has been adequately discussed in a previous study which reported that plasma

TE

concentrations of atorvastatin did not correlate with the reduction in LDL-C [25]. Therefore, the

evaluated.

EP

pharmacodynamics and pharmacokinetics of the selected SIM-NLCs and the drug suspension were

CC

After 24 h of poloxamer injection into the rats, there was ca. 5-fold increase in the TC level of the hyperlipidemic control group and the different treatment groups as compared to normal

A

group (data not shown). The rats were orally dosed with multiple doses of drug suspension and formulations after 12 h of the poloxamer injection for three days (dose = 5 mg/kg/day). Percentages of the initial (before drug administration) TC and non-HDL-C levels were estimated to evaluate the efficacy of the treatment in each group. Percentages of initial TC and non-HDL-C levels after oral administration of the SIM-NLCs (F4 and F6) and SIM suspension are presented on Figure 5I. The 20

TC level after 12 h was significantly higher (p < 0.001) in the control group as compared to the treated groups, which indicate the ability of the SIM-NLCs and the drug suspension to prevent the increase in TC levels (Figure 5, IA). After 36 h of the treatment, there was a further increase in the

SC RI PT

TC levels of the control rats, and rats treated with the SIM suspension and SIM-NLCs-F6. Interestingly, in the case of F4 formulation, the TC significantly decreased (p < 0.001). After 66 h of the treatment, F4 reduced the TC level to a highly significant level (p < 0.01), as compared to F6 and the drug suspension. Similar significant decrease (p < 0.001) in the non-HDL-C level was also observed for F4-treated group, as compared to F6 and the SIM suspension-treated groups (Figure

U

5, IB). The greater lipid lowering activity of the F4 (1.5% w/v Pluronic F-68) as compared to F6

N

(1% w/v Pluronic F-68) might be attributed to the higher Pluronic F-68 concentration of F4. The

A

presence of Pluronic F-68 might enhance the absorption of NLCs, due to its effect on the intestinal

M

epithelial permeability [53]. Furthermore, F4 has smaller particle size that might increase the absorption and dissolution rate, and thus improving the drug bioavailability [34].

D

For further investigation of the sustained antihyperlipidemic effect of the SIM-NLCs in

TE

comparison to the SIM suspension, single dose pharmacodynamic study was evaluated and the

EP

percentages of the initial TC and non-HDL-C levels were measured (Figure 5II). After 2 h of the treatment, the TC and non-HDL-C levels were significantly higher (p < 0.001) in the SIM

CC

suspension-treated group, as compared to the SIM-NLCs-treated group (F4). Moreover, the enhanced efficacy of the F4 remained detectable during the 24 h of the treatment period. On the

A

contrary, SIM suspension resulted in a detectable increase in TC and non-HDL-C levels above the baseline level, and this increase was continued during the 24 h of the treatment. The sustained antihyperlipidemic activity of the SIM-NLCs might lower the dose and frequency of administration, and thus improving patient tolerability and decreasing side effects of the drug.

21

3.10. Pharmacokinetic study Selected SIM-NLCs (F4) and SIM suspension were orally administrated as a single dose to male

SC RI PT

Wistar rats. SIM plasma concentrations were measured using a previously reported HPLC method, with some modifications [34]. SIM was completely separated as a sharp peak at a retention time of ca. 8.2 min without any interfering peaks. SIM plasma concentrations after oral administration of SIM-NLCs and the drug suspension are presented on Figure 6. The plasma concentration-time data were subjected to a non-compartmental analysis to determine the mean pharmacokinetic parameters

U

(Supplementary Information, Table S7).

N

The SIM plasma concentrations were remarkably higher after administration of SIM-NLCs, as

A

compared to those observed for SIM suspension. The Cmax for SIM-NLCs was significant higher

M

than the SIM suspension (89.86 ± 4.11 vs. 45.73 ± 12.15 ng/mL, p < 0.05). After 24 h of oral administration of SIM–NLCs, the SIM plasma concentration was 34.54 ± 1.52 ng/mL, whereas

D

SIM suspension resulted in a plasma concentration of 9.71 ± 1.55 ng/mL. The higher plasma

TE

concentrations observed following the oral administration of SIM-NLCs as compared to the drug

EP

suspension reflect the enhanced systemic absorption of SIM when incorporated into the NLCs. Similar enhancement of systemic absorption of the same drug was previously reported using other

CC

drug delivery systems such as solid lipid nanocarriers [7] and self microemulsifying drug delivery system [54]. In the case of SIM-NLCs, the pre-systemic hepatic effect can be largely avoided via

A

absorption through the lymphatic pathway [7]. Improved solubilization of SIM due to the transformation from crystalline to amorphous form is another reason for the increased systemic absorption [55]. The other factors that facilitate the absorption in the intestinal milieu are the high dispersibility of the NLCs and small particle size of the NLC formulation (< 200 nm) that resulted in a great increase in the surface area of the particles. Furthermore, NLCs could protect the drug 22

from enzymatic degradation by reducing the exposure of the drug to the intestinal bacteria, as well as, protecting the drug from the enzymatic degradation during the absorption process, and thus allowing for a longer residence time in the gastrointestinal tract [56]. In general, improved

SC RI PT

pharmacokinetic profile was observed when SIM was loaded into the NLCs (Supplementary Information, Table S7). The SIM-NLCs exhibited a delayed Tmax (2 h), as compared to that of SIM suspension (1 h), which could be attributed to the slow and sustained release of the drug from the NLCs. Longer elimination half-life of SIM-NLCs (35.04 ± 2.86 h) was also observed as compared to the SIM suspension (10.20 ± 0.47 h), which might aid in reducing the dose and

U

frequency of administration. The extent of SIM absorption from NLC (AUC0-∞) was significantly

N

higher (p < 0.001) than the AUC0-∞ of the SIM suspension.

A

The oral bioavailability of SIM after its incorporation into solid lipid nanoparticles was

M

improved by 3.37-fold [5]. Similarly, increase in SIM oral bioavailability from self microemulsifying drug delivery system (1.5-fold increase) [54] and nanosuspension (2.2-fold

D

increase) [13] have been also reported previously. Our group has utilized various strategies to

TE

improve stability and delivery efficiency of several drugs [14,57-61]. In the current study, the

EP

relative bioavailability of SIM-NLCs was 4-fold higher than for the drug suspension, indicating significant enhancement of oral delivery and bioavailability of SIM when incorporated into the

CC

NLCs. The rate and extent of systemic absorption of SIM were significantly increased, and thus improving the oral bioavailability of SIM. It is worth mentioning that no mortalities have been

A

observed in the treatment group and the hyperlipidemic control group in the in vivo experiments.

23

4. Conclusions In the present study, SIM-NLCs were successfully prepared and evaluated for their abilities to enhance oral delivery of SIM as compared to the drug suspension. Several in vitro characterizations

SC RI PT

of SIM-NLCs were carried out. SIM-NLCs of small particle size, high entrapment efficiency, sustained in vitro drug release, high stability and low surfactant concentration were selected for further characterizations. The in vivo pharmacodynamic study of the SIM-NLCs demonstrated a sustained and greater lipid lowering activity as compared to the free drug suspension. There was a 4-fold increase in the oral bioavailability of SIM-NLCs in comparison to the SIM suspension. The

U

developed NLCs might provide efficient nanodevices to enhance the oral delivery and prolong the

N

therapeutic effect of SIM, and thus improving the patient compliance by eliminating the need for

A

frequent dosing of the drug.

M

5. Disclosure

The authors report no conflicts of interest in this work.

D

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Figures

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N U SC RI PT A M ED PT CC E

Figure 1. In vitro release profiles of simvastatin from nanostructured lipid carriers, as compared to the drug suspension: (A) Different oleic acid concentrations and (B) Different surfactant (Pluronic F-68) concentrations. Data are presented as means ±

A

SD (n = 3).

29

SC RI PT U N A M D TE

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Figure 2. Transmission electron micrograph (A), scanning electron micrograph (B) and

CC

Intensity-, volume- and number-averaged hydrodynamic diameter histograms of the SIM-NLCs (F4) (C). The intensity-, volume- and number-averaged hydrodynamic diameters of SIM-NLCs

A

(F4) demonstrated on Figure 2C are 185.2, 175.9 and 120.9 nm, respectively.

30

SC RI PT U N A M

Figure 3. FT-IR spectra of the pure drug (SIM) (A), stearic acid (B), Pluronic F-68 (C), physical

A

CC

EP

TE

D

mixture (SIM/stearic acid) (D), physical mixture (SIM/Pluronic F-68 ) (E), F4 (F) and F6 (G).

I

31

SC RI PT U N

A

CC

EP

TE

D

M

A

II

Figure 4. DSC analysis (I) and X-ray diffraction patterns (II) of the pure drug (SIM) (A), stearic acid (B), Pluronic F-68 (C), physical mixture (SIM/stearic acid) (D), physical mixture (SIM/Pluronic F-68) (E), F4 (F) and F6 (G).

32

SC RI PT U N A M D TE EP

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Figure 5. (I) Changes in cholesterol levels in rats after administration of simvastatin suspension and simvastatin-nanostructured lipid carriers (F4 and F6) over 3 days. (II) Changes in the

A

cholesterol levels in rats after single dose administration of simvastatin suspension and simvastatin-nanostructured lipid carriers (F4). (A) Total cholesterol and (B) Non-high density lipoprotein-cholesterol. *p < 0.05 when compared to the drug suspension; ** p < 0.001 when compared to the drug suspension. Data are presented as means ± SD (n = 4).

33

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Figure 6. Plasma concentration–time profiles of SIM after single oral dose (20 mg/kg) of SIM

A

CC

EP

TE

D

M

A

N

suspensions and SIM-NLCs. Values are presented as means ± SD (n=3).

34